http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Lgdeer&year=&month=2010.igem.org - User contributions [en]2024-03-29T14:11:45ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Peking/ModelingTeam:Peking/Modeling2010-10-28T02:22:18Z<p>Lgdeer: /* Introduction */</p>
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Modeling Home</font></font></font><br />
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=Introduction=<br />
The biological systems are full of noises, and the response of bioreporters can be represented by a Hill Function. Noises will be significantly amplified and the output detected will be invalid at the ends of dose response curve.(Figure 1(a)) Therefore, The detection is limited at a narrow range. In order to solve the problem, we seek to transform the Hill Function curve to a straight line. As Figure 1(a) shows, the noise is reducted, and the sensitivity and detectable-range-characteristic of biosensors will be significantly improved. <br><br><br />
Aimed at finding a genetic circuit that can perform the transformation, a simple and universal one for practice convenience, we adopted the process of [[Team:Peking/Notebook/Vocabulary#Reverse engineering|Reverse engineering]] which in our work means to enumerate all possible networks and analyzed whether they function well, thus getting the right topology. In details, we first express the target functionalities in quantitative characters and set numerical standards according to our expectations, next define the network for simplest circuit search and derive equations precisely describing it, then calculate the characters using the equations and compare them with standards, then further analyze the comparison results and find the simplest functional network. Finally, to fully understand it and provide enough information for application, we analyze the mechanism and the parameter preferences, as well as exploring whether the very circuit is necessary for performing the function well.<br />
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Here we name our target functionality as Input-Output Alignment(IOA). In order to define IOA precisely for need of calculation, we considered most important characters of IOA and adopted Pearson Correlation Coefficient r to represent Input-Output Linear Relationship in the overall search work ( when r>0.99 we consider the network topology having the IOA function ), and also, regulated two levels for the initial and ultimate output concentration for the second character – the output range in further search work.(Figure 1(b))<br />
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<font size=1.5><b>Figure 1</b> (a) The transformation reducts noises and make output more recognizable.(b)Factors for selection of IOA network topologies. r is the Pearson Correlation Coefficient and the output range is HIGHLEVEL minus LOWLEVEL.</font> <br />
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=Calculating Process=<br />
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In this part, we will demostrate our calculating process in three sections--Network enumeration, Equations set up and network topologies’ analysis.<br />
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<a href="https://2010.igem.org/Team:Peking/Modeling/CalculationProcess">== Learn more ==</a><br />
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=Analyses and Results=<br />
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Here we analyzed the results we obtained and found the final right topology.<br />
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<a href="https://2010.igem.org/Team:Peking/Modeling/Analysis">== Learn more ==</a><br />
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=Advanced Model=<br />
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When the range of input is very large, the IOA networks no longer meet out needs and so we tried to find one semilog-linear topology in this section.<br />
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<a href="https://2010.igem.org/Team:Peking/Modeling/Advanced_Model">== Learn more ==</a><br />
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=References=<br />
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1 Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. & Walter, P. (2008). Molecular Biology of the Cell (Garland, New York).<br><br />
2 Nicolas E. Buchler, Ulrich Gerland, Terence Hwa (2005). Nonlinear protein degradation and the function of genetic circuits, PNAS 102, 9559-9564.<br><br />
3 John D. Helmann, Barry T. Ballard, Christopher T. Walsh (1998). The MerR Metalloregulatory Protein Binds Mercuric Ion as a Tricoordinate, Metal-Bridged Dimer. Science 247, 946-948.<br><br />
4 Diana. M, Ralston and Thomas V. O Halloran (1990). Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex. PNAS 87, 3846-3850.<br><br />
5 Uri Alon (2007). An Introduction to Systems Biology—Design Principles of Biological Circuits[M], Chapman&Hall/CRC.<br />
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6 Wickner, S., Maurizi, M. R. & Gottesman, S.(1999). Posttranslational Quality Control: Folding, Refolding, and Degrading Proteins. Science 286, 1888–1893.<br><br />
7 W. Ma, A. Trusina, H. El-Samad, W.A. Lim, C. Tang (2009). Defining network topologies that can achieve biochemical adaptation. Cell 138, 760–773.<br><br />
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<b>Download the model part materials</b><br />
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<a href="https://static.igem.org/mediawiki/2010/7/73/PKU_Model_Part_One.pdf"><font color=000000><img src="https://static.igem.org/mediawiki/2010/9/91/PKU_Adobe_Reader_Logo.jpg" width=20><b><i>PART ONE</i></b></font></a><br />
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<a href="https://static.igem.org/mediawiki/2010/b/bc/Wiki_semilog.pdf"><font color=000000><img src="https://static.igem.org/mediawiki/2010/9/91/PKU_Adobe_Reader_Logo.jpg" width=20><b><i>PART TWO</i></b></font></a><br />
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Any problem, please contact with <b>Zhenzhen Yin</b><i> evamail.pku AT gmail.com </i>Or <b>Yuheng Lu</b> <i>lgdeer AT gmail.com</i><br />
<b>For more clear pictures, please click on it.</b><br />
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</html></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Application/KitOperationTeam:Peking/Project/Application/KitOperation2010-10-28T02:18:20Z<p>Lgdeer: </p>
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Application|Application]] > [[Team:Peking/Project/Application/KitOperation|Kit Operation]]<html><br />
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Here is a general view of how our heavy metal decontamination biokit functions. Our biokit consists of two parts: the bioreporter and the bioabsorbent. We can firstly determine the heavy metal pollution level by the bioreporter, and then clean the contaminated water exploiting the bioabsorbent.<br />
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When heavy metal emerges, a tri-node response will be switched on in our biosensor (Fig.1). Different strains with parameter variations between the nodes will have different response threshold to heavy metal ions, such as mercury. <br />
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'''Fig 1. Structure of Tri-node response system. According to the results of modeling, we designed the specific genetic circuit to realize the linear response function. Node A is a generator of MerR. For Node B, the gene is the activator which can activate the psid promoter. Node C is GFP whose expression was driven by Psid (activated by activator) and PmerT (activated by MerR). '''<br />
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'''Fig 2. Our bioreporter with tri-node response system will possess a linear transfer function (right) rather than the natural hill function (left). '''<br />
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Before wiki freezing, data collecting of the final result (linear transfer function transformed from hill function) were still under progressing. Primary result demonstrated that it worked as expected, which will be show at Jamboree.<br />
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If the transfer function of bioreporter response represents linear , the working range of the bioreporter will be expanded, and the error rating will be reduced. This type of bioreporter will be excellent for in lab accurate measurement or heavy metal pollution assessment. <br />
To determine the pollution level in field, we developed an assay easy to understand and accessible to the general public, called traffic light bioassay. By using bacteria strains with different sensitivity threshold to mercury, the heavy metal concentration can be easily determined by the number of reporter strains reacting to a sample, which is representative to different heavy metal concentration range (Fig.3). <br />
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'''Fig 3. The schematic sketch of how the biosensor functions.''' <br />
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'''Fig 4. The primary result of traffic light bioassay, which was performed in the 96-well plate. The response of whole-cell bioreporter incubated with simulated mercury containing polluted water was recorded by digital image at 10h, 15h, 20h and 30h. 3 replicates of 1 biosensor strain behaved very similarly with respect to indicating the mercury concentration range in a wide time window. Surprisingly, the biosensor strain we selected was able to response to 7*10^-9M mercury (II) with repeatability and the color contrast of mercury concentration near the threshold seemed to be good. '''<br />
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After the heavy metal concentration determined, it’s time for our bioabsorbent to decontaminate the heavy metal ions from water. Firstly, bacteria switch on MBP generating device and facilitation module to get ready-to-use (Fig 5A). Then bioabsorbent will be applied to the contaminated water, to absorb heavy metal ions, for instance, mercury (II) with high performance (Fig 5B). At the same time, a genetic cascade will amplify the input (presence of mercury) and Ag43 will be expressed finally with a time delay and high intensity. When the bacteria sedimentate, post treatment will be ready to be conducted (Fig 5C). <br />
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'''Fig. 5 The schematic of how our bioabsorbent functions. A: bioabsorbent gets ready-to-use; B: bioabsorbent absorbs heavy metal ions from water; C: bioabsorbent autoagretate at a population level.'''<br />
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To better demonstrate the function and performance of our bioreporter, we used the characterized indicator for direct visualization of mercury detoxification and bacterial aggregation process, using method described at MBP Expression Page of our wiki. <br />
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'''Fig. 6 Direct visualization of mercury detoxification and bacterial aggregation process. Water-soluble metal indicator with colorimetric selectivity for heavy metals was exploited to indicate the mercury concentration in the water. The lower limit of metal concentration for color transition was 0.8×10-5M. (A) The indicator represented rosy color, indicating that high concentration of mercury existed (for more details, see our MBP Expression Page). (B) When applied with our mercury bioabsorbent, after shaking cultured at 37℃ for 10 min, evident color change emerged, indicating that the absorption was under progressing. (C) 30 min later, bacteria aggregated even under slight shaking cultivating at 37℃ in an aslant tube, forming a visible pellet at the bottom. It’s notable that mercury had been decontaminated because the significant color change of the indicator.'''<br />
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To conclude the above work, we have developed a extensible method to construct heavy metal decontamination kits consisting of efficient bioreporters and bioabsorbents both for in field application and in lab use. Compared with the technologies we have now, this biological engineering approach gives advantages to its bioavailability assessment, relatively low cost and less resource consumption. More importantly, it clearly points the way of future development. As a revolutionary progress, we believe, the conceptual advancement if of great value when the potential of bio-decontamination is unveiled. At present, it is highly probable that bioremediation is confined in the laboratory study, taking possible defects of mass application and public panic over transgenic products into consideration. However, as long as this promising field is constantly being probed into, we are firmly convinced that, there will be more experts devoted into relevant research, thus optimizing the bio-decontamination method. This is not remote, but imminent; for the biological science and technology is marked by tremendously rapid and ever-accelerating change. Hopefully, we are involved in the exciting improvement and contribute to the rescuing of the environment and human beings ourselves. <br />
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</html></div>Lgdeerhttp://2010.igem.org/Team:Peking/Notebook/ZZYinTeam:Peking/Notebook/ZZYin2010-10-28T02:15:20Z<p>Lgdeer: /* 10.20-10.24 */</p>
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Zhenzhen Yin's Notes</font></font></font><br />
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Using computional methods to search for proper topology whose input and output have a linear correlationship. Additionally They conduct modeling to explain and simulate the operation mechanism of MerR family TFs on their cognate promoters.<br />
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=='''Contents'''==<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZZYin#June| June, 2010]]</span><br />
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZZYin#July| July, 2010]]</span><br />
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/JZZYin#August| August, 2010]]</span><br />
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZZYin#September| September, 2010]]</span><br />
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZZYin#October| October, 2010]]</span><br />
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==June==<br />
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===6.19-6.22===<br />
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*Paper reading, learning about Stochastic Models and network types such as bifan networks<br />
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==July==<br />
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===7.5-7.7===<br />
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*Paper Reading: Negative feedback that improves information transmission in yeast signaling &one master thesis of Long Yan, Peking University.<br />
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*Learn about wiki language.<br />
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===7.9-7.11===<br />
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*Construct the wiki website.<br />
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===7.12===<br />
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*Meet with Qi Ouyang and Xiaomeng Zhang<br />
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**Discuss about Reverse Engineering Method and decide to adopt it for designing our project.<br />
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**Decide to search for input-output linear function for making up the shortages of current biosensors.<br />
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*Discuss with Yuheng Lu and Haoqian Zhang<br />
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**Decide to use the tri-node network and the essence of each TF Node is still to be discussed.<br />
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*Discuss with Yuheng Lu<br />
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**Decide to use TF as the essence of each TF node.<br />
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**Start to derive the ODEs.<br />
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**Try to search for the right form of equations to describe the combinational regulation of TFs.<br />
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*Paper reading: On schemes of combinatorial transcription logic <br />
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**Try to find the answer to the problem discussed today.<br />
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===7.14-7.19===<br />
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*Continue paper reading and search for some other materials and papers about combinational regulation.<br />
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*Derive the ODEs to describe the tri-node network. <br />
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*Fix the LINUX system on my laptop and start to familiar with coding under LINUX, study about vim operator, gcc and gdb.<br />
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===7.31===<br />
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*Meet with Xiaomeng Zhang<br />
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**Discuss the equations derived to see whether it can describe our network precisely.<br />
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**Discuss how to derive the equations describing the binding of Hg to MerR dimer.<br />
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**Discuss whether the hypotheses of our equations fit for the real condition.<br />
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*Discuss with Yuheng Lu<br />
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**Develop our equations and discuss about the paper.<br />
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**Find out that the calculation may be too much for our computer that has 16 CPUs in the laboratory so decide to take a more simple form to only consider the combinational regulation instead of also considering the interactions between TFs and RNAP.<br />
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==August==<br />
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*Derive one set of equations and discuss with Yuheng Lu to revise the equation.<br />
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*After derivation, get the ODEs meet all of our requirements, and so the equations are obtained.<br />
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*Examine again the ODEs to see whether there’s any problem with them.<br />
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===8.3===<br />
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*Meet with Xiaomeng Zhang<br />
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**Examine again our ODEs<br />
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*Start to code the object function and read the makefile codes. <br />
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*Code the object function and other related function codes.<br />
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*Put the codes together with Yuheng Lu’s. <br />
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*Examine the codes.<br />
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===8.16=== <br />
*Meet with Xiaomeng Zhang<br />
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**Examine our codes.<br />
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**Discuss running the program on laboratory’s computer in details.<br />
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===8.17-8.19===<br />
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*Examine the codes again and again with gdb because there’re some bugs when running on the computer.<br />
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===8.21===<br />
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*Try to run the program and find some bugs still present.<br />
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*Revise and examine the codes.<br />
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===8.22===<br />
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*Run again and examine a small part of the results, find them fit the expectation.<br />
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===8.23-8.25===<br />
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*Wait for the program running on the computer.<br />
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===8.26-9.3===<br />
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*Get our results and start to analyze it with Matlab and Excel and SPSS.<br />
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==September==<br />
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===9.4=== <br />
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*Meet with Qi Ouyang <br />
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**Get the guidance to revise our object function for significance in field use<br />
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*Revise the object function and run again the program on computer.<br />
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*Wait for the program running on the computer.<br />
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*Get the results and analyze them.<br />
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*Write wiki modeling part.<br />
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===9.22===<br />
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*Meet with Qi Ouyang<br />
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**Get some advice about how to revise the wiki modeling part.<br />
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==October==<br />
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===9.26-10.10===<br />
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*Revise wiki and draw the figures with Matlab and origin and PowerPoint that used in wiki.<br />
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*Further analyze the second results.<br />
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===10.14===<br />
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*Work on some drawings need in the biosensor wiki part.<br />
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===10.16===<br />
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*Work on the Modelling Part Presentation’s PPT<br />
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*Meet with members and Discuss about the PPT.<br />
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*Prepare and present the PPT for the first time.<br />
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===10.20-10.24===<br />
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*Revise the PPT and the lecture notes again and again.<br />
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===10.25-10.27===<br />
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*Upload wiki materials.<br />
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===10.27-11.4===<br />
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*Prepare for the presentation. <br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Parts/ExistedTeam:Peking/Parts/Existed2010-10-28T02:10:31Z<p>Lgdeer: </p>
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__NOTOC__<br />
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<br><br><br />
<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Parts Characterization</font></font></font><br />
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[[Team:Peking/Parts|Parts]] > [[Team:Peking/Parts/Existed|Existing Part's Improvement or Characterization]] <html><br />
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=Characterization of Existing Parts on Registry=<br />
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<br />
==Content==<br />
*[[Team:Peking/Parts/Existed#CrtEBI Characterization| CrtEBI Characterization]]<br />
<br />
*[[Team:Peking/Parts/Existed#CrtEBIY Characterization| CrtEBIY Characterization]]<br />
<br />
*[[Team:Peking/Parts/Existed#Reference| Reference]]<br />
<br />
==CrtEBI Characterization==<br />
Biobrick BBa_K274100 is the lycopene-producing device submitted by team Cambridge 2009. It is used to produce lycopene which is red pigment can be detected by the naked eye. <br />
<br />
However, we find that BBa_K274100 represented a significant leakage expression when exploited as a reporter gene and even bacteria bearing BBa_K274100 only could also represent significant color change, compared with the blank. We speculate that it’s because of a putative promoter upstream, resulting in the leaky expression of CrtEBI. In order to verify the speculation, we further characterize this biobrick. This biobrick was suffixed to the constitutive promoter BBa_J23103. On the other hand, we did something fantastic: The biobrick was suffixed to the terminator BBa_B0015. Terminator upstream was expected to reduce the basal level of lycopene production, thus to verify our speculation. Resulted two new biobricks are shown in Figure 1.<br />
<br />
[[Image:Fig_0.JPG]]<br><br />
'''Figure 1 Biobricks we constructed to characterize biobrick BBa_K274100. The left construct denotes Constitutive promoter BBa_J23103-CrtEBI and on the right is an interesting biobrick—Terminator BBa_B0015-CrtEBI, namely a terminator was prefixed to CrtEBI, expected to reduce the leaky expression. '''<br />
<br />
<br />
After the two biobricks was constructed, they were transformed to JM109, BL21 (DE3) and DH5α, respectively. Then we compared their color with the corresponding strains that bear plasmid with only CrtEBI as the insert, at different time intervals. <br />
<br />
After cultivated at 37℃ for 9 hours, as shown in Figure 2, bacteria bearing BBa_J23103-CrtEBI and naked CrtEBI represented a light red color while bacteria bearing Terminator-CrtEBI and blank vector appeared white (Fig 2). We can’t collect bacteria of strain BL21 (DE3) at this time because it was slow-growing in this experiment for some reason. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/4/4b/Im107.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/4/4b/Im107.jpg" width=350></a></html><br><br />
Constitutive promoter BBa_J23103-CrtEBI <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/0/01/Im108.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/0/01/Im108.jpg" width=350></a></html><br><br />
Terminator BBa_B0015-CrtEBI <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/b/b4/Im109.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/b/b4/Im109.jpg" width=350 ></a></html><br><br />
Naked CrtEBI<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/df/Im110.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/d/df/Im110.jpg" width=350></a></html><br><br />
Blank<br />
<br />
'''Fig. 2 Color contrast after 9 hours’ cultivation (for each photo, from the left to the right are JM109, BL21 and DH5α).'''<br />
<br />
<br />
After 12 hours’ incubation, strain BL21 (DE3) appeared white in all cases. Besides, constitutive promoter BBa_J23103-CrtEBI appeared red while CrtEBI still represented light red. The terminator-CrtEBI appeared light red in strain JM109 and still white in DH5α. The results are shown in Figure 3.<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/6b/Im111.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/6b/Im111.jpg" width=350></a></html><br><br />
Constitutive promoter BBa_J23103-CrtEBI<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/0/03/Img112.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/0/03/Img112.jpg" width=350 ></a></html><br><br />
Terminator BBa_B0015-CrtEBI<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/7/77/Img113.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/7/77/Img113.jpg" width=350 ></a></html><br><br />
Naked CrtEBI<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/0/0b/Img114.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/0/0b/Img114.jpg" width=350></a></html><br><br />
Blank<br />
<br />
'''Fig 3. Color contrast after 12 hours’ cultivation (for each photo, from the left to the right are JM109, BL21 and DH5α). '''<br />
<br />
<br />
After 15 hours, as was shown in Figure 4, the situation was the same as 12 hours. The only difference was that strain BL21 (DE3) appeared red color. BL21 (DE3) constitutive promoter BBa_J23103-CrtEBI appeared red while naked CrtEBI appeared light red. BL21 (DE3) terminator-CrtEBI represented white like the blank.<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/3/3f/Im115.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/3/3f/Im115.jpg" width=350></a></html><br><br />
Constitutive promoter BBa_J23103-CrtEBI <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/b/b5/Im116.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/b/b5/Im116.jpg" width=350 ></a></html><br><br />
Terminator BBa_B0015-CrtEBI<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/b/b8/Im117.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/b/b8/Im117.jpg"width=350 ></a></html><br><br />
Naked CrtEBI <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/0/01/Im118.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/0/01/Im118.jpg" width=350 ></a></html><br><br />
Blank<br />
<br />
'''Fig 4. Color contrast after 15 hours’ cultivation (for each photo, from the left to the right are JM109, BL21 and DH5α).'''<br />
<br />
<br />
After 24 hours, the situation was the same as 15 hours as was shown in Figure 5. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Img119.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/9/9c/Img119.jpg" width=350></a></html><br><br />
Constitutive promoter BBa_J23103-CrtEBI <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/1/17/Img120.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/1/17/Img120.jpg" width=350 ></a></html><br><br />
Terminator BBa_B0015-CrtEBI<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/b/b7/Img121.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/b/b7/Img121.jpg" width=350 ></a></html><br><br />
Naked CrtEBI<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/7/7a/Img122.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/7/7a/Img122.jpg" width=350></a></html><br><br />
Blank<br />
<br />
'''Fig 5. Color contrast after 24 hours’ cultivation (for each photo, from the left to the right are JM109, BL21 and DH5α).'''<br />
<br />
<br />
A conclusion from these results was that CrtEBI has expression leakage and the constitutive promoter can enhance the expression while the terminator can prevent the leakage for some extent. From our viewpoint, 2 factors are responsible for the significant color leakage together.<br />
<br />
First of all, transcription factor-DNA interaction may result in this phenomenon. From the energy landscape for the TF Cro on the bacteriophage λDNA (Figure 6), it is obvious that many sites on the genome can have a strong interaction with TF. Maybe there are several such sites at the start of CrtEBI and CrtEBIY, or their upstream non-coding sequence. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/5d/Img123.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/5/5d/Img123.jpg" ></a></html><br><br />
'''Figure 6 Energy landscape for the TF Cro on the bacteriophage λDNA(Gerland et al., 2002). The landscape appears to be random, with different strength.'''<br />
<br />
<br />
Secondly, the lycopene biosynthesis is an enzymatic reaction. Lycopene biosynthesis needs three enzymes which are GGPP synthase (produced by CrtE), phytoene synthase (produced by CrtB) and phytoene desaturase (produced by CrtI). All the Km values of these enzymes are showed in Table 1 (Takaya et al., 2003; Neudert et al., 1998; Raisig et al., 1996). From the Km value, it is obvious that a small amount leakage of enzyme will lead to a large amount of product. <br />
<br />
Table 1 Km value for GGPP synthase, phytoene synthase, phytoene desaturase<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/4/4f/Img124.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/4/4f/Img124.jpg" ></a></html><br><br />
<br />
[[Team:Peking/Parts/Existed#Content| TOP]]<br />
<br />
==CrtEBIY Characterization==<br />
Biobrick K274200 is the β-carotene-producing device submitted by team Cambridge 2009. It is used to produceβ-carotene which is a yellow pigment which can be detected by the naked eye. <br />
However, we find that BBa_K274200 represented a significant leakage expression when exploited as a reporter gene and even bacteria bearing BBa_K274200 only could also represent significant color change, compared with the blank. We speculate that it’s because of a putative promoter upstream, resulting in the leaky expression of CrtEBIY. In order to verify the speculation, we further characterize this biobrick. This biobrick was suffixed to the constitutive promoter BBa_J23103. Resulted new biobrick is shown in Figure 7. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/f/fd/Fig_1-pku.JPG" target="blank"><img src="https://static.igem.org/mediawiki/2010/f/fd/Fig_1-pku.JPG" width=300></a></html><br><br />
'''Figure 1. The biobrick we constructed to characterize biobrick BBa_K274200. It is Constitutive promoter BBa_J23103 driving CrtEBIY. '''<br />
<br />
<br />
After the biobrick was constructed, it was transformed to JM109, BL21 (DE3) and DH5α, respectively. Then we compared their color with the corresponding strains that bear plasmid with only CrtEBIY as the insert. <br />
<br />
After cultivated at 37℃ for 12 hours, as shown in Figure 8, bacteria bearing BBa_J23103-CrtEBIY and naked CrtEBIY represented a yellow color and blank vector appeared white (Fig 2).<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/68/Im102.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/68/Im102.jpg"width=350 ></a></html><br><br />
Naked CrtEBIY <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/3/3d/Im103.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/3/3d/Im103.jpg" width=350></a></html><br><br />
Constitutive promoter BBa_J23103-CrtEBIY<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/4/46/Im105.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/4/46/Im105.jpg" width=350 ></a></html><br><br />
Blank<br />
<br />
'''Figure 2 Color contrast after 12 hours’ cultivation (for each photo, from the left to the right are JM109, DH5α and BL21)'''<br />
<br />
<br />
From these pictures above, we can see that both strains bearing constitutive promoter-CrtEBIY and strains bearing naked CrtEBIY appeared yellow. This demonstrated that CrtEBIY has a serious leakage, and the cause of leakage is probably the same as CrtEBI. It is noteworthy that CrtEBIY pathway need one more enzyme (Lycopene Cyclase, produced by CrtY) than CrtEBI and the Km for Lycopene Cyclase is 1.8μM (Schnurr et al., 1996), so the leakage was more serious as expected.<br />
<br />
We intended to use CrtEBI and CrtEBIY as our bioreporter at first. After our characterization, we decided to use CrtEBI for the serious leakage of CrtEBIY. However, we found a serious leakage when CrtEBI was combined with the promoter PmerT and PpbrA. Leakage was serious regardless of the prefixed promoter in our experiment. For this reason, we chose other reporter gene, such as LacZ alpha instead of CrtEBI.<br />
<br />
[[Team:Peking/Parts/Existed#Content| TOP]]<br />
<br />
==Reference==<br />
Gerland, U., Moroz, JD., Hwa, T.( 2002). Physical constraints and functional characteristics of transcription factor–DNA interaction. Proc Natl Acad Sci USA 99, 12015-12020.<br />
<br />
Tomoko, N., Kenji, T., Mitsuhiro, I., Nobuhide, D., Hiroshi, Y. (2007). Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis (OGAB). Appl. Environ. Microbiol. 73, 1355–1361<br />
<br />
Takaya A., Zhang Y.W., Asawatreratanakul K., Wititsuwannakul D., Wititsuwannakul R., Takahashi S., and Koyama T. (2003). Cloning, expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis, Biochim. Biophys. Acta, 1625(2), 214-220<br />
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Neudert U., Martinez-Ferez I., Fraser P. D. , Sandmann G.(1998). Expression of an active phytoene synthase from Erwinia uredoÍora and biochemical properties of the enzyme. Biochim. Biophys. Acta 1392, 51–58<br />
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Raisig A., Bartley G., Scolnik P., Sandmann G. (1996) Purification in an active state and properties of the 3-step phytoene desaturase from Rhodobacter capsulatus overexpressed in Escherichia coli. J Biochem 119, 559–564<br />
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Schnurr, G., Misawa, N. and Sandmann, G. (1996). Expression, purification and properties of lycopene cyclase from Erwinia uredovora. J Biochem 315, 869–874<br />
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
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Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
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[[Image:FIG_1.png|600px|center]]<br />
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'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
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==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
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[[Image:Fig2.png|450px|center]]<br />
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'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
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The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
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[[Image:FIG_3.png|center]]<br />
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'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
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We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
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==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
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DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
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[[Image:Fig 4.png|500px|center]]<br />
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'''Figure 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''<br />
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Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. <br />
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As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously. <br />
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[[Image:Fig 5.png|500px|center]]<br />
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'''Figure 5. Procedure of DsbA-MBP construction.'''<br />
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As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.<br />
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[[Image:Fig 6.png|500px|center]]<br />
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'''Figure 6. Standardization procedure of DsbA-MBP.'''<br />
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DsbA and MBD gene was amplified by PCR from pET-39b (+)-DsbA-MBP, with the primer containing T7 promoter, RBS and SD restriction sites, as shown in Fig 6. The PCR product was digested with EcoR I / Pst I and then cloned into EcoR I / Pst I double digested pSB1K3, to achieve the goal of standardization of the fusion protein (Fig. 6).<br />
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===RESULT===<br />
Periplasmic Expression DsbA-MBPs was verified by SDS-PAGE and western blotting (Fig 7).<br />
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[[Image:Fig_7.png|600px|center]]<br />
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[[Image:Fig_7b.png|600px|center]]<br />
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'''Figure 7 Result of SDS-PAGE and Western blotting'''<br />
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Over-expression band in SDS-PAGE result and bands specific to his-tag in western blot result were found at about 40 kD. The presence of these bands confirmed that we have got the expected protein expressed in periplasm. But there are large amount of proteins detected in lysate pellet, this was because of the high intensity of T7 promoter. When induced with 0.5 mM IPTG, our target protein were expressed so rapidly that some of them cannot fold properly and accumulated in the inclusion bodies. This problem can be solved by using lower IPTG concentration, lower induction temperature or shorter induction time.<br />
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[[Image:Fig_8.png|500px|center]]<br />
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'''Fig 8. Lpp-OmpA-MBP was designed as a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA, which can be anchored to the outer membrane. '''<br />
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Lpp-OmpA(46-159) fusions were proved to be an effective surface display system to anchor a variety of proteins such as ,B-lactamase [6], a cellulose binding protein, alkaline phosphatase and a single chain Fv antibody fragment(scFv)[7] on the external surface of E. coli. For OmpA, targeting and correct assembly into the outer membrane appear to be distinct events. Extensive studies on it suggest that the region between residue 154 and 180 is crucial for localization on the membrane, while the region between residue 46 and 159 ,the fragment constitute our fusion protein, contains five transmembrane β-strands of an eight-stranded β-barrel, providing an sufficient region to assemble into the membrane[6].<br />
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Additionally, a signal peptide is also needed to mediate the proper localization of the fusion protein on the outer membrane. The N-termini of the outer membrane lipoprotein (Lpp) can serve as the signal peptide, which is considered to be firmly associated with the membrane due to its extreme hydrophobicity [6]. To conclude this, the fusion protein of Lpp-OmpA , with their target and assembly function, can serve as a powerful tool in surface display [9].<br />
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===CONSTRUCTION===<br />
We constructed a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and our metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA. <br />
To test the expression of the fusion protein and the function, it was essential to construct both the standard plasmid and commercial plasmid. <br />
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Firstly, we used PCR to obtain Lpp-OmpA gene from the template of PSD-MBD which was provided by Anne O Summers as a gift. The primers were designed based the literature (Fig 9). And the MBP gene was also obtained by PCR. Then the two fragment were cut with EcoRI(or NdeI), SalI and SalI, PstI (or XhoI), respectively, as inserts and the standard plasmid PSB1A2 was cut with EcoRI and PstI(for the commercial plasmid PET21a, the enzymes were NdeI and XhoI) as the vector. Ultimately, three fragments (Lpp-OmpA, MBP and the vector) were combined to get the standard and commercial plasmid bearing Lpp-OmpA-MBP, as is shown in Fig 9 and Fig 10.<br />
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[[Image:Fig_9.png|450px|center]]<br />
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'''Fig 9. Procedures of the construction of standard plasmid. '''<br />
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[[Image:Fig_10.png|450px|center]]<br />
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'''Fig 10 Procedures of Construction of Commercial Plasmid. '''<br />
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After the construction of the plasmid with the fusion protein gene Lpp-OmpA-MBP, We prefixed T7 promoter and BBa_B0030 upstream of Lpp-OmpA-MBP. Additionally, a strong terminator BBa_B0015 was suffixed. <br />
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[[Image:Fig_11.png|600px|center]]<br />
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'''Fig 11. Results of SDS-PAGE and Western Blotting.''' The overexpression band in SDS-PAGE and specific band in western blotting comfirmed the correct expression and localization of our fusion protein. Specific band cannot be detected in the cytosol which indicates the excellent translocation efficiency of OmpA. <br />
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[[Image:Fig_12.png|500px|center]]<br />
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'''Fig 12. Overview of various localization of MBP. '''<br />
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In summary, as is shown in Fig 12, MBP will be translocated in three different compartments in cell, directly into cytosol, into periplasm with the help of DsbA, and display on the surface using OmpA fusion. We would also like to compare the effects to evaluate the different efficacy of these modules, as is shown below.<br />
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==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
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2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
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3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
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4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
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5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
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6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
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7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
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8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
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9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Biosensor/PromoterCharacterizationTeam:Peking/Project/Biosensor/PromoterCharacterization2010-10-27T14:54:15Z<p>Lgdeer: /* reference */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Promoter Characterization</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Biosensor|Biosensor]] > [[Team:Peking/Project/Biosensor/PromoterCharacterization|Promoter Characterization]]<html><br />
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PmerT is a promoter from Tn21 mercury resistance (mer) operon. Transposon 21 itself is carried by plasmid NR1 (Nakaya, Nakamura et al. 1960), which was originally isolated from Shigella flexneri in Japan in the late 1950s (Liebert, Hall et al. 1999). <br />
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The mer operon of Tn21 consists of two tightly overlapped, divergently oriented promoters – PR and PTPCAD.(Park, Wireman et al. 1992). PR is the promoter of the regulatory protein gene, merR, and PTPCAD is for the transcription of the structural gene – merPTCAD. They are called merOP as a whole. MerR, as a regulatory protein, always binds to merOP as a homodimer and enhances the occupancy of PTPCAD by RNA polymerase regardless of the presence of Hg(II), although only after Hg(II)’s binding can the dimer stop preventing the formation of the open complex by RNA polymerase. Also, MerR repress its own expression independently of Hg(II). In our design, merR was isolated from the operon and assembled with constitutive promoters of certain strength to maintain its expression intensity at certain level. For the same reason, the divergent promoter PR was also removed by deletion of its -35 region (Fig 1).<br />
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'''Fig 1 DNA sequence of the Tn21 mer operator promoter region. The MerR binding site on PmerT is marked by a box. The -35 and -10 regions for both PmerR and PmerTPAD are marked with boxes, and the dyad symmetrical DNA sequence that MerR recognizes and binds to is marked with arrows under the DNA sequence. (A) The divergently oriented promoters are marked by blue box and purple box, respectively. (B) In our project, the expression intensity of MerR should be maintained exogenously, so the divergent promoter PR (of MerR transcript) was also removed by deletion of its -35 region. The resulted promoter sequence is marked with a dark purple box Modified from (Hobman, Wilkie et al. 2005)'''<br />
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As shown in Fig 1, the key sequence for MerR’s binding is a region of interrupted dyad symmetry (19bp) located between the -35 and -10 haxamers of PTPCAD (The top strand). And the structure of PR (botton strand) is similar to PTPCAD in a divergent orientation. The -10 hexamers of PTPCAD and PR actually overlap by four bases. (Park, Wireman et al. 1992). <br />
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'''Fig. 2. A generalized mechanism of MerR family regulator transcriptional activation. (A) The dimeric MerR regulator binds to the operator region of the promoter and recruits RNA polymerase, forming a ternary complex. Transcription is slightly repressed because the apo-MerR regulator dimer has bent the promoter DNA such that RNA polymerase does not contact it properly. (B) Upon binding the cognate metal ions (shown as cyan circles) the metallated MerR homodimer causes a realignment of the promoter such that RNA polymerase contacts the -35 and -10 sequences leading to open complex formation and transcription. Modified from Brown et al.'''<br />
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The model of MerR’s action is shown in Fig 2. In (A), merR (showed as green dimer downside the DNA strand) bound to PTPCAD and recruited RNA polymerase but was unable to form an open complex. Hg (II)’s binding to the dimer changed its conformation and transcription was initiated. (Brown, Stoyanov et al. 2003)<br />
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By annealing PmerT-forward (5’-3’) and PmerT-reverse (5’-3’) primers, PTPCAD carrying a sticky end of EcoRI and SpeI was cloned upstream of BBa_E0840, a GFP generator. With exogenous expression of MerR in bacteria, GFP’s expression could be induced by Hg (II) in a dose response manner. The PTPCAD-E0840 was then cloned into pSB3K3 backbone and the BBa_J23103 (constitutive promoter)-merR into pSB1A3.<br />
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===Protocols for promoter characterization===<br />
'''An overnight culture of bacteria carrying the two plasmids pSB3K3 and pSB1A3 were grown in LB broth with ampicillin and kanamycin at 37°C was reactivated by diluting the culture in a ratio of 1:100 with fresh LB. When OD600 reached 0.4-0.6, the bacteria was disposed to several EP tubes, each owning 500uL, and different dose of Mercuric chloride solution was spplied with 3 duplicates and the final concentration varied from 0 to 1.0E-6 mol/L. 100uL of the 500uL was added to the black-96-well plates for GFP intensity’s measurement and another 100uL was added to the transparent-96-well plates for OD600 measurement. In-plate culture fluorescence and OD600 was recorded at 20min intervals from 0 to 275min and the GFP intensity of 20min before inducement was also measured. Temperature was constant at 37°C. '''<br />
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'''Fig. 3. Time and dose response of GFP and OD 600. The data was measured every 20 minutes since 20 minutes before supplement of different dose of Hg(II) to the wells and the plates was incubated in the shaker at 37℃during the interval of measurement. For both plates, the volume of LB medium with bacteria was 100uL per well. (a) GFP intensity was measured by Tecan Microplate Reader with excitation wavelength at 470nm and emission wavelength at 509nm. A black 96-well plate was used to minimize the interference of different well. (b) OD 600 was also measured by Tecan Microplate Reader in a transparent 96-well plate, since the depth of 100uL in the well did not reach 1 centimeter, the OD 600 value here was smaller than that was measured by a spectrophotometer.'''<br />
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As shown in Fig 3, the basal level of PTPCAD is relatively low. At the concentration of 5.0E-8 mol/L, the expression of GFP was observed. With longer induction time and higher concentration of Hg (II), the fluorescent intensity dramatically increased in a narrow concentration range (Fig. 3a). After the normalization by OD 600, the trend did not change obviously (figure not shown). Around 120min later, bacteria growth reached stationary phase (Fig. 3b) while GFP still accumulated rapidly (Fig. 3a). When the concentration of Hg(II) was higher than 1.0E-6 mol/uL, GFP expression level grew slower which might be explained by the saturation of MerR by Hg(II) and the cytotoxity of mercury. <br />
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'''Fig 4. Dose response curve of MerR/PmerT to mercury in vivo. Note that the GFP expression intensity was induced from 10% to the maximum across a narrow range – 7 fold change in Hg (II) concentration. The basal level is low and the activation fold is dramatic. Also the metal reorganization specificity of MerR was demonstrated by the result that lead (II) could not activate significant GFP expression in vivo. '''<br />
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Besides, it is obvious that the MerR dependent regulation worked as a hypersensitive switch which represented negligible basal level expression of GFP, but induced from 10% to 90% of maximum in response across a range of Hg (II) concentration as narrow as 7-fold change (Fig 4). When the expression was fully induced, it was even below the threshold at which Hg (II) adversely affects the growth rate of bacterial cells. Actually, the hypersensitive switch behavior is postulated to arise from the unusual metal binding mechanism of MerR. Therefore, the molecular basis of the hypersensitivity switch will be discussed thoroughly in Bioabsorbent part of our wiki. <br />
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===Reference===<br />
Brown, N. L., J. V. Stoyanov, et al. (2003). "The MerR family of transcriptional regulators." FEMS Microbiol Rev 27(2-3): 145-163.<br />
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Hobman, J. L., J. Wilkie, et al. (2005). "A design for life: prokaryotic metal-binding MerR family regulators." Biometals 18(4): 429-436.<br />
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Liebert, C. A., R. M. Hall, et al. (1999). "Transposon Tn21, flagship of the floating genome." Microbiol Mol Biol Rev 63(3): 507-522.<br />
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Nakaya, R., A. Nakamura, et al. (1960). "Resistance transfer agents in Shigella." Biochem Biophys Res Commun 3: 654-659.<br />
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Park, S. J., J. Wireman, et al. (1992). "Genetic analysis of the Tn21 mer operator-promoter." J Bacteriol 174(7): 2160-2171.<br />
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[[https://2010.igem.org/Team:Peking/Project/Biosensor/PromoterCharacterization TOP]]<br />
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Aiming to analyze the PmerT promoter further, construction of merOP libriay (Random mutagenesis of the merOP) was also conducted.<br />
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Previous footprint studies [Lee, et al, 1993, Park et al, 1992] had suggested that there are three distinct domains within the palindromic merO. Mutants in domain I, which consists of the four highly conserved inner bases (---GTAC---GTAC---) of the seven-base interrupted dyad, generally allow constitutive transcription at PR. Mutants in dyad domain II, which located on either side of domain I, are semiconstitutive but support additional Hg(II)-induced open complex formation at PTPCAD (Fig 1). In our project, we planned to analyze the behavior of merO thoroughly, and it could be expected that mutation at the dyad domain II would change the Ka of MerR-DNA interactions. Thus we mutated the semiconserved sites and left the conserved sites constant.<br />
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The approach is generally based upon degenerate primer PCR, with the combination of a ‘DNA shuffling’ procedure, that is performed on the target DNA sequence; the resulting library of variants is then screened for the desired feature, and selected isolates are subjected to a repeated procedure. <br />
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We performed a degenerate primer PCR on the merTPCAD wild-type promoter region of the alterable sites. The resulting PCR fragments, each potentially containing one or more random mutation sites at a restricted location, were cloned into pSB3K3 and transformed into DH5 alpha competent cells, yielding approximately 100 colonies (Fig. 2). <br />
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The merTPCAD (PmerT) variants library was screened and primarily characterized in 96-well plates and the performance of each individual construct was compared with that of the merT wild-type promoter. <br />
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Then secondary characterization of the best performing candidates in primary characterization was then performed in 96-well plates by induction of mercury concentration gradient of 10^-5M, 10^-6M and 10^-7M for 2 hours, followed by recording GFP intensity of each well by Microplate Reader. The result of secondary characterization showed Fig. 3.<br />
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'''Fig.4 Dose response curves of each final mutants. The fitting result was shown by a solid line. Promoter sequences were shown in Table 1. Corresponding graph data were presented in Table 2. '''<br />
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Among all these candidates, mutant1, 3, 25, 44, 85, 88 (also shown in Fig 3) were selected for the final careful characterization (Fig 4) during which a higher concentration resolution was exploited. Then the dose-response curves were fitted by Hill function (Table 2.) <br />
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It can be observed that mutations at the semiconserved region of PmerT promoter significantly influenced the response behavior of MerR/PmerT pair. It is probably because that the mutations alter the binding affinity between MerR and PmerT promoter, which can also be deduced from Table 1. As a result of this, different sensitivity (higher or lower than the wild-type) PmerT promoters were gained by us (Table 1 and Table 2). <br />
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<html><a href="https://static.igem.org/mediawiki/2010/a/a3/Pc11.png"target="blank"><img src="https://static.igem.org/mediawiki/2010/a/a3/Pc11.png" width=650 ></a></html><br><br />
In summary, After initially characterizing the dose-time response manner of MerR based reporter system, we constructed a saturated mutagenesis library at MerR binding site, a dyad sequence between -10 and -35 region of promoter PmerT. Shift of switch point and maxima of dose response curve was observed in dyad-sequence mutants, which shows that the binding affinity between TF and its operator site greatly influence bacterial sensitivity to mercury.<br />
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===Reference===<br />
Lee, I. W., Park, S. J., and Summers, A. O. (1993) In Vivo DNA-Protein Interactions at the Divergent Mercury Resistance(mer) Promoters. JBC. 268(4). 2632-9<br />
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Park, S. J., Wireman, J., and Summers, A. O. (1992) Genetic analysis of the Tn21 mer operator-promoter. J. Bacteriol. 174(6), 2160-71.<br />
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Lee, I. W., Park, S. J., and Summers, A. O. (1993) In Vivo DNA-Protein Interactions at the Divergent Mercury Resistance(mer) Promoters. JBC. 268(4). 2632-9<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Biosensor/PromoterCharacterizationTeam:Peking/Project/Biosensor/PromoterCharacterization2010-10-27T14:53:24Z<p>Lgdeer: /* reference */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Promoter Characterization</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Biosensor|Biosensor]] > [[Team:Peking/Project/Biosensor/PromoterCharacterization|Promoter Characterization]]<html><br />
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PmerT is a promoter from Tn21 mercury resistance (mer) operon. Transposon 21 itself is carried by plasmid NR1 (Nakaya, Nakamura et al. 1960), which was originally isolated from Shigella flexneri in Japan in the late 1950s (Liebert, Hall et al. 1999). <br />
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The mer operon of Tn21 consists of two tightly overlapped, divergently oriented promoters – PR and PTPCAD.(Park, Wireman et al. 1992). PR is the promoter of the regulatory protein gene, merR, and PTPCAD is for the transcription of the structural gene – merPTCAD. They are called merOP as a whole. MerR, as a regulatory protein, always binds to merOP as a homodimer and enhances the occupancy of PTPCAD by RNA polymerase regardless of the presence of Hg(II), although only after Hg(II)’s binding can the dimer stop preventing the formation of the open complex by RNA polymerase. Also, MerR repress its own expression independently of Hg(II). In our design, merR was isolated from the operon and assembled with constitutive promoters of certain strength to maintain its expression intensity at certain level. For the same reason, the divergent promoter PR was also removed by deletion of its -35 region (Fig 1).<br />
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'''Fig 1 DNA sequence of the Tn21 mer operator promoter region. The MerR binding site on PmerT is marked by a box. The -35 and -10 regions for both PmerR and PmerTPAD are marked with boxes, and the dyad symmetrical DNA sequence that MerR recognizes and binds to is marked with arrows under the DNA sequence. (A) The divergently oriented promoters are marked by blue box and purple box, respectively. (B) In our project, the expression intensity of MerR should be maintained exogenously, so the divergent promoter PR (of MerR transcript) was also removed by deletion of its -35 region. The resulted promoter sequence is marked with a dark purple box Modified from (Hobman, Wilkie et al. 2005)'''<br />
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As shown in Fig 1, the key sequence for MerR’s binding is a region of interrupted dyad symmetry (19bp) located between the -35 and -10 haxamers of PTPCAD (The top strand). And the structure of PR (botton strand) is similar to PTPCAD in a divergent orientation. The -10 hexamers of PTPCAD and PR actually overlap by four bases. (Park, Wireman et al. 1992). <br />
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'''Fig. 2. A generalized mechanism of MerR family regulator transcriptional activation. (A) The dimeric MerR regulator binds to the operator region of the promoter and recruits RNA polymerase, forming a ternary complex. Transcription is slightly repressed because the apo-MerR regulator dimer has bent the promoter DNA such that RNA polymerase does not contact it properly. (B) Upon binding the cognate metal ions (shown as cyan circles) the metallated MerR homodimer causes a realignment of the promoter such that RNA polymerase contacts the -35 and -10 sequences leading to open complex formation and transcription. Modified from Brown et al.'''<br />
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The model of MerR’s action is shown in Fig 2. In (A), merR (showed as green dimer downside the DNA strand) bound to PTPCAD and recruited RNA polymerase but was unable to form an open complex. Hg (II)’s binding to the dimer changed its conformation and transcription was initiated. (Brown, Stoyanov et al. 2003)<br />
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By annealing PmerT-forward (5’-3’) and PmerT-reverse (5’-3’) primers, PTPCAD carrying a sticky end of EcoRI and SpeI was cloned upstream of BBa_E0840, a GFP generator. With exogenous expression of MerR in bacteria, GFP’s expression could be induced by Hg (II) in a dose response manner. The PTPCAD-E0840 was then cloned into pSB3K3 backbone and the BBa_J23103 (constitutive promoter)-merR into pSB1A3.<br />
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===Protocols for promoter characterization===<br />
'''An overnight culture of bacteria carrying the two plasmids pSB3K3 and pSB1A3 were grown in LB broth with ampicillin and kanamycin at 37°C was reactivated by diluting the culture in a ratio of 1:100 with fresh LB. When OD600 reached 0.4-0.6, the bacteria was disposed to several EP tubes, each owning 500uL, and different dose of Mercuric chloride solution was spplied with 3 duplicates and the final concentration varied from 0 to 1.0E-6 mol/L. 100uL of the 500uL was added to the black-96-well plates for GFP intensity’s measurement and another 100uL was added to the transparent-96-well plates for OD600 measurement. In-plate culture fluorescence and OD600 was recorded at 20min intervals from 0 to 275min and the GFP intensity of 20min before inducement was also measured. Temperature was constant at 37°C. '''<br />
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'''Fig. 3. Time and dose response of GFP and OD 600. The data was measured every 20 minutes since 20 minutes before supplement of different dose of Hg(II) to the wells and the plates was incubated in the shaker at 37℃during the interval of measurement. For both plates, the volume of LB medium with bacteria was 100uL per well. (a) GFP intensity was measured by Tecan Microplate Reader with excitation wavelength at 470nm and emission wavelength at 509nm. A black 96-well plate was used to minimize the interference of different well. (b) OD 600 was also measured by Tecan Microplate Reader in a transparent 96-well plate, since the depth of 100uL in the well did not reach 1 centimeter, the OD 600 value here was smaller than that was measured by a spectrophotometer.'''<br />
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As shown in Fig 3, the basal level of PTPCAD is relatively low. At the concentration of 5.0E-8 mol/L, the expression of GFP was observed. With longer induction time and higher concentration of Hg (II), the fluorescent intensity dramatically increased in a narrow concentration range (Fig. 3a). After the normalization by OD 600, the trend did not change obviously (figure not shown). Around 120min later, bacteria growth reached stationary phase (Fig. 3b) while GFP still accumulated rapidly (Fig. 3a). When the concentration of Hg(II) was higher than 1.0E-6 mol/uL, GFP expression level grew slower which might be explained by the saturation of MerR by Hg(II) and the cytotoxity of mercury. <br />
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'''Fig 4. Dose response curve of MerR/PmerT to mercury in vivo. Note that the GFP expression intensity was induced from 10% to the maximum across a narrow range – 7 fold change in Hg (II) concentration. The basal level is low and the activation fold is dramatic. Also the metal reorganization specificity of MerR was demonstrated by the result that lead (II) could not activate significant GFP expression in vivo. '''<br />
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Besides, it is obvious that the MerR dependent regulation worked as a hypersensitive switch which represented negligible basal level expression of GFP, but induced from 10% to 90% of maximum in response across a range of Hg (II) concentration as narrow as 7-fold change (Fig 4). When the expression was fully induced, it was even below the threshold at which Hg (II) adversely affects the growth rate of bacterial cells. Actually, the hypersensitive switch behavior is postulated to arise from the unusual metal binding mechanism of MerR. Therefore, the molecular basis of the hypersensitivity switch will be discussed thoroughly in Bioabsorbent part of our wiki. <br />
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===Reference===<br />
Brown, N. L., J. V. Stoyanov, et al. (2003). "The MerR family of transcriptional regulators." FEMS Microbiol Rev 27(2-3): 145-163.<br />
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Hobman, J. L., J. Wilkie, et al. (2005). "A design for life: prokaryotic metal-binding MerR family regulators." Biometals 18(4): 429-436.<br />
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Liebert, C. A., R. M. Hall, et al. (1999). "Transposon Tn21, flagship of the floating genome." Microbiol Mol Biol Rev 63(3): 507-522.<br />
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Nakaya, R., A. Nakamura, et al. (1960). "Resistance transfer agents in Shigella." Biochem Biophys Res Commun 3: 654-659.<br />
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Park, S. J., J. Wireman, et al. (1992). "Genetic analysis of the Tn21 mer operator-promoter." J Bacteriol 174(7): 2160-2171.<br />
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[[https://2010.igem.org/Team:Peking/Project/Biosensor/PromoterCharacterization TOP]]<br />
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Aiming to analyze the PmerT promoter further, construction of merOP libriay (Random mutagenesis of the merOP) was also conducted.<br />
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Previous footprint studies [Lee, et al, 1993, Park et al, 1992] had suggested that there are three distinct domains within the palindromic merO. Mutants in domain I, which consists of the four highly conserved inner bases (---GTAC---GTAC---) of the seven-base interrupted dyad, generally allow constitutive transcription at PR. Mutants in dyad domain II, which located on either side of domain I, are semiconstitutive but support additional Hg(II)-induced open complex formation at PTPCAD (Fig 1). In our project, we planned to analyze the behavior of merO thoroughly, and it could be expected that mutation at the dyad domain II would change the Ka of MerR-DNA interactions. Thus we mutated the semiconserved sites and left the conserved sites constant.<br />
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The approach is generally based upon degenerate primer PCR, with the combination of a ‘DNA shuffling’ procedure, that is performed on the target DNA sequence; the resulting library of variants is then screened for the desired feature, and selected isolates are subjected to a repeated procedure. <br />
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We performed a degenerate primer PCR on the merTPCAD wild-type promoter region of the alterable sites. The resulting PCR fragments, each potentially containing one or more random mutation sites at a restricted location, were cloned into pSB3K3 and transformed into DH5 alpha competent cells, yielding approximately 100 colonies (Fig. 2). <br />
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The merTPCAD (PmerT) variants library was screened and primarily characterized in 96-well plates and the performance of each individual construct was compared with that of the merT wild-type promoter. <br />
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Then secondary characterization of the best performing candidates in primary characterization was then performed in 96-well plates by induction of mercury concentration gradient of 10^-5M, 10^-6M and 10^-7M for 2 hours, followed by recording GFP intensity of each well by Microplate Reader. The result of secondary characterization showed Fig. 3.<br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9e/Pc9.png"target="blank"><img src="https://static.igem.org/mediawiki/2010/9/9e/Pc9.png" width=650 ></a></html><br><br />
'''Fig.4 Dose response curves of each final mutants. The fitting result was shown by a solid line. Promoter sequences were shown in Table 1. Corresponding graph data were presented in Table 2. '''<br />
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Among all these candidates, mutant1, 3, 25, 44, 85, 88 (also shown in Fig 3) were selected for the final careful characterization (Fig 4) during which a higher concentration resolution was exploited. Then the dose-response curves were fitted by Hill function (Table 2.) <br />
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It can be observed that mutations at the semiconserved region of PmerT promoter significantly influenced the response behavior of MerR/PmerT pair. It is probably because that the mutations alter the binding affinity between MerR and PmerT promoter, which can also be deduced from Table 1. As a result of this, different sensitivity (higher or lower than the wild-type) PmerT promoters were gained by us (Table 1 and Table 2). <br />
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<html><a href="https://static.igem.org/mediawiki/2010/a/a3/Pc11.png"target="blank"><img src="https://static.igem.org/mediawiki/2010/a/a3/Pc11.png" width=650 ></a></html><br><br />
In summary, After initially characterizing the dose-time response manner of MerR based reporter system, we constructed a saturated mutagenesis library at MerR binding site, a dyad sequence between -10 and -35 region of promoter PmerT. Shift of switch point and maxima of dose response curve was observed in dyad-sequence mutants, which shows that the binding affinity between TF and its operator site greatly influence bacterial sensitivity to mercury.<br />
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===reference===<br />
Lee, I. W., Park, S. J., and Summers, A. O. (1993) In Vivo DNA-Protein Interactions at the Divergent Mercury Resistance(mer) Promoters. JBC. 268(4). 2632-9<br />
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Park, S. J., Wireman, J., and Summers, A. O. (1992) Genetic analysis of the Tn21 mer operator-promoter. J. Bacteriol. 174(6), 2160-71.<br />
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Lee, I. W., Park, S. J., and Summers, A. O. (1993) In Vivo DNA-Protein Interactions at the Divergent Mercury Resistance(mer) Promoters. JBC. 268(4). 2632-9<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Biosensor/BioreporterTeam:Peking/Project/Biosensor/Bioreporter2010-10-27T14:52:20Z<p>Lgdeer: /* Traffic-Light Bioassay For Application Ease */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Bioreporter</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Biosensor|Biosensor]] > [[Team:Peking/Project/Biosensor/Bioreporter|Bioreporter]]<html><br />
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=Traffic-Light Bioassay For Application Ease=<br />
&nbsp;&nbsp;&nbsp;&nbsp;Traditional bioassays with biosensor bacteria are usually calibrated with analyte solutions of known concentrations that are analyzed along with the samples of interest (Chakraborty et al., 2008; Hansen and Sorensen, 2000). This is done as bioreporter output (fluorescence or colour) does not only depend on the target concentration, but also on the incubation time and physiological activity of the cells in the assay. Comparing the biosensor output with standardized colour tables in the field application seems rather difficult and error-prone. <br>&nbsp;&nbsp;&nbsp;&nbsp;To solve this hard truth, a new approach called ‘traffic light’ heavy metal bioassay was then developed. Previous work has shown that the “traffic light” bioassay could work independently of external calibration of the bioreporter output, thus to control assay variations and to improve application ease(Anke Wackwitz, 2008). Actually, an internal calibration based on the use of multiple isogenic bioreporter cell lines with the same output but drastically different sensitivity at a given heavy metal concentration is proceeded during this bioassy.<br><br />
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'''Fig 1. The traffic light bioassay uses, for example, four isogenic strains with the same reporter output but differing in the compound concentration threshold at which their reporter circuit is activated. The number of reporter strains reacting to a sample (rather than their reporter signal intensity per se) is then representative for the compound concentration range. Adapted from (van der Meer and Belkin)'''<br><br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Specially speaking, we pyramided the information collected during the Promoter Characterization, Operation Characterization and Modeling. It has been shown that the expression level of MerR and the binding affinity of MerR to the cognate promoter dyad sequence both significantly determinate the bacterial sensitivity to mercury — higher MerR expression intensity and lower binding affinity of MerR to DNA target would result in a less sensitive bacterial mercury sensor and vice versa. In order to verify this, we constructed a reporter system similar to the ones we used before, as shown in Fig 2C.(<html><a href="https://2010.igem.org/Team:Peking/Project/Biosensor/PromoterCharacterization">to read more…</a></html>). <br><br />
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'''Fig 2. Result of pyramiding. According to the information collected in promoter characterization and operation characterization, both the expression level of MerR and the semiconserved region of MerR binding site could determine the threshold of mercury sensitivity. (A) Each combination was carefully characterized. All of the dose response curves represented as a hill function. (B) When comparing these combinations, we can easily find that both of the factors worked as expected: for instance, BBa_J23101+Mutant 3 represents a higher threshold than that of BBa_J23101+Mutant 88 and BBa_J23103+Mutant 88 has a higher threshold than BBa_J23101+Mutant 88. (C) The genetic construction of the system used for characterization. '''<br />
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&nbsp;&nbsp;&nbsp;&nbsp;Therefore, 4 isogenic biosensor strains with the same reporter output (beta-galactosidase) but differing in the mercury concentration threshold at which their reporter circuit is activated was constructed as is shown in Fig 3. <br><br />
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&nbsp;&nbsp;&nbsp;&nbsp;Interestingly, when we employed full length LacZ gene (BBa_I732017) as the reporter gene for design in Fig 3, a significant leakage expression emerged which was as high as we could not tell the difference in intensity of the beta-galactosidase activity between experimental group and the control. However, when using LacZ alpha fragment instead, we found that the leakage expression became negligible – Hg (II) induction would significantly activate the beta-galactosidase activity compared with the control and bacteria bearing the mercury sensing device behaved in a dose-response manner in response to mercury concentration gradient (Fig 4), which implied that when separated into 2 peptide fragments, the enzymatic activity of LacZ (beta-galactosidase) decreased remarkably and the decrement could be rescued by higher expression level of the alpha fragment. It was probably because the alpha-complementary process is mediated by intra-molecular non-covalent interactions. In comparison with the full length LacZ, alpha-complement forms the correct conformation for beta-galactosidase activity in lower possibility. Therefore, mercury sensing device exploiting full length LacZ as reporter gene can not actually sense the mercury. However, it may act as the positive control in the traffic light assay, which exhibits beta-galactosidase activity whether mercury is in presence or not (Fig 3). <br><br />
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'''Fig 3. Scheme of construction of 4 isogenic biosensor strains. It was a pyramiding process during which the phenotypes of bacterial reporter developed previously in our project were combined, in order to implement strains with different mercury sensitivity. (E) BBa_J23103, BBa_J23101 and BBa_J23117 belong to a constitutive promoter library from partsregistry, among which BBa_J23103 is a very weak one, BBa_J23117 is medium and BBa_J23101 is the second strongest in the library. (A)Expression of MerR driven by weak constitutive promoter BBa_J23103 on low copy number plasmid backbone pSB3K3 was pyramided with wild type PmerT which is the most mercury-sensitive promoter combined with LacZ alpha fragment as the reporter gene on plasmid backbone pSB1A3. It is an extreme to confer the bioreporter the most sensitivity, based on our previous results. (B) Strong promoter BBa_J23101 drive the high expression intensity of MerR on plasmid backbone pSB1A2, while PmerT 88 on pSB3K3 is the most insensitive mercury-response promoter developed before. Reporter gene is still lacZ alpha fragment. This is the other extreme of MerR expression intensity, aiming to endow the bacteria the most insensitivity. (C) Promoter BBa_J23117 is medium compared with other 2 constitutive promoters, and the same with PmerT 03 which was screened out previously. This mercury sensing device is expected to be more sensitive than (A) and less than (B). (D) This device is similar to (A), except the reporter gene was replaced by LacZ full length gene. As mentioned in the context, when acting as the reporter gene, full length LacZ represents a significantly leakage expression, giving output regardless of the input, of which we took the advantage to regard it as the positive control in traffic light bioassay. '''<br><br><br />
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&nbsp;&nbsp;&nbsp;&nbsp;With completion of 4 isogenic biosensor strain construction, traffic light bioassay was performed in 96-well plates to allow visual signal detection by digital imaging (Fig 4), rather than other detection methods which are time-comsuming or need costly instruments. For each biosensor strain the bioassay was performed in triplicate with 24 different mercury concentrations ranging from 10^-9 M to 10^-5 M.<br><br />
''fig 4''<br><br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Results demonstrated that the bioreporter system using combinations of these biosensor strains to define mercury concentration ranges at which none, one or more biosensor strains gave qualitative (yes/no) visible signals that were relatively independent of incubation time or bioreporter activity. The discriminated concentration ranges would fit very well with the current permissive (e.g. World Health Organization) levels of mercury in common aquatic environment, such as drinking water.<br><br><br />
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==Reference==<br />
Anke Wackwitz, H.H., Antonis Chatzinotas, Uta Breuer, Christelle Vogne, Jan Roelof Van Der Meer (2008). Internal arsenite bioassay calibration using multiple bioreporter cell lines. Microbial Biotechnology 1, 149-157.<br>Chakraborty, T., Babu, P.G., Alam, A., and Chaudhari, A. (2008). GFP expressing bacterial biosensor to measure lead contamination in aquatic environment. Current Science 94, 800-805.<br>Hansen, L.H., and Sorensen, S.J. (2000). Versatile biosensor vectors for detection and quantification of mercury. FEMS Microbiol Lett 193, 123-127.<br>van der Meer, J.R., and Belkin, S. Where microbiology meets microengineering: design and applications of reporter bacteria. Nat Rev Microbiol 8, 511-522.<br><br />
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
<br />
DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
<br />
[[Image:Fig 4.png|500px|center]]<br />
<br />
'''Figure 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''<br />
<br />
Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. <br />
<br />
As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously. <br />
<br />
[[Image:Fig 5.png|500px|center]]<br />
<br />
'''Figure 5. Procedure of DsbA-MBP construction.'''<br />
<br />
As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.<br />
<br />
[[Image:Fig 6.png|500px|center]]<br />
<br />
'''Figure 6. Standardization procedure of DsbA-MBP.'''<br />
<br />
DsbA and MBD gene was amplified by PCR from pET-39b (+)-DsbA-MBP, with the primer containing T7 promoter, RBS and SD restriction sites, as shown in Fig 6. The PCR product was digested with EcoR I / Pst I and then cloned into EcoR I / Pst I double digested pSB1K3, to achieve the goal of standardization of the fusion protein (Fig. 6).<br />
<br />
===RESULT===<br />
Periplasmic Expression DsbA-MBPs was verified by SDS-PAGE and western blotting (Fig 7).<br />
<br />
[[Image:Fig_7.png|600px|center]]<br />
<br />
[[Image:Fig_7b.png|600px|center]]<br />
<br />
'''Figure 7 Result of SDS-PAGE and Western blotting'''<br />
<br />
Over-expression band in SDS-PAGE result and bands specific to his-tag in western blot result were found at about 40 kD. The presence of these bands confirmed that we have got the expected protein expressed in periplasm. But there are large amount of proteins detected in lysate pellet, this was because of the high intensity of T7 promoter. When induced with 0.5 mM IPTG, our target protein were expressed so rapidly that some of them cannot fold properly and accumulated in the inclusion bodies. This problem can be solved by using lower IPTG concentration, lower induction temperature or shorter induction time.<br />
<br />
[[Image:Fig_8.png|500px|center]]<br />
<br />
'''Fig 8. Lpp-OmpA-MBP was designed as a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA, which can be anchored to the outer membrane. '''<br />
<br />
Lpp-OmpA(46-159) fusions were proved to be an effective surface display system to anchor a variety of proteins such as ,B-lactamase [6], a cellulose binding protein, alkaline phosphatase and a single chain Fv antibody fragment(scFv)[7] on the external surface of E. coli. For OmpA, targeting and correct assembly into the outer membrane appear to be distinct events. Extensive studies on it suggest that the region between residue 154 and 180 is crucial for localization on the membrane, while the region between residue 46 and 159 ,the fragment constitute our fusion protein, contains five transmembrane β-strands of an eight-stranded β-barrel, providing an sufficient region to assemble into the membrane[6].<br />
<br />
Additionally, a signal peptide is also needed to mediate the proper localization of the fusion protein on the outer membrane. The N-termini of the outer membrane lipoprotein (Lpp) can serve as the signal peptide, which is considered to be firmly associated with the membrane due to its extreme hydrophobicity [6]. To conclude this, the fusion protein of Lpp-OmpA , with their target and assembly function, can serve as a powerful tool in surface display [9].<br />
<br />
===CONSTRUCTION===<br />
We constructed a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and our metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA. <br />
To test the expression of the fusion protein and the function, it was essential to construct both the standard plasmid and commercial plasmid. <br />
<br />
Firstly, we used PCR to obtain Lpp-OmpA gene from the template of PSD-MBD which was provided by Anne O Summers as a gift. The primers were designed based the literature (Fig 9). And the MBP gene was also obtained by PCR. Then the two fragment were cut with EcoRI(or NdeI), SalI and SalI, PstI (or XhoI), respectively, as inserts and the standard plasmid PSB1A2 was cut with EcoRI and PstI(for the commercial plasmid PET21a, the enzymes were NdeI and XhoI) as the vector. Ultimately, three fragments (Lpp-OmpA, MBP and the vector) were combined to get the standard and commercial plasmid bearing Lpp-OmpA-MBP, as is shown in Fig 9 and Fig 10.<br />
<br />
[[Image:Fig_9.png|450px|center]]<br />
<br />
'''Fig 9. Procedures of the construction of standard plasmid. '''<br />
<br />
[[Image:Fig_10.png|450px|center]]<br />
<br />
'''Fig 10 Procedures of Construction of Commercial Plasmid. '''<br />
<br />
After the construction of the plasmid with the fusion protein gene Lpp-OmpA-MBP, We prefixed T7 promoter and BBa_B0030 upstream of Lpp-OmpA-MBP. Additionally, a strong terminator BBa_B0015 was suffixed. <br />
<br />
[[Image:Fig_11.png|600px|center]]<br />
<br />
'''Fig 11. Results of SDS-PAGE and Western Blotting.''' The overexpression band in SDS-PAGE and specific band in western blotting comfirmed the correct expression and localization of our fusion protein. Specific band cannot be detected in the cytosol which indicates the excellent translocation efficiency of OmpA. <br />
<br />
[[Image:Fig_12.png|500px|center]]<br />
<br />
'''Fig 12. Overview of various localization of MBP. '''<br />
<br />
In summary, as is shown in Fig 12, MBP will be translocated in three different compartments in cell, directly into cytosol, into periplasm with the help of DsbA, and display on the surface using OmpA fusion. We would also like to compare the effects to evaluate the different efficacy of these modules, as is shown below.<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
<br />
DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
<br />
[[Image:Fig 4.png|500px|center]]<br />
<br />
'''Figure 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''<br />
<br />
Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. <br />
<br />
As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously. <br />
<br />
[[Image:Fig 5.png|500px|center]]<br />
<br />
'''Figure 5. Procedure of DsbA-MBP construction.'''<br />
<br />
As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.<br />
<br />
[[Image:Fig 6.png|500px|center]]<br />
<br />
'''Figure 6. Standardization procedure of DsbA-MBP.'''<br />
<br />
DsbA and MBD gene was amplified by PCR from pET-39b (+)-DsbA-MBP, with the primer containing T7 promoter, RBS and SD restriction sites, as shown in Fig 6. The PCR product was digested with EcoR I / Pst I and then cloned into EcoR I / Pst I double digested pSB1K3, to achieve the goal of standardization of the fusion protein (Fig. 6).<br />
<br />
===RESULT===<br />
Periplasmic Expression DsbA-MBPs was verified by SDS-PAGE and western blotting (Fig 7).<br />
<br />
[[Image:Fig_7.png|600px|center]]<br />
<br />
[[Image:Fig_7b.png|600px|center]]<br />
<br />
'''Figure 7 Result of SDS-PAGE and Western blotting'''<br />
<br />
Over-expression band in SDS-PAGE result and bands specific to his-tag in western blot result were found at about 40 kD. The presence of these bands confirmed that we have got the expected protein expressed in periplasm. But there are large amount of proteins detected in lysate pellet, this was because of the high intensity of T7 promoter. When induced with 0.5 mM IPTG, our target protein were expressed so rapidly that some of them cannot fold properly and accumulated in the inclusion bodies. This problem can be solved by using lower IPTG concentration, lower induction temperature or shorter induction time.<br />
<br />
[[Image:Fig_8.png|500px|center]]<br />
<br />
'''Fig 8. Lpp-OmpA-MBP was designed as a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA, which can be anchored to the outer membrane. '''<br />
<br />
Lpp-OmpA(46-159) fusions were proved to be an effective surface display system to anchor a variety of proteins such as ,B-lactamase [6], a cellulose binding protein, alkaline phosphatase and a single chain Fv antibody fragment(scFv)[7] on the external surface of E. coli. For OmpA, targeting and correct assembly into the outer membrane appear to be distinct events. Extensive studies on it suggest that the region between residue 154 and 180 is crucial for localization on the membrane, while the region between residue 46 and 159 ,the fragment constitute our fusion protein, contains five transmembrane β-strands of an eight-stranded β-barrel, providing an sufficient region to assemble into the membrane[6].<br />
<br />
Additionally, a signal peptide is also needed to mediate the proper localization of the fusion protein on the outer membrane. The N-termini of the outer membrane lipoprotein (Lpp) can serve as the signal peptide, which is considered to be firmly associated with the membrane due to its extreme hydrophobicity [6]. To conclude this, the fusion protein of Lpp-OmpA , with their target and assembly function, can serve as a powerful tool in surface display [9].<br />
<br />
===CONSTRUCTION===<br />
We constructed a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and our metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA. <br />
To test the expression of the fusion protein and the function, it was essential to construct both the standard plasmid and commercial plasmid. <br />
<br />
Firstly, we used PCR to obtain Lpp-OmpA gene from the template of PSD-MBD which was provided by Anne O Summers as a gift. The primers were designed based the literature (Fig 9). And the MBP gene was also obtained by PCR. Then the two fragment were cut with EcoRI(or NdeI), SalI and SalI, PstI (or XhoI), respectively, as inserts and the standard plasmid PSB1A2 was cut with EcoRI and PstI(for the commercial plasmid PET21a, the enzymes were NdeI and XhoI) as the vector. Ultimately, three fragments (Lpp-OmpA, MBP and the vector) were combined to get the standard and commercial plasmid bearing Lpp-OmpA-MBP, as is shown in Fig 9 and Fig 10.<br />
<br />
[[Image:Fig_9.png|600px|center]]<br />
<br />
'''Fig 9. Procedures of the construction of standard plasmid. '''<br />
<br />
[[Image:Fig_10.png|600px|center]]<br />
<br />
'''Fig 10 Procedures of Construction of Commercial Plasmid. '''<br />
<br />
After the construction of the plasmid with the fusion protein gene Lpp-OmpA-MBP, We prefixed T7 promoter and BBa_B0030 upstream of Lpp-OmpA-MBP. Additionally, a strong terminator BBa_B0015 was suffixed. <br />
<br />
[[Image:Fig_11.png|600px|center]]<br />
<br />
'''Fig 11. Results of SDS-PAGE and Western Blotting.''' The overexpression band in SDS-PAGE and specific band in western blotting comfirmed the correct expression and localization of our fusion protein. Specific band cannot be detected in the cytosol which indicates the excellent translocation efficiency of OmpA. <br />
<br />
[[Image:Fig_12.png|500px|center]]<br />
<br />
'''Fig 12. Overview of various localization of MBP. '''<br />
<br />
In summary, as is shown in Fig 12, MBP will be translocated in three different compartments in cell, directly into cytosol, into periplasm with the help of DsbA, and display on the surface using OmpA fusion. We would also like to compare the effects to evaluate the different efficacy of these modules, as is shown below.<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:39:31Z<p>Lgdeer: /* RESULT */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
<br />
DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
<br />
[[Image:Fig 4.png|500px|center]]<br />
<br />
'''Figure 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''<br />
<br />
Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. <br />
<br />
As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously. <br />
<br />
[[Image:Fig 5.png|500px|center]]<br />
<br />
'''Figure 5. Procedure of DsbA-MBP construction.'''<br />
<br />
As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.<br />
<br />
[[Image:Fig 6.png|500px|center]]<br />
<br />
'''Figure 6. Standardization procedure of DsbA-MBP.'''<br />
<br />
DsbA and MBD gene was amplified by PCR from pET-39b (+)-DsbA-MBP, with the primer containing T7 promoter, RBS and SD restriction sites, as shown in Fig 6. The PCR product was digested with EcoR I / Pst I and then cloned into EcoR I / Pst I double digested pSB1K3, to achieve the goal of standardization of the fusion protein (Fig. 6).<br />
<br />
===RESULT===<br />
Periplasmic Expression DsbA-MBPs was verified by SDS-PAGE and western blotting (Fig 7).<br />
<br />
[[Image:Fig_7.png|600px|center]]<br />
<br />
[[Image:Fig_7b.png|600px|center]]<br />
<br />
'''Figure 7 Result of SDS-PAGE and Western blotting'''<br />
<br />
Over-expression band in SDS-PAGE result and bands specific to his-tag in western blot result were found at about 40 kD. The presence of these bands confirmed that we have got the expected protein expressed in periplasm. But there are large amount of proteins detected in lysate pellet, this was because of the high intensity of T7 promoter. When induced with 0.5 mM IPTG, our target protein were expressed so rapidly that some of them cannot fold properly and accumulated in the inclusion bodies. This problem can be solved by using lower IPTG concentration, lower induction temperature or shorter induction time.<br />
<br />
[[Image:Fig_8.png|500px|center]]<br />
<br />
'''Fig 8. Lpp-OmpA-MBP was designed as a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA, which can be anchored to the outer membrane. '''<br />
<br />
Lpp-OmpA(46-159) fusions were proved to be an effective surface display system to anchor a variety of proteins such as ,B-lactamase [6], a cellulose binding protein, alkaline phosphatase and a single chain Fv antibody fragment(scFv)[7] on the external surface of E. coli. For OmpA, targeting and correct assembly into the outer membrane appear to be distinct events. Extensive studies on it suggest that the region between residue 154 and 180 is crucial for localization on the membrane, while the region between residue 46 and 159 ,the fragment constitute our fusion protein, contains five transmembrane β-strands of an eight-stranded β-barrel, providing an sufficient region to assemble into the membrane[6].<br />
<br />
Additionally, a signal peptide is also needed to mediate the proper localization of the fusion protein on the outer membrane. The N-termini of the outer membrane lipoprotein (Lpp) can serve as the signal peptide, which is considered to be firmly associated with the membrane due to its extreme hydrophobicity [6]. To conclude this, the fusion protein of Lpp-OmpA , with their target and assembly function, can serve as a powerful tool in surface display [9].<br />
<br />
===CONSTRUCTION===<br />
We constructed a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and our metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA. <br />
To test the expression of the fusion protein and the function, it was essential to construct both the standard plasmid and commercial plasmid. <br />
<br />
Firstly, we used PCR to obtain Lpp-OmpA gene from the template of PSD-MBD which was provided by Anne O Summers as a gift. The primers were designed based the literature (Fig 9). And the MBP gene was also obtained by PCR. Then the two fragment were cut with EcoRI(or NdeI), SalI and SalI, PstI (or XhoI), respectively, as inserts and the standard plasmid PSB1A2 was cut with EcoRI and PstI(for the commercial plasmid PET21a, the enzymes were NdeI and XhoI) as the vector. Ultimately, three fragments (Lpp-OmpA, MBP and the vector) were combined to get the standard and commercial plasmid bearing Lpp-OmpA-MBP, as is shown in Fig 9 and Fig 10.<br />
<br />
[[Image:Fig_11.png|600px|center]]<br />
<br />
'''Fig 11. Results of SDS-PAGE and Western Blotting.''' The overexpression band in SDS-PAGE and specific band in western blotting comfirmed the correct expression and localization of our fusion protein. Specific band cannot be detected in the cytosol which indicates the excellent translocation efficiency of OmpA. <br />
<br />
[[Image:Fig_12.png|500px|center]]<br />
<br />
'''Fig 12. Overview of various localization of MBP. '''<br />
<br />
In summary, as is shown in Fig 12, MBP will be translocated in three different compartments in cell, directly into cytosol, into periplasm with the help of DsbA, and display on the surface using OmpA fusion. We would also like to compare the effects to evaluate the different efficacy of these modules, as is shown below.<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:33:13Z<p>Lgdeer: /* RESULT */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
<br />
DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
<br />
[[Image:Fig 4.png|500px|center]]<br />
<br />
'''Figure 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''<br />
<br />
Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. <br />
<br />
As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously. <br />
<br />
[[Image:Fig 5.png|500px|center]]<br />
<br />
'''Figure 5. Procedure of DsbA-MBP construction.'''<br />
<br />
As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.<br />
<br />
[[Image:Fig 6.png|500px|center]]<br />
<br />
'''Figure 6. Standardization procedure of DsbA-MBP.'''<br />
<br />
DsbA and MBD gene was amplified by PCR from pET-39b (+)-DsbA-MBP, with the primer containing T7 promoter, RBS and SD restriction sites, as shown in Fig 6. The PCR product was digested with EcoR I / Pst I and then cloned into EcoR I / Pst I double digested pSB1K3, to achieve the goal of standardization of the fusion protein (Fig. 6).<br />
<br />
===RESULT===<br />
Periplasmic Expression DsbA-MBPs was verified by SDS-PAGE and western blotting (Fig 7).<br />
<br />
[[Image:Fig_7.png|600px|center]]<br />
<br />
[[Image:Fig_7b.png|600px|center]]<br />
<br />
'''Figure 7 Result of SDS-PAGE and Western blotting'''<br />
<br />
Over-expression band in SDS-PAGE result and bands specific to his-tag in western blot result were found at about 40 kD. The presence of these bands confirmed that we have got the expected protein expressed in periplasm. But there are large amount of proteins detected in lysate pellet, this was because of the high intensity of T7 promoter. When induced with 0.5 mM IPTG, our target protein were expressed so rapidly that some of them cannot fold properly and accumulated in the inclusion bodies. This problem can be solved by using lower IPTG concentration, lower induction temperature or shorter induction time.<br />
<br />
[[Image:Fig_8.png|500px|center]]<br />
<br />
'''Fig 8. Lpp-OmpA-MBP was designed as a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA, which can be anchored to the outer membrane. '''<br />
<br />
Lpp-OmpA(46-159) fusions were proved to be an effective surface display system to anchor a variety of proteins such as ,B-lactamase [6], a cellulose binding protein, alkaline phosphatase and a single chain Fv antibody fragment(scFv)[7] on the external surface of E. coli. For OmpA, targeting and correct assembly into the outer membrane appear to be distinct events. Extensive studies on it suggest that the region between residue 154 and 180 is crucial for localization on the membrane, while the region between residue 46 and 159 ,the fragment constitute our fusion protein, contains five transmembrane β-strands of an eight-stranded β-barrel, providing an sufficient region to assemble into the membrane[6].<br />
<br />
Additionally, a signal peptide is also needed to mediate the proper localization of the fusion protein on the outer membrane. The N-termini of the outer membrane lipoprotein (Lpp) can serve as the signal peptide, which is considered to be firmly associated with the membrane due to its extreme hydrophobicity [6]. To conclude this, the fusion protein of Lpp-OmpA , with their target and assembly function, can serve as a powerful tool in surface display [9].<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:26:05Z<p>Lgdeer: /* Periplasmic Translocation of MBP */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
<br />
DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
<br />
[[Image:Fig 4.png|500px|center]]<br />
<br />
'''Figure 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''<br />
<br />
Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. <br />
<br />
As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously. <br />
<br />
[[Image:Fig 5.png|500px|center]]<br />
<br />
'''Figure 5. Procedure of DsbA-MBP construction.'''<br />
<br />
As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.<br />
<br />
[[Image:Fig 6.png|500px|center]]<br />
<br />
'''Figure 6. Standardization procedure of DsbA-MBP.'''<br />
<br />
DsbA and MBD gene was amplified by PCR from pET-39b (+)-DsbA-MBP, with the primer containing T7 promoter, RBS and SD restriction sites, as shown in Fig 6. The PCR product was digested with EcoR I / Pst I and then cloned into EcoR I / Pst I double digested pSB1K3, to achieve the goal of standardization of the fusion protein (Fig. 6).<br />
<br />
===RESULT===<br />
Periplasmic Expression DsbA-MBPs was verified by SDS-PAGE and western blotting (Fig 7).<br />
<br />
[[Image:Fig_7.png|600px|center]]<br />
<br />
[[Image:Fig_7b.png|600px|center]]<br />
<br />
'''Figure 7 Result of SDS-PAGE and Western blotting'''<br />
<br />
Over-expression band in SDS-PAGE result and bands specific to his-tag in western blot result were found at about 40 kD. The presence of these bands confirmed that we have got the expected protein expressed in periplasm. But there are large amount of proteins detected in lysate pellet, this was because of the high intensity of T7 promoter. When induced with 0.5 mM IPTG, our target protein were expressed so rapidly that some of them cannot fold properly and accumulated in the inclusion bodies. This problem can be solved by using lower IPTG concentration, lower induction temperature or shorter induction time.<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
<br />
DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
<br />
[[Image:Fig 4.png|500px|center]]<br />
<br />
'''Figure 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''<br />
<br />
Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. <br />
<br />
As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously. <br />
<br />
[[Image:Fig 5.png|500px|center]]<br />
<br />
'''Figure 5. Procedure of DsbA-MBP construction.'''<br />
<br />
As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.<br />
<br />
[[Image:Fig 6.png|500px|center]]<br />
<br />
'''Figure 6. Standardization procedure of DsbA-MBP.'''<br />
===RESULT===<br />
Periplasmic Expression DsbA-MBPs was verified by SDS-PAGE and western blotting (Fig 7).<br />
<br />
[[Image:Fig_7.png|600px|center]]<br />
<br />
[[Image:Fig_7b.png|600px|center]]<br />
<br />
'''Figure 7 Result of SDS-PAGE and Western blotting'''<br />
<br />
Over-expression band in SDS-PAGE result and bands specific to his-tag in western blot result were found at about 40 kD. The presence of these bands confirmed that we have got the expected protein expressed in periplasm. But there are large amount of proteins detected in lysate pellet, this was because of the high intensity of T7 promoter. When induced with 0.5 mM IPTG, our target protein were expressed so rapidly that some of them cannot fold properly and accumulated in the inclusion bodies. This problem can be solved by using lower IPTG concentration, lower induction temperature or shorter induction time.<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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</div></div>Lgdeerhttp://2010.igem.org/File:Fig_7b.pngFile:Fig 7b.png2010-10-27T14:23:02Z<p>Lgdeer: </p>
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<div></div>Lgdeerhttp://2010.igem.org/File:Fig_7.pngFile:Fig 7.png2010-10-27T14:21:43Z<p>Lgdeer: </p>
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<div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:20:36Z<p>Lgdeer: /* Periplasmic Translocation of MBP */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
<br />
DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
<br />
[[Image:Fig 4.png|500px|center]]<br />
<br />
'''Figure 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''<br />
<br />
Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. <br />
<br />
As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously. <br />
<br />
[[Image:Fig 5.png|500px|center]]<br />
<br />
'''Figure 5. Procedure of DsbA-MBP construction.'''<br />
<br />
As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.<br />
<br />
[[Image:Fig 6.png|500px|center]]<br />
<br />
'''Figure 6. Standardization procedure of DsbA-MBP.'''<br />
===RESULT===<br />
Periplasmic Expression DsbA-MBPs was verified by SDS-PAGE and western blotting (Fig 7).<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:19:13Z<p>Lgdeer: /* Periplasmic Translocation of MBP */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
<br />
DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
<br />
[[Image:Fig 4.png|500px|center]]<br />
<br />
'''Figure 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''<br />
<br />
Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. <br />
<br />
As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously. <br />
<br />
[[Image:Fig 5.png|500px|center]]<br />
<br />
'''Figure 5. Procedure of DsbA-MBP construction.'''<br />
<br />
As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.<br />
<br />
[[Image:Fig 6.png|500px|center]]<br />
<br />
'''Figure 6. Standardization procedure of DsbA-MBP.'''<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:16:16Z<p>Lgdeer: /* Periplasmic Translocation of MBP */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
<br />
DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
<br />
[[Image:Fig 4.png|500px|center]]<br />
<br />
'''Figure 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''<br />
<br />
Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins. <br />
<br />
As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously. <br />
<br />
[[Image:Fig 5.png|500px|center]]<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing.<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<div></div>Lgdeerhttp://2010.igem.org/File:Fig_4.pngFile:Fig 4.png2010-10-27T14:13:08Z<p>Lgdeer: </p>
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<div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:12:10Z<p>Lgdeer: /* METAL BINDING PEPTIDE (MBP) CONSTRUCTION */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==Periplasmic Translocation of MBP==<br />
Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal. <br />
<br />
DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
<br />
B<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing.<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:09:58Z<p>Lgdeer: /* Cytoplasmic Expression of MBP */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
<br />
B<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:09:05Z<p>Lgdeer: /* Cytoplasmic Expression of MBP */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3) <br />
<br />
[[Image:FIG_3.png|center]]<br />
<br />
'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''<br />
<br />
MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
<br />
'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
<br />
B<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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</div><br />
<br />
<br />
<div id="bottomwhite"><br />
<a href="#top"><img src="https://static.igem.org/mediawiki/2010/8/87/Top.png" width="100px" height="75px"alt="go back to top"></a><br />
</div></div>Lgdeerhttp://2010.igem.org/File:FIG_3.pngFile:FIG 3.png2010-10-27T14:07:26Z<p>Lgdeer: </p>
<hr />
<div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:06:12Z<p>Lgdeer: </p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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</html><br />
As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png|600px|center]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
[[Image:Fig2.png|450px|center]]<br />
<br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br><br />
'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''<br />
<br />
<br />
Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.<br />
<br />
MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
<br />
'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
<br />
B<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<div id="bottomwhite"><br />
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</div></div>Lgdeerhttp://2010.igem.org/File:Fig2.pngFile:Fig2.png2010-10-27T14:05:20Z<p>Lgdeer: </p>
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<div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T14:03:04Z<p>Lgdeer: </p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
[[Image:FIG_1.png]]<br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" width=650 ></a></html><br><br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br><br />
'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''<br />
<br />
<br />
Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.<br />
<br />
MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
<br />
'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
<br />
B<br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<div id="bottomwhite"><br />
<a href="#top"><img src="https://static.igem.org/mediawiki/2010/8/87/Top.png" width="100px" height="75px"alt="go back to top"></a><br />
</div></div>Lgdeerhttp://2010.igem.org/File:FIG_1.pngFile:FIG 1.png2010-10-27T14:01:47Z<p>Lgdeer: </p>
<hr />
<div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T13:59:08Z<p>Lgdeer: /* Cytoplasmic Expression of MBP */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
<br><br><br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html><br />
[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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<div id="middlewhite"><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" width=650 ></a></html><br><br />
'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br><br />
'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''<br />
<br />
<br />
Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.<br />
<br />
MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
<br />
'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
<br />
B<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<div id="bottomwhite"><br />
<a href="#top"><img src="https://static.igem.org/mediawiki/2010/8/87/Top.png" width="100px" height="75px"alt="go back to top"></a><br />
</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T13:57:32Z<p>Lgdeer: /* STRUCTURE ANALYSIS */</p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
<br><br><br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html><br />
[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
</div><br />
<div id="middlewhite"><br />
</html><br />
As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==Cytoplasmic Expression of MBP==<br />
To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" width=650 ></a></html><br><br />
'''Figure 1. The results of 3D structure modeling for MerR based on data of CueR. (A) Crystal structure of CueR[2], which represent the overall structure of the MerR family. The ribbon diagram depicts one monomer in color and the other in gray, with DNA-binding domain in blue, the dimerization helix in red, the metal binding domain in purple and the metal ion in cyan. (B) The predicted three dimensional conformation of Hg (II) bound MerR structure by our 3D modeling. Two monomers are indicated in green and cyan, respectively. (C) and (D) were obtained after rotation of the structure in (B) by 90° about the z-axis and x- axis, respectively. The C-terminal metal binding domain and the N-terminal DNA binding domain are likely to be modular for each other. '''<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br><br />
'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''<br />
<br />
<br />
Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.<br />
<br />
MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
<br />
'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
<br />
B<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T13:45:02Z<p>Lgdeer: </p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
'''Figure1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''<br />
<br />
==STRUCTURE ANALYSIS==<br />
As described before, MerR family transcription factors can be separated into two modular domains: the metal binding domain and DNA binding domain. The signature of the family is a helix-turn-helix (HTH) motif followed by a coiled-coil region, namely, a similar N-terminal helix-turn-helix DNA binding domains and a C-terminal effector binding domains that are specific to the effector (eg. metal ions)to be recognized[1] (Fig. 1A, Fig 3). These two domains communicate with each other by the intervening region (hinge). The crystal structure of several members of MerR family have been solved which provides us evidence for MBP design. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" width=650 ></a></html><br><br />
'''Figure 1. The results of 3D structure modeling for MerR based on data of CueR. (A) Crystal structure of CueR[2], which represent the overall structure of the MerR family. The ribbon diagram depicts one monomer in color and the other in gray, with DNA-binding domain in blue, the dimerization helix in red, the metal binding domain in purple and the metal ion in cyan. (B) The predicted three dimensional conformation of Hg (II) bound MerR structure by our 3D modeling. Two monomers are indicated in green and cyan, respectively. (C) and (D) were obtained after rotation of the structure in (B) by 90° about the z-axis and x- axis, respectively. The C-terminal metal binding domain and the N-terminal DNA binding domain are likely to be modular for each other. '''<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br><br />
'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''<br />
<br />
<br />
Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.<br />
<br />
MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
<br />
'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
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B<br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T13:42:11Z<p>Lgdeer: </p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1). <br />
<br />
<br />
==STRUCTURE ANALYSIS==<br />
As described before, MerR family transcription factors can be separated into two modular domains: the metal binding domain and DNA binding domain. The signature of the family is a helix-turn-helix (HTH) motif followed by a coiled-coil region, namely, a similar N-terminal helix-turn-helix DNA binding domains and a C-terminal effector binding domains that are specific to the effector (eg. metal ions)to be recognized[1] (Fig. 1A, Fig 3). These two domains communicate with each other by the intervening region (hinge). The crystal structure of several members of MerR family have been solved which provides us evidence for MBP design. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" width=650 ></a></html><br><br />
'''Figure 1. The results of 3D structure modeling for MerR based on data of CueR. (A) Crystal structure of CueR[2], which represent the overall structure of the MerR family. The ribbon diagram depicts one monomer in color and the other in gray, with DNA-binding domain in blue, the dimerization helix in red, the metal binding domain in purple and the metal ion in cyan. (B) The predicted three dimensional conformation of Hg (II) bound MerR structure by our 3D modeling. Two monomers are indicated in green and cyan, respectively. (C) and (D) were obtained after rotation of the structure in (B) by 90° about the z-axis and x- axis, respectively. The C-terminal metal binding domain and the N-terminal DNA binding domain are likely to be modular for each other. '''<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br><br />
'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''<br />
<br />
<br />
Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.<br />
<br />
MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
<br />
'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
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B<br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<div id="bottomwhite"><br />
<a href="#top"><img src="https://static.igem.org/mediawiki/2010/8/87/Top.png" width="100px" height="75px"alt="go back to top"></a><br />
</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T13:41:25Z<p>Lgdeer: </p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
</div><br />
<div id="middlewhite"><br />
</html><br />
As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).<br />
<br />
However, since MerR is a transcription regulator, over-expression of MerR in bacteria may lead to some unpredictable side effect. Earlier work suggested that the truncated peptide only consisting of the metal binding domain can form a stable dimer with its mercury binding affinity remained [ ]; and DNA binding domain and metal binding domain can function individually [ ]. Based on all these above and carefully structure analysis of MerR via 3D structure modeling, we directly tandemed two copies of metal binding domain of MerR together, to implement a mercury metal binding peptide (MBP) (Fig 2). <br />
<br />
<br />
==STRUCTURE ANALYSIS==<br />
As described before, MerR family transcription factors can be separated into two modular domains: the metal binding domain and DNA binding domain. The signature of the family is a helix-turn-helix (HTH) motif followed by a coiled-coil region, namely, a similar N-terminal helix-turn-helix DNA binding domains and a C-terminal effector binding domains that are specific to the effector (eg. metal ions)to be recognized[1] (Fig. 1A, Fig 3). These two domains communicate with each other by the intervening region (hinge). The crystal structure of several members of MerR family have been solved which provides us evidence for MBP design. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" width=650 ></a></html><br><br />
'''Figure 1. The results of 3D structure modeling for MerR based on data of CueR. (A) Crystal structure of CueR[2], which represent the overall structure of the MerR family. The ribbon diagram depicts one monomer in color and the other in gray, with DNA-binding domain in blue, the dimerization helix in red, the metal binding domain in purple and the metal ion in cyan. (B) The predicted three dimensional conformation of Hg (II) bound MerR structure by our 3D modeling. Two monomers are indicated in green and cyan, respectively. (C) and (D) were obtained after rotation of the structure in (B) by 90° about the z-axis and x- axis, respectively. The C-terminal metal binding domain and the N-terminal DNA binding domain are likely to be modular for each other. '''<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br><br />
'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''<br />
<br />
<br />
Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.<br />
<br />
MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
<br />
'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
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B<br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T13:39:03Z<p>Lgdeer: </p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between (Details can be seen in the MBP Construction Part).<br />
<br />
However, since MerR is a transcription regulator, over-expression of MerR in bacteria may lead to some unpredictable side effect. Earlier work suggested that the truncated peptide only consisting of the metal binding domain can form a stable dimer with its mercury binding affinity remained [ ]; and DNA binding domain and metal binding domain can function individually [ ]. Based on all these above and carefully structure analysis of MerR via 3D structure modeling, we directly tandemed two copies of metal binding domain of MerR together, to implement a mercury metal binding peptide (MBP) (Fig 2). <br />
<br />
<br />
==STRUCTURE ANALYSIS==<br />
As described before, MerR family transcription factors can be separated into two modular domains: the metal binding domain and DNA binding domain. The signature of the family is a helix-turn-helix (HTH) motif followed by a coiled-coil region, namely, a similar N-terminal helix-turn-helix DNA binding domains and a C-terminal effector binding domains that are specific to the effector (eg. metal ions)to be recognized[1] (Fig. 1A, Fig 3). These two domains communicate with each other by the intervening region (hinge). The crystal structure of several members of MerR family have been solved which provides us evidence for MBP design. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" width=650 ></a></html><br><br />
'''Figure 1. The results of 3D structure modeling for MerR based on data of CueR. (A) Crystal structure of CueR[2], which represent the overall structure of the MerR family. The ribbon diagram depicts one monomer in color and the other in gray, with DNA-binding domain in blue, the dimerization helix in red, the metal binding domain in purple and the metal ion in cyan. (B) The predicted three dimensional conformation of Hg (II) bound MerR structure by our 3D modeling. Two monomers are indicated in green and cyan, respectively. (C) and (D) were obtained after rotation of the structure in (B) by 90° about the z-axis and x- axis, respectively. The C-terminal metal binding domain and the N-terminal DNA binding domain are likely to be modular for each other. '''<br />
<br />
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<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br><br />
'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''<br />
<br />
<br />
Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.<br />
<br />
MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
<br />
'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
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B<br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<div id="bottomwhite"><br />
<a href="#top"><img src="https://static.igem.org/mediawiki/2010/8/87/Top.png" width="100px" height="75px"alt="go back to top"></a><br />
</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpressionTeam:Peking/Project/Bioabsorbent/MBPExpression2010-10-27T13:37:59Z<p>Lgdeer: </p>
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Construction</font></font></font><br />
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Bioabsorbent|Bioabsorbent]] > [[Team:Peking/Project/Bioabsorbent/MBPExpression|MBP Expression]]<html><br />
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Early biophysical work shows that homodimer is the active form of MerR. The metal binding domain lies at the C-terminal half of the protein, as predicted by earlier genetic, biochemical, and biophysical work, while the Helix-Turn-Helix DNA binding domain locates at the N-terminal (Fig 1A). To be specific, MerR can response to as low as 10-9 M Hg (II) even in the presence of 1 to 5 mM competing thiol ligands[ ] ,which indicates that MerR may act as an effective mercury accumulator in aquatic environment. <br />
<br />
However, since MerR is a transcription regulator, over-expression of MerR in bacteria may lead to some unpredictable side effect. Earlier work suggested that the truncated peptide only consisting of the metal binding domain can form a stable dimer with its mercury binding affinity remained [ ]; and DNA binding domain and metal binding domain can function individually [ ]. Based on all these above and carefully structure analysis of MerR via 3D structure modeling, we directly tandemed two copies of metal binding domain of MerR together, to implement a mercury metal binding peptide (MBP) (Fig 2). <br />
<br />
<br />
==STRUCTURE ANALYSIS==<br />
As described before, MerR family transcription factors can be separated into two modular domains: the metal binding domain and DNA binding domain. The signature of the family is a helix-turn-helix (HTH) motif followed by a coiled-coil region, namely, a similar N-terminal helix-turn-helix DNA binding domains and a C-terminal effector binding domains that are specific to the effector (eg. metal ions)to be recognized[1] (Fig. 1A, Fig 3). These two domains communicate with each other by the intervening region (hinge). The crystal structure of several members of MerR family have been solved which provides us evidence for MBP design. <br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" width=650 ></a></html><br><br />
'''Figure 1. The results of 3D structure modeling for MerR based on data of CueR. (A) Crystal structure of CueR[2], which represent the overall structure of the MerR family. The ribbon diagram depicts one monomer in color and the other in gray, with DNA-binding domain in blue, the dimerization helix in red, the metal binding domain in purple and the metal ion in cyan. (B) The predicted three dimensional conformation of Hg (II) bound MerR structure by our 3D modeling. Two monomers are indicated in green and cyan, respectively. (C) and (D) were obtained after rotation of the structure in (B) by 90° about the z-axis and x- axis, respectively. The C-terminal metal binding domain and the N-terminal DNA binding domain are likely to be modular for each other. '''<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br><br />
'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''<br />
<br />
<br />
Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.<br />
<br />
MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
<br />
'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
<br />
==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
<br />
A<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
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B<br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
<br />
'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
<br />
To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
<br />
Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
<br />
<br />
==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
<br />
3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
<br />
8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
<br />
9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
<br />
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<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Construction</font></font></font><br />
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Early biophysical work shows that homodimer is the active form of MerR. The metal binding domain lies at the C-terminal half of the protein, as predicted by earlier genetic, biochemical, and biophysical work, while the Helix-Turn-Helix DNA binding domain locates at the N-terminal (Fig 1A). To be specific, MerR can response to as low as 10-9 M Hg (II) even in the presence of 1 to 5 mM competing thiol ligands[ ] ,which indicates that MerR may act as an effective mercury accumulator in aquatic environment. <br />
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However, since MerR is a transcription regulator, over-expression of MerR in bacteria may lead to some unpredictable side effect. Earlier work suggested that the truncated peptide only consisting of the metal binding domain can form a stable dimer with its mercury binding affinity remained [ ]; and DNA binding domain and metal binding domain can function individually [ ]. Based on all these above and carefully structure analysis of MerR via 3D structure modeling, we directly tandemed two copies of metal binding domain of MerR together, to implement a mercury metal binding peptide (MBP) (Fig 2). <br />
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==STRUCTURE ANALYSIS==<br />
As described before, MerR family transcription factors can be separated into two modular domains: the metal binding domain and DNA binding domain. The signature of the family is a helix-turn-helix (HTH) motif followed by a coiled-coil region, namely, a similar N-terminal helix-turn-helix DNA binding domains and a C-terminal effector binding domains that are specific to the effector (eg. metal ions)to be recognized[1] (Fig. 1A, Fig 3). These two domains communicate with each other by the intervening region (hinge). The crystal structure of several members of MerR family have been solved which provides us evidence for MBP design. <br />
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<html><a href="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" width=650 ></a></html><br><br />
'''Figure 1. The results of 3D structure modeling for MerR based on data of CueR. (A) Crystal structure of CueR[2], which represent the overall structure of the MerR family. The ribbon diagram depicts one monomer in color and the other in gray, with DNA-binding domain in blue, the dimerization helix in red, the metal binding domain in purple and the metal ion in cyan. (B) The predicted three dimensional conformation of Hg (II) bound MerR structure by our 3D modeling. Two monomers are indicated in green and cyan, respectively. (C) and (D) were obtained after rotation of the structure in (B) by 90° about the z-axis and x- axis, respectively. The C-terminal metal binding domain and the N-terminal DNA binding domain are likely to be modular for each other. '''<br />
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<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br><br />
'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''<br />
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Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.<br />
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MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide. <br />
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<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html><br />
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'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''<br />
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==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==<br />
To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).<br />
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A<br />
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<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html><br />
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B<br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html><br />
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'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''<br />
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To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing. <br />
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Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing. <br />
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==Reference==<br />
1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
<br />
2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.<br />
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3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.<br />
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4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.<br />
<br />
5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.<br />
<br />
6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.<br />
<br />
7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448<br />
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8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.<br />
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9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719<br />
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Calculating Process</font></font></font><br />
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=Network Enumeration=<br />
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<p>We use three nodes as a minimal framework: one node that receives input( A in Figure 2 ), a second node that transmits output( C in Figure 2 ), and a third node that can play diverse regulatory roles( B in Figure 2 ). There are 9 direct links among the three nodes and there are altogether 3^9=19,683 three-node topologies. With 3,645 topologies that have no direct or indirect links from the input to the output occluded, there remain a total of 16,038 possible three-node topologies that contain at least one direct or indirect causal link from the input node to the output node. For each topology, we sampled 10,000 sets of network parameters with the method of latin hypercube sampling (LHS, Figure 3). In all, we have analyzed a total of 16,038*10,000 different circuits. This search resulted in an exhaustive circuit function map used to extract core topological motifs essential for IOA.</html><br />
[[Image:Network.png|245px|center]]<br><br />
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<b><font size=1.5>Figure 2 Three-node network with all of its possible directed links(Ref. 7) </b><br />
There are altogether 9 possible links and the input here is Hg(Ⅱ), and the concentration of C is taken as output.<br></html><br />
[[Image:LHS.png|315px|center]]<br><br />
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<b>Figure 3 Latin Hypercube Sampling</b></font><br />
When sampling a function of N variables, the range of each variable is divided into M equally probable intervals, M sample points are then placed to satisfy the Latin Hypercube requirements. Then each sample is the Only one in each axis-aligned hyperplane containing it.</font><br />
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=Equations Set Up=<br />
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Our model is based on mainly the following statements:<br><br />
<br>(1) The nodes are restricted to TF nodes so that the links stand for TF-TF interactions via DNA. The expression level is quantified by the equilibrium binding probability P of TF binding on its site and the maximum expression rate constant β, and we adopt a constant λ to modify P to make different TFs equal status. When it comes to several TF factors, we use the multiplication of their λP or 1-λP to indicate their interactions.<br><br />
<br>(2) We take into consideration only the transcription and translation process because other reactions such as signal-transduction activities typically operate much faster and can be considered to be approximately at steady state on the slow timescales of transcription networks. Also the TF activity levels can be considered to be at steady state within the equations that describe network dynamics on the slow timescale of changes in protein levels. So that the equations contain only the accumulation and degradation of the protein products ( here the TFs). <br><br />
<br>(3) It has been observed that one ordinary gene usually has a nonzero expression level with no TFs on its binding site. We propose that one repressor will lower the initial expression level and one activator will shift it, further on, each TF has its unique contribution to the final expression level,that is, they shift or lower the expression level to different extents according to their own properties.<br><br />
<br>&nbsp;&nbsp;Consider first the simplest condition under which there is only one link from a node to another. (A<img src="https://static.igem.org/mediawiki/2010/8/88/Jiantou.png" width="13">C, Figure 4)<br><br />
It is widely accepted that the possibility of TF binding to the binding site in promoter is<br><img src="https://static.igem.org/mediawiki/2010/0/02/Function1.png" width="120">(X*: the effective concentration of one TF; Kd : the dissociation constant )<br><br><br />
&nbsp;&nbsp;According to hypothesis (1)&(3), the link from A(node1) to C(node3) can be translated as<br><br><br />
<img src="https://static.igem.org/mediawiki/2010/c/cc/Function2.png" width="670"><br><br><br />
( <img src="https://static.igem.org/mediawiki/2010/4/4b/Beita0.png" width="15">:the basal translation rate factor;<img src="https://static.igem.org/mediawiki/2010/d/d4/Beitam.png" width="15">:the effective translation rate factor.)<br />
<br><br />
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&nbsp;&nbsp;As <img src="https://static.igem.org/mediawiki/2010/1/15/Function3.png" width="177">(Figure 5(a))<br />
<br><br><br />
&nbsp;&nbsp;Thus <img src="https://static.igem.org/mediawiki/2010/0/00/Function4.png" width="386">(Figure 5(b))(The subscript of X is the node number.) <br />
<br><br>&nbsp;&nbsp;Actually, the first component of the equation is the fundamental expression level of the network in which the <img src="https://static.igem.org/mediawiki/2010/a/ac/1-lamdaP.png" width="45"> is the possibility that the TF is off the DNA target site, and the second component of the equation is the effective expression level in which <img src="https://static.igem.org/mediawiki/2010/7/70/LamdaP.png" width="25"> is the possibility that TF is on its site.<br />
<br><br><br />
&nbsp;&nbsp;As to general conditions, there are<br><br />
<img src="https://static.igem.org/mediawiki/2010/c/cc/Function5.png" width="554"> <br><br><br />
&nbsp;&nbsp;And in order to make the equations fit hypothesis 3,we deduced the proper range of <img src="https://static.igem.org/mediawiki/2010/a/aa/Lamda.png" width="14">, that is, no regulation <img src="https://static.igem.org/mediawiki/2010/d/da/Function6.png" width="33"> , activation <img src="https://static.igem.org/mediawiki/2010/7/78/Function7.png" width="53"> and repression <img src="https://static.igem.org/mediawiki/2010/2/22/Function8.png" width="132">.The choice of other Parameter values and their reference is in Table 1.<br />
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[[Image:Simple_network.png|center|50px]]<br />
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<font size=1.5><br><b>Figure 4 simplest network</b> There is only one link from node A to node C and we don’t regulate the property of the link. Actually, whether it is activation, repression or no regulation is represented by λ.<br />
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[[Image:MerR+Hg+DNA.png|650px|center]]<br />
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<font size=1.5><br><b>Figure 5 MerR interacts with Hg(Ⅱ)& MerR-Hg binding to DNA</b> (a)The orange poor arc sector stands for the MerR monomer and the orange major arc sector stands for the MerR dimer. The blue ball is Hg ion that can be bound by the MerR dimer, and only the complex can serve as Transcription Factor that promotes the expression of downstream genes. We list the balance equations, derive the calculation function of X* as above, and substitute this function to the ODE equations used in our model. (b) As the MerR dimer is only slightly repress DNA transcription, we regrad Kd' << K1*Kd, so neglecting the competitive binding of MerR dimer to DNA.<br />
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<font size=1.5><br><b>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Table 1 Physiological range of the model parameters</b></font><br />
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[[Image:Table_1.png|500px|center]]<br />
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=Network Topologies’ Analysis=<br />
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Aiming at getting the values of r for each circuit, we need to numerically simulate the ODE equations to get the steady-state concentration of output node C under each input concentration, and then making linear fit of input and output concentrations. As our input concentration range is 10-9~10-5, we select points that have the same logarithmic distance intervals, then simulate output evolution curve to get the steady concentration one point by one. We choose the fourth-order Runge-Kutta method to solve the ODE equations and so as to save calculation time, we adopt Implicit Runge-Kutta algorithm to get the output steady concentration when Input=10-9M and set the very concentration as initial value for the Newton-Raphson method for following different Input concentration. And considering the possibility of bistable network topology, we calculate the two directions (positive sequence and the reverse ) that Input concentration changes to avoid wrongly supposing it as one IOA function circuit.<br />
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Calculating Process</font></font></font><br />
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[[Team:Peking/Modeling|Modeling]] > [[Team:Peking/Modeling/CalculationProcess|Calculation Process]] <html><br />
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=Network Enumeration=<br />
<html><br />
<p>We use three nodes as a minimal framework: one node that receives input( A in Figure 2 ), a second node that transmits output( C in Figure 2 ), and a third node that can play diverse regulatory roles( B in Figure 2 ). There are 9 direct links among the three nodes and there are altogether 3^9=19,683 three-node topologies. With 3,645 topologies that have no direct or indirect links from the input to the output occluded, there remain a total of 16,038 possible three-node topologies that contain at least one direct or indirect causal link from the input node to the output node. For each topology, we sampled 10,000 sets of network parameters with the method of latin hypercube sampling (LHS, Figure 3). In all, we have analyzed a total of 16,038*10,000 different circuits. This search resulted in an exhaustive circuit function map used to extract core topological motifs essential for IOA.</html><br />
[[Image:Network.png|245px|center]]<br><br />
<html><br />
<b><font size=1.5>Figure 2 Three-node network with all of its possible directed links(Ref. 7) </b><br />
There are altogether 9 possible links and the input here is Hg(Ⅱ), and the concentration of C is taken as output.<br></html><br />
[[Image:LHS.png|315px|center]]<br><br />
<html><font size=1.5><br />
<b>Figure 3 Latin Hypercube Sampling</b></font><br />
When sampling a function of N variables, the range of each variable is divided into M equally probable intervals, M sample points are then placed to satisfy the Latin Hypercube requirements. Then each sample is the Only one in each axis-aligned hyperplane containing it.</font><br />
</p><br />
</html><br />
[[https://2010.igem.org/Team:Peking/Modeling TOP]]<br />
<br />
=Equations Set Up=<br />
<html><br />
Our model is based on mainly the following statements:<br><br />
<br>(1) The nodes are restricted to TF nodes so that the links stand for TF-TF interactions via DNA. The expression level is quantified by the equilibrium binding probability P of TF binding on its site and the maximum expression rate constant β, and we adopt a constant λ to modify P to make different TFs equal status. When it comes to several TF factors, we use the multiplication of their <img src="https://static.igem.org/mediawiki/2010/7/70/LamdaP.png" width="25"> or <img src="https://static.igem.org/mediawiki/2010/a/ac/1-lamdaP.png" width="45"> to indicate their interactions.<br><br />
<br>(2) We take into consideration only the transcription and translation process because other reactions such as signal-transduction activities typically operate much faster and can be considered to be approximately at steady state on the slow timescales of transcription networks. Also the TF activity levels can be considered to be at steady state within the equations that describe network dynamics on the slow timescale of changes in protein levels. So that the equations contain only the accumulation and degradation of the protein products ( here the TFs). <br><br />
<br>(3) It has been observed that one ordinary gene usually has a nonzero expression level with no TFs on its binding site. We propose that one repressor will lower the initial expression level and one activator will shift it, further on, each TF has its unique contribution to the final expression level,that is, they shift or lower the expression level to different extents according to their own properties.<br><br />
<br>&nbsp;&nbsp;Consider first the simplest condition under which there is only one link from a node to another. (A<img src="https://static.igem.org/mediawiki/2010/8/88/Jiantou.png" width="13">C, Figure 4)<br><br />
It is widely accepted that the possibility of TF binding to the binding site in promoter is<br><img src="https://static.igem.org/mediawiki/2010/0/02/Function1.png" width="120">(X*: the effective concentration of one TF; Kd : the dissociation constant )<br><br><br />
&nbsp;&nbsp;According to hypothesis (1)&(3), the link from A(node1) to C(node3) can be translated as<br><br><br />
<img src="https://static.igem.org/mediawiki/2010/c/cc/Function2.png" width="670"><br><br><br />
( <img src="https://static.igem.org/mediawiki/2010/4/4b/Beita0.png" width="15">:the basal translation rate factor;<img src="https://static.igem.org/mediawiki/2010/d/d4/Beitam.png" width="15">:the effective translation rate factor.)<br />
<br><br />
<br><br />
&nbsp;&nbsp;As <img src="https://static.igem.org/mediawiki/2010/1/15/Function3.png" width="177">(Figure 5(a))<br />
<br><br><br />
&nbsp;&nbsp;Thus <img src="https://static.igem.org/mediawiki/2010/0/00/Function4.png" width="386">(Figure 5(b))(The subscript of X is the node number.) <br />
<br><br>&nbsp;&nbsp;Actually, the first component of the equation is the fundamental expression level of the network in which the <img src="https://static.igem.org/mediawiki/2010/a/ac/1-lamdaP.png" width="45"> is the possibility that the TF is off the DNA target site, and the second component of the equation is the effective expression level in which <img src="https://static.igem.org/mediawiki/2010/7/70/LamdaP.png" width="25"> is the possibility that TF is on its site.<br />
<br><br><br />
&nbsp;&nbsp;As to general conditions, there are<br><br />
<img src="https://static.igem.org/mediawiki/2010/c/cc/Function5.png" width="554"> <br><br><br />
&nbsp;&nbsp;And in order to make the equations fit hypothesis 3,we deduced the proper range of <img src="https://static.igem.org/mediawiki/2010/a/aa/Lamda.png" width="14">, that is, no regulation <img src="https://static.igem.org/mediawiki/2010/d/da/Function6.png" width="33"> , activation <img src="https://static.igem.org/mediawiki/2010/7/78/Function7.png" width="53"> and repression <img src="https://static.igem.org/mediawiki/2010/2/22/Function8.png" width="132">.The choice of other Parameter values and their reference is in Table 1.<br />
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[[Image:Simple_network.png|center|50px]]<br />
<html><br />
<font size=1.5><br><b>Figure 4 simplest network</b> There is only one link from node A to node C and we don’t regulate the property of the link. Actually, whether it is activation, repression or no regulation is represented by λ.<br />
<br></font><br />
</html><br />
[[Image:MerR+Hg+DNA.png|650px|center]]<br />
<br><br />
<html><br />
<font size=1.5><br><b>Figure 5 MerR interacts with Hg(Ⅱ)& MerR-Hg binding to DNA</b> (a)The orange poor arc sector stands for the MerR monomer and the orange major arc sector stands for the MerR dimer. The blue ball is Hg ion that can be bound by the MerR dimer, and only the complex can serve as Transcription Factor that promotes the expression of downstream genes. We list the balance equations, derive the calculation function of X* as above, and substitute this function to the ODE equations used in our model. (b) As the MerR dimer is only slightly repress DNA transcription, we regrad Kd' << K1*Kd, so neglecting the competitive binding of MerR dimer to DNA.<br />
<br></font><br />
</html><br />
<html><br />
<font size=1.5><br><b>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Table 1 Physiological range of the model parameters</b></font><br />
<br><br />
</html><br />
[[Image:Table_1.png|500px|center]]<br />
<br><br />
<br />
[[https://2010.igem.org/Team:Peking/Modeling TOP]]<br />
<br />
=Network Topologies’ Analysis=<br />
<html><br />
Aiming at getting the values of r for each circuit, we need to numerically simulate the ODE equations to get the steady-state concentration of output node C under each input concentration, and then making linear fit of input and output concentrations. As our input concentration range is 10-9~10-5, we select points that have the same logarithmic distance intervals, then simulate output evolution curve to get the steady concentration one point by one. We choose the fourth-order Runge-Kutta method to solve the ODE equations and so as to save calculation time, we adopt Implicit Runge-Kutta algorithm to get the output steady concentration when Input=10-9M and set the very concentration as initial value for the Newton-Raphson method for following different Input concentration. And considering the possibility of bistable network topology, we calculate the two directions (positive sequence and the reverse ) that Input concentration changes to avoid wrongly supposing it as one IOA function circuit.<br />
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<br />
[[https://2010.igem.org/Team:Peking/Modeling TOP]]<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Modeling/CalculationProcessTeam:Peking/Modeling/CalculationProcess2010-10-27T13:04:21Z<p>Lgdeer: /* Equations Set Up */</p>
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<div>{{:Team:Peking/Headermodel}}<br />
{{:Team:Peking/boxes}}<br />
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Calculating Process</font></font></font><br />
<br><br><br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html><br />
[[Team:Peking/Modeling|Modeling]] > [[Team:Peking/Modeling/CalculationProcess|Calculation Process]] <html><br />
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<div id="middlewhite"><br />
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<br />
=Network Enumeration=<br />
<html><br />
<p>We use three nodes as a minimal framework: one node that receives input( A in Figure 2 ), a second node that transmits output( C in Figure 2 ), and a third node that can play diverse regulatory roles( B in Figure 2 ). There are 9 direct links among the three nodes and there are altogether 3^9=19,683 three-node topologies. With 3,645 topologies that have no direct or indirect links from the input to the output occluded, there remain a total of 16,038 possible three-node topologies that contain at least one direct or indirect causal link from the input node to the output node. For each topology, we sampled 10,000 sets of network parameters with the method of latin hypercube sampling (LHS, Figure 3). In all, we have analyzed a total of 16,038*10,000 different circuits. This search resulted in an exhaustive circuit function map used to extract core topological motifs essential for IOA.</html><br />
[[Image:Network.png|245px|center]]<br><br />
<html><br />
<b><font size=1.5>Figure 2 Three-node network with all of its possible directed links(Ref. 7) </b><br />
There are altogether 9 possible links and the input here is Hg(Ⅱ), and the concentration of C is taken as output.<br></html><br />
[[Image:LHS.png|315px|center]]<br><br />
<html><font size=1.5><br />
<b>Figure 3 Latin Hypercube Sampling</b></font><br />
When sampling a function of N variables, the range of each variable is divided into M equally probable intervals, M sample points are then placed to satisfy the Latin Hypercube requirements. Then each sample is the Only one in each axis-aligned hyperplane containing it.</font><br />
</p><br />
</html><br />
[[https://2010.igem.org/Team:Peking/Modeling TOP]]<br />
<br />
=Equations Set Up=<br />
<html><br />
Our model is based on mainly the following statements:<br><br />
<br>(1) The nodes are restricted to TF nodes so that the links stand for TF-TF interactions via DNA. The expression level is quantified by the equilibrium binding probability P of TF binding on its site and the maximum expression rate constant <img src="https://static.igem.org/mediawiki/2010/4/49/Beita.png" width="16">, and we adopt a constant <img src="https://static.igem.org/mediawiki/2010/a/aa/Lamda.png" width="16"> to modify P to make different TFs equal status. When it comes to several TF factors, we use the multiplication of their <img src="https://static.igem.org/mediawiki/2010/7/70/LamdaP.png" width="25"> or <img src="https://static.igem.org/mediawiki/2010/a/ac/1-lamdaP.png" width="45"> to indicate their interactions.<br><br />
<br>(2) We take into consideration only the transcription and translation process because other reactions such as signal-transduction activities typically operate much faster and can be considered to be approximately at steady state on the slow timescales of transcription networks. Also the TF activity levels can be considered to be at steady state within the equations that describe network dynamics on the slow timescale of changes in protein levels. So that the equations contain only the accumulation and degradation of the protein products ( here the TFs). <br><br />
<br>(3) It has been observed that one ordinary gene usually has a nonzero expression level with no TFs on its binding site. We propose that one repressor will lower the initial expression level and one activator will shift it, further on, each TF has its unique contribution to the final expression level,that is, they shift or lower the expression level to different extents according to their own properties.<br><br />
<br>&nbsp;&nbsp;Consider first the simplest condition under which there is only one link from a node to another. (A<img src="https://static.igem.org/mediawiki/2010/8/88/Jiantou.png" width="13">C, Figure 4)<br><br />
It is widely accepted that the possibility of TF binding to the binding site in promoter is<br><img src="https://static.igem.org/mediawiki/2010/0/02/Function1.png" width="120">(X*: the effective concentration of one TF; Kd : the dissociation constant )<br><br><br />
&nbsp;&nbsp;According to hypothesis (1)&(3), the link from A(node1) to C(node3) can be translated as<br><br><br />
<img src="https://static.igem.org/mediawiki/2010/c/cc/Function2.png" width="670"><br><br><br />
( <img src="https://static.igem.org/mediawiki/2010/4/4b/Beita0.png" width="15">:the basal translation rate factor;<img src="https://static.igem.org/mediawiki/2010/d/d4/Beitam.png" width="15">:the effective translation rate factor.)<br />
<br><br />
<br><br />
&nbsp;&nbsp;As <img src="https://static.igem.org/mediawiki/2010/1/15/Function3.png" width="177">(Figure 5(a))<br />
<br><br><br />
&nbsp;&nbsp;Thus <img src="https://static.igem.org/mediawiki/2010/0/00/Function4.png" width="386">(Figure 5(b))(The subscript of X is the node number.) <br />
<br><br>&nbsp;&nbsp;Actually, the first component of the equation is the fundamental expression level of the network in which the <img src="https://static.igem.org/mediawiki/2010/a/ac/1-lamdaP.png" width="45"> is the possibility that the TF is off the DNA target site, and the second component of the equation is the effective expression level in which <img src="https://static.igem.org/mediawiki/2010/7/70/LamdaP.png" width="25"> is the possibility that TF is on its site.<br />
<br><br><br />
&nbsp;&nbsp;As to general conditions, there are<br><br />
<img src="https://static.igem.org/mediawiki/2010/c/cc/Function5.png" width="554"> <br><br><br />
&nbsp;&nbsp;And in order to make the equations fit hypothesis 3,we deduced the proper range of <img src="https://static.igem.org/mediawiki/2010/a/aa/Lamda.png" width="14">, that is, no regulation <img src="https://static.igem.org/mediawiki/2010/d/da/Function6.png" width="33"> , activation <img src="https://static.igem.org/mediawiki/2010/7/78/Function7.png" width="53"> and repression <img src="https://static.igem.org/mediawiki/2010/2/22/Function8.png" width="132">.The choice of other Parameter values and their reference is in Table 1.<br />
</html><br />
<br><br />
[[Image:Simple_network.png|center|50px]]<br />
<html><br />
<font size=1.5><br><b>Figure 4 simplest network</b> There is only one link from node A to node C and we don’t regulate the property of the link. Actually, whether it is activation, repression or no regulation is represented by λ.<br />
<br></font><br />
</html><br />
[[Image:MerR+Hg+DNA.png|650px|center]]<br />
<br><br />
<html><br />
<font size=1.5><br><b>Figure 5 MerR interacts with Hg(Ⅱ)& MerR-Hg binding to DNA</b> (a)The orange poor arc sector stands for the MerR monomer and the orange major arc sector stands for the MerR dimer. The blue ball is Hg ion that can be bound by the MerR dimer, and only the complex can serve as Transcription Factor that promotes the expression of downstream genes. We list the balance equations, derive the calculation function of X* as above, and substitute this function to the ODE equations used in our model. (b) As the MerR dimer is only slightly repress DNA transcription, we regrad Kd' << K1*Kd, so neglecting the competitive binding of MerR dimer to DNA.<br />
<br></font><br />
</html><br />
<html><br />
<font size=1.5><br><b>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Table 1 Physiological range of the model parameters</b></font><br />
<br><br />
</html><br />
[[Image:Table_1.png|500px|center]]<br />
<br><br />
<br />
[[https://2010.igem.org/Team:Peking/Modeling TOP]]<br />
<br />
=Network Topologies’ Analysis=<br />
<html><br />
Aiming at getting the values of r for each circuit, we need to numerically simulate the ODE equations to get the steady-state concentration of output node C under each input concentration, and then making linear fit of input and output concentrations. As our input concentration range is 10-9~10-5, we select points that have the same logarithmic distance intervals, then simulate output evolution curve to get the steady concentration one point by one. We choose the fourth-order Runge-Kutta method to solve the ODE equations and so as to save calculation time, we adopt Implicit Runge-Kutta algorithm to get the output steady concentration when Input=10-9M and set the very concentration as initial value for the Newton-Raphson method for following different Input concentration. And considering the possibility of bistable network topology, we calculate the two directions (positive sequence and the reverse ) that Input concentration changes to avoid wrongly supposing it as one IOA function circuit.<br />
</html><br />
<br />
[[https://2010.igem.org/Team:Peking/Modeling TOP]]<br />
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</div></div>Lgdeerhttp://2010.igem.org/Team:Peking/Notebook/ZRLiuTeam:Peking/Notebook/ZRLiu2010-10-27T10:35:15Z<p>Lgdeer: /* October */</p>
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__NOTOC__<br />
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Zairan Liu's Notes</font></font></font><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<a href="https://2010.igem.org/Team:Peking/Team/ZRLiu"><img src="https://static.igem.org/mediawiki/2010/a/af/Rr.jpg" width="40px" alt="goto her page"id="imggreen"> </a><br />
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I am responsible for the construction of metal binding peptide periplasm display module for Pb(II). This module aims at binding Pb(II) ions in the periplasmic space using the engineered anti-parallel coiled coil which is transported with the help of DsbA signal sequence. During the process several other intermediate plasmids are also constructed. Furthermore, I contribute to the characterization of Pc promoters of different intensity.<br />
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=='''Contents'''==<br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#July| July, 2010]]</span><br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#August| August, 2010]]</span><br />
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#September| September, 2010]]</span><br />
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#October| October, 2010]]</span><br />
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<br />
==July==<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
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|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.5|5]]<br />
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|- <br />
|style="text-align:center"| 25<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.26|26]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.27|27]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.30|30]]<br />
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[<html><a href="#top">TOP</a></html>]<br />
===7.5===<br />
Purification of digested PCR product: merP, merT, and merC<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (merP / merT / merC digested with EcoRI and PstI): 7μL<br />
<br />
Vector (pSB1A2 backbone digested with EcoRI and PstI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
===7.6===<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
Digestion (20μL):<br />
<br />
pSB1A2: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
PCR to get merP, merT and merC fragments by Phusion (20μL)<br />
<br />
5*phusionHF buffer: 4μL<br />
<br />
2.5mM dNTPs: 1.6μL<br />
<br />
Polymerase: 0.2μL<br />
<br />
Primer_For:1μL<br />
<br />
Primer_Rev: 1μL<br />
<br />
Template: 0.5μL<br />
<br />
ddH2O: 11.7μL<br />
<br />
<br />
===7.7===<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
merT: 351bp<br />
<br />
merP: 256bp <br />
<br />
merC: 423bp<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (merP / merT / merC digested with EcoRI and PstI): 7μL<br />
<br />
Vector (pSB1A2 backbone digested with EcoRI and PstI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
===7.8===<br />
Pick up 3 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
[7.9-7.19 Field Practices @ Yantai & Beijing (^_<)]<br />
[7.20-7.21 Home @ Nanjing (#_#)]<br />
[7.22-7.24 World Exhibit @ Shanghai (*~*)Orz]<br />
[7.25 Home @ Nanjing (#_#)]<br />
<br />
===7.26===<br />
<br />
Design the PbrR Metal Binding Peptide (MBP) Construction<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by PFUEasyMix (20μL)<br />
<br />
EasyMix: 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Primer_For_N: 0.5μL<br />
<br />
Primer_Rev_N:0.5μL<br />
<br />
Template (pbrR): 0.5μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for MBP_STD<br />
<br />
EasyMix: 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Primer_For_C: 0.5μL<br />
<br />
Primer_Rev_C:0.5μL<br />
<br />
Template (pbrR): 0.5μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for MBP_STD<br />
<br />
Linker are designed into Primer_Rev_N and Primer_For_C<br />
<br />
<br />
===7.27===<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
N C<br />
<br />
Digestion:<br />
<br />
N terminal: EcoRI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+PstI+NEBuffer3<br />
<br />
Purification of digestion product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with EcoRI and BspEI): 3μL <br />
<br />
Insert2 (C terminal digested with BspEI and PstI): 3μL<br />
<br />
Vector (pSB1K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
<br />
===7.28===<br />
<br />
Transformation: OmniMAX<br />
<br />
Pick up 3 colonies from the agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
===7.29===<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR_MBP_STD (pSB1K3)<br />
<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by EasyPFU (50μL)<br />
<br />
10*EasyPFU buffer: 5μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_For_N’:1μL<br />
<br />
Primer_Rev_N’: 1μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
ddH2O: 36.8μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for MBP_COM<br />
<br />
10*EasyPFU buffer: 5μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_For_C’:1μL<br />
<br />
Primer_Rev_C’: 1μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
ddH2O: 36.8μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for MBP_COM<br />
<br />
Linker are designed into Primer_Rev_N’ and Primer_For_C’<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Digestion: <br />
<br />
N terminal: NdeI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+XhoI+NEBuffer3<br />
<br />
<br />
===7.30===<br />
Purification of digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with NdeI and BspEI): 3μL<br />
<br />
Insert2 (C terminal digested with BspEI and XhoI): 3μL<br />
<br />
Vector (pET21a backbone digested with NdeI and XhoI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===7.31===<br />
Pick up 3 colonies from the agar plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR_MBP_COM (pET21a)<br />
<br />
==August==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
|style="text-align:center"| Sat<br />
|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| 1<br />
|style="text-align:center"| 2<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.3|3]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.4|4]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.5|5]]<br />
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|style="text-align:center"| 7<br />
|- <br />
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|style="text-align:center"| 9<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.10|10]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.11|11]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.12|12]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.13|13]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.14|14]]<br />
|- <br />
|style="text-align:center"| 15<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.16|16]]<br />
|style="text-align:center"| 17<br />
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|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|}<br />
[<html><a href="#top">TOP</a></html>]<br />
===8.3===<br />
Design the DsbA-MBP Construction<br />
<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For_N: 2μL<br />
<br />
Primer_Rev_N:2μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for DsbA-MBP<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For_C: 2μL<br />
<br />
Primer_Rev_C:2μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for DsbA-MBP<br />
<br />
Linker are designed into Primer_Rev_N and Primer_For_C<br />
<br />
Electrophoresis to verify<br />
<br />
Purification of PCR product<br />
<br />
Digestion: <br />
<br />
N terminal: XbaI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+XhoI+NEBuffer3<br />
<br />
===8.4===<br />
Purification of digested product<br />
<br />
Digestion (20μL):<br />
<br />
pET39b: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
XhoI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with XbaI and BspEI): 3μL<br />
<br />
Insert2 (C terminal digested with BspEI and XhoI): 3μL<br />
<br />
Vector (pET39b backbone digested with SpeI and XhoI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===8.5===<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
NdeI: 1μL<br />
<br />
XhoI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
NdeI(1030) XhoI(174) MBP(abt 500bp)<br />
<br />
Correct band=1030-174+500=1356<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: DsbA-MBP (pET39b)<br />
<br />
<br />
===8.10===<br />
Design and the T7-RBS-DsbA-MBP Construction<br />
<br />
<br />
1st Round of Nest PCR by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For (T7-RBS-DsbA-F1): 2μL<br />
<br />
Primer_Rev (PbrR-MBP-C’-R):2μL<br />
<br />
Template (DsbA-MBP): 0.2μL<br />
<br />
=>RBS is prefixed to DsbA-MBP<br />
<br />
Electrophoresis<br />
<br />
<br />
<br />
FAILED!!!(TAT)<br />
<br />
<br />
REPEAT-PCR with Gradient<br />
<br />
Gradient: T=60℃ G=5<br />
<br />
Electrophoresis<br />
<br />
<br />
Gel extraction<br />
<br />
===8.11===<br />
2nd Round of Nest PCR by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For (T7-RBS-DsbA-F2): 2μL<br />
<br />
Primer_Rev (PbrR-MBP-C’-R):2μL<br />
<br />
Template (1st Round of Nest PCR product (RBS-DsbA-MBP)): 0.2μL<br />
<br />
=>T7 is prefixed to 1st Round of Nest PCR product (RBS-DsbA-MBP)<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
<br />
===8.12===<br />
Digestion (20μL):<br />
<br />
2nd Round of Nest PCR product (T7-RBS-DsbA-MBP): 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
SpeI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of digested product<br />
<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
SpeI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (T7-RBS-DsbA-MBP digested with EcoRI and SpeI): 6μL<br />
<br />
Vector (pSB1K3 backbone digested with EcoRI and SpeI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===8.13===<br />
Pick up 5 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
===8.14===<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be two bands; one is about 3000bp and the other is 600bp<br />
<br />
Verification: PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be one band, which is about 1200bp<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: DsbA-MBP (pET39b)<br />
<br />
<br />
===8.16===<br />
FAILED (T~T)<br />
<br />
Why?<br />
<br />
Because the reverse primer cannot specifically distinguish the C terminal from the N terminal of the MBP, new primer <br />
including the sequence of His-tag is designed in order to recognize the C terminal only<br />
<br />
<br />
Hand over this work to Donghai Liang<br />
<br />
[8.17-8.31] Physical Training<br />
<br />
==September==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
|style="text-align:center"| Sat<br />
|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.1|1]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.2|2]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.3|3]]<br />
|style="text-align:center"| 4<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.5|5]] <br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.6|6]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.7|7]]<br />
|style="text-align:center"|8<br />
|style="text-align:center"| 9<br />
|style="text-align:center"| 10<br />
|style="text-align:center"| 11<br />
|style="text-align:center"| 12<br />
|- <br />
|style="text-align:center"| 13<br />
|style="text-align:center"|14<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.15|15]]<br />
|style="text-align:center"| 16<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.17|17]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.18|18]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.19|19]]<br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.20|20]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.21|21]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.22|22]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.23|23]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.24|24]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.25|25]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.26|26]]<br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.27|27]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.28|28]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.30|30]]<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|}<br />
[<html><a href="#top">TOP</a></html>]<br />
===9.1===<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Digestion (20μL):<br />
<br />
pbrR_MBP_STD (pSB1A2): 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (PbrR_MBP_STD digested with EcoRI and SpeI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and SpeI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
<br />
===9.2===<br />
<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be one band, which is about 500bp<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR-MBP (pSB1C3)<br />
<br />
<br />
<br />
===9.3===<br />
<br />
Construct the concentration of Hg(II) ions for induction<br />
<br />
Final Concentration/M Volume/μL Original Concentration/M<br />
<br />
1. 5*10-5 25 10-3<br />
<br />
2. 3*10-5 15 10-3<br />
<br />
3. 10-5 5 10-3<br />
<br />
4. 8*10-6 4 10-3<br />
<br />
5. 5*10-6 2.5 10-3<br />
<br />
6. 3*10-6 1.5 10-3<br />
<br />
7. 10-6 0.5 10-3<br />
<br />
8. 8*10-7 4 10-4<br />
<br />
9. 5*10-7 2.5 10-4<br />
<br />
10. 3*10-7 1.5 10-4<br />
<br />
11. 10-7 0.5 10-4<br />
<br />
12. 8*10-8 4 10-5<br />
<br />
13. 5*10-8 2.5 10-5<br />
<br />
14. 3*10-8 1.5 10-5<br />
<br />
15. 10-8 0.5 10-5<br />
<br />
16. 8*10-9 0.4 10-5<br />
<br />
17. 5*10-9 25 10-7<br />
<br />
18. 3*10-9 15 10-7<br />
<br />
19. 10-9 5 10-7<br />
<br />
20. 0 0 0<br />
<br />
<br />
<br />
===9.5===<br />
<br />
Learn the protocol for preparation of competent cells for transformation for bi-transformation<br />
<br />
Pick up one colony from each agar plate: J23109 and J23112<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===9.6===<br />
<br />
Re-activate the culture of J23109 and J23112 in fresh LB with ampicilin and kanamyclin<br />
<br />
Measure the value of OD600<br />
<br />
Induced with Hg(II) ions of different concentration for 2hrs<br />
<br />
Centrifugation at 5000r for 5min<br />
<br />
Resuspension with PBS<br />
<br />
Measure the value of OD600 and GFP with ELISA<br />
<br />
<br />
<br />
===9.7===<br />
<br />
Learn about Western Blot with Boxuan Zhao<br />
<br />
<br />
<br />
===9.15===<br />
<br />
Design the traffic light assay<br />
<br />
LacZ full length (RBS: B0034) BBa_I1732017 2_3K 3093bp pSB1A2<br />
<br />
LacZ α fragment BBa_I732006 1_23H 234bp pSB1AK3<br />
<br />
Plasmid1: 1_18I MerR (pSB3K3)<br />
<br />
Plasmid2: PmerT - LacZ full length / LacZ α fragment (pSB1C3)<br />
<br />
X-GAL assay<br />
<br />
<br />
<br />
Positive Transformation of 2_3K and 1_23H: TansMAX<br />
<br />
<br />
<br />
===9.17===<br />
<br />
Pick up 2 colonies from each agar plate: 2_3K and 1_23H<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Get the plasmids: 2_3K (LacZ full length_ pSB1A2)<br />
<br />
1_23H (LacZ α fragment_ pSB1AK3)<br />
<br />
<br />
<br />
===9.18===<br />
<br />
Digestion (20μL):<br />
<br />
LacZ full length: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
LacZα fragment: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
<br />
FAILED(T_T)<br />
<br />
Wrong enzyme used<br />
<br />
<br />
<br />
===9.19===<br />
<br />
Digestion (20μL):<br />
<br />
LacZ full length: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
LacZ α fragment: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
The band for LacZ full length is clear but not for LacZ α fragment<br />
<br />
Change method<br />
<br />
PCR from 1_23H by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for LacZ α fragment: 250bp<br />
<br />
<br />
<br />
===9.20===<br />
<br />
Digestion (20μL):<br />
<br />
RBS_pSB1A2: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer2): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Get the RBS_pSB1A2 backbone digested with SpeI and PstI<br />
<br />
Digestion (20μL):<br />
<br />
PmerT_pSB1C3: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer2): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Get the PmerT_pSB1C3 backbone digested with SpeI and PstI<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (RBS_pSB1A2 backbone digested with SpeI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (PmerT_pSB1C3 backbone digested with SpeI and PstI): 2μL<br />
<br />
Transformation of RBS-LacZ α fragment (pSB1A2)<br />
<br />
<br />
<br />
===9.21===<br />
<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
FAILED (>.<||)<br />
<br />
Forgot to digest the PCR product<br />
<br />
<br />
<br />
Digestion (20μL):<br />
<br />
1_23H PCR product: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of the digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (RBS_pSB1A2 backbone digested with SpeI and PstI): 2μL<br />
<br />
Transformation of RBS-LacZ α fragment (pSB1A2)<br />
<br />
Transformation of PmerT-LacZ full length (pSB1C3)<br />
<br />
<br />
<br />
===9.22===<br />
<br />
Pick up 6 colonies from each agar plate of RBS-LacZ α fragment (pSB1A2) and PmerT-LacZ full length (pSB1C3)<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-prep of PmerT-LacZ full length (pSB1C3)<br />
<br />
Digestion (20μL):<br />
<br />
PmerT-LacZ full length (pSB1C3): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 6 are of correct size<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: PmerT-LacZ full length (pSB1C3)<br />
<br />
<br />
<br />
Mini-prep of RBS-LacZ α fragment (pSB1A2)<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment (pSB1A2): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 4, 5, 6 are all of correct size<br />
<br />
<br />
<br />
===9.23===<br />
<br />
PCR to get RBS-LacZ α fragment by FastPFU (50μL)<br />
<br />
5*phusionHF buffer: 10μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.2μL<br />
<br />
ddH2O: 32μL<br />
<br />
Electrophoresis <br />
<br />
Excise the band of 250bp<br />
<br />
Gel Extraction<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (PmerT_pSB1C3 backbone digested with SpeI and PstI): 2μL<br />
<br />
<br />
<br />
===9.24===<br />
<br />
Transformation: Trans5α<br />
<br />
Pick up 6 colonies from the agar plate: PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
Grow the culture for overnight<br />
<br />
<br />
<br />
===9.25===<br />
<br />
Mini-prep of PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
Digestion (20μL):<br />
<br />
PmerT- RBS-LacZ α fragment (pSB1C3): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 4, 5, 6 are all of correct size<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
<br />
<br />
===9.26===<br />
<br />
Construct the mutant promoter P88 and P3 into the traffic light system<br />
<br />
<br />
<br />
===9.27===<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 1.5μL<br />
<br />
Primer_Rev_P88:1.5μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 4μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 1μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 1.5μL<br />
<br />
Primer_Rev_P3:1.5μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 4μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 1μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 3μL<br />
<br />
Primer_Rev_P88: 3μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 2μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 3μL<br />
<br />
Primer_Rev_P3: 3μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 2μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===9.28===<br />
<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
REPEAT<br />
<br />
<br />
<br />
===9.29===<br />
<br />
Still no recognizable colonies grew on the agar plates (TAT)<br />
<br />
Change the ratio of different components<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 15μL<br />
<br />
Primer_Rev_P88:15μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 3.5μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 1.5μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 10μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 15μL<br />
<br />
Primer_Rev_P3:15μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 3.5μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 1.5μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 10μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 7μL<br />
<br />
Primer_Rev_P88: 7μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1.7μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 3.3μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 7μL<br />
<br />
Primer_Rev_P3: 7μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1.7μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 3.3μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===9.30===<br />
<br />
Still no recognizable colonies grew on the agar plates (TT_TT)<br />
<br />
Hand over this work to Ying Sheng<br />
<br />
==October==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
|style="text-align:center"| Sat<br />
|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"|-<br />
|style="text-align:center"|-<br />
|style="text-align:center"|1<br />
|style="text-align:center"|2<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.3|3]] <br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.4|4]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.5|5]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.6|6]]<br />
|style="text-align:center"|7<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.8|8]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.9|9]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.10|10]]<br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.11|11]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.12|12]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.13|13]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.14|14]]<br />
|style="text-align:center"| 15<br />
|style="text-align:center"|16<br />
|style="text-align:center"| 17<br />
|- <br />
|style="text-align:center"| 18<br />
|style="text-align:center"| 19<br />
|style="text-align:center"|20<br />
|style="text-align:center"|21<br />
|style="text-align:center"|22<br />
|style="text-align:center"|23<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.24|24]]<br />
|- <br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.25|25]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.26|26]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.27|27]]<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|}<br />
[<html><a href="#top">TOP</a></html>]<br />
===10.3===<br />
<br />
Prepare PARTS<br />
<br />
<br />
===10.4===<br />
<br />
Change backbone<br />
<br />
(1) T7-RBS-DsbA-MBP_pSB1K3=>T7-RBS-DsbA-MBP_pSB1C3<br />
<br />
(2) RBS-LacZ α fragment _pSB1A2=> RBS-LacZ α fragment _pSB1C3 <br />
<br />
<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
T7-RBS-DsbA-MBP_pSB1K3: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment _pSB1A2: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (T7-RBS-DsbA-MBP digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Mach-T1<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Mach-T1<br />
<br />
<br />
<br />
===10.5===<br />
<br />
Pick up 3 colonies from each agar plates<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: T7-RBS-DsbA-MBP (pSB1C3) and RBS-LacZ α fragment (pSB1C3)<br />
<br />
SUBMIT the plasmids<br />
<br />
Positive Transformation of T7-RBS-DsbA-MBP (pSB1C3) and RBS-LacZ α fragment (pSB1C3)<br />
<br />
<br />
<br />
===10.6===<br />
<br />
Pick up 1 colonies from each agar plates<br />
<br />
Grow the culture for 12hrs<br />
<br />
SUBMIT the culture preserved with glycerol<br />
<br />
<br />
<br />
Brief Characterization of PmerT-LacZ full length and PmerT-RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M<br />
<br />
(pictures see in PDF)<br />
<br />
<br />
<br />
===10.8===<br />
<br />
DNA sequencing shows that P88 / P3-LacZ α fragment (pSB1C3) are correct<br />
<br />
Positive Transformation<br />
<br />
Pick up one colony from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.9===<br />
<br />
Digestion (20μL):<br />
<br />
J23114_pSB3K3: 10μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
PCR from P88-LacZ α fragment_pSB1C3 by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for P88-RBS-LacZ α fragment: 350bp<br />
<br />
PCR from P3-LacZ α fragment_pSB1C3 by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for P3-RBS-LacZ α fragment: 350bp<br />
<br />
Digestion (20μL):<br />
<br />
P88-RBS-LacZ α fragment PCR product: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
P3-RBS-LacZ α fragment PCR product: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of the digested PCR product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P88-RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P3-RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===10.10===<br />
<br />
Pick up 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
Pick up one colony from plates: 1_18E and J23117<br />
<br />
Grow the culture for 12hrs<br />
<br />
<br />
<br />
<br />
<br />
===10.11===<br />
<br />
Fail in cultivating overnight culture<br />
<br />
PHAGE appeared??? (>…<)<br />
<br />
Pick up another 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
===10.12===<br />
<br />
Fail in cultivating overnight culture again (T_T)<br />
<br />
Pick up another 5 colonies from each plate<br />
<br />
Grow the culture in fresh LB with 1/10 kanamyclin for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
===10.13===<br />
<br />
Mini-prep<br />
<br />
Get the plasmids: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
P3-RBS-LacZα fragment_pSB3K3<br />
<br />
Re-activate the culture: 1_18E and J23117<br />
<br />
Make them competent cells for bi-transformation<br />
<br />
Transformation:<br />
<br />
Competent cell 1_18E: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
Competent cell J23117: P3-RBS-LacZα fragment_pSB3K3<br />
<br />
<br />
<br />
===10.14===<br />
<br />
Pick up 3 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.15===<br />
<br />
Brief Characterization of P88-RBS-LacZ α fragment and P3- RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M<br />
<br />
<br />
<br />
NO COLOR CHANGE (! _ !) <br />
<br />
<br />
<br />
[10.16-10.23] Prepare for GRE test<br />
<br />
<br />
<br />
===10.24===<br />
<br />
Pick up 6 colonies from each agar plate: J23109_pSB3K3<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.25===<br />
<br />
Mini-prep<br />
<br />
Get the plasmid: J23109_pSB3K3<br />
<br />
Digestion (20μL):<br />
<br />
J23109_pSB3K3: 10μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P88-RBS-LacZα fragment_pSB3K3): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P3-RBS-LacZα fragment_pSB3K3): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: TransT1-phage [14:00-0:00]<br />
<br />
Pick up one colony from plates: 1_18E and J23117 <br />
<br />
Grow the culture for 12hrs [19:30-7:30]<br />
<br />
<br />
<br />
===10.26===<br />
<br />
Pick up 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs [0:30-12:30]<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
Re-activate the culture of 1_18E and J23117 [7:30-11:30]<br />
<br />
Make them competent cells for bi-transformation [12:00-13:00]<br />
<br />
<br />
<br />
Mini-prep [12:30-13:30] Helped by Haoqian Zhang<br />
<br />
Get the plasmids P88-RBS-LacZα fragment_pSB3K3<br />
<br />
P3-RBS-LacZα fragment_pSB3K3<br />
<br />
<br />
<br />
Transformation: TransT1-phage [13:30-15:00]<br />
<br />
Competent cell 1_18E: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
Competent cell J23117: P3-RBS-LacZα fragment_pSB3K3<br />
<br />
[15:00-1:00]<br />
<br />
===10.27===<br />
<br />
Pick up 3 colonies on each agar plate<br />
<br />
Grow the culture for 12hrs [1:30-13:30]<br />
<br />
Re-activate the culture [13:30-15:30] <br />
<br />
<br />
<br />
Brief Characterization of PmerT-LacZ full length and PmerT-RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M [16:00-10:00]<br />
<br />
<br />
<br />
<br />
<br />
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<a href="https://2010.igem.org/Team:Peking/Team/ZRLiu"><font color=#FFFFFF>==go to her page==</font></a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
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</html></div>Lgdeerhttp://2010.igem.org/Team:Peking/Notebook/ZRLiuTeam:Peking/Notebook/ZRLiu2010-10-27T10:34:43Z<p>Lgdeer: /* October */</p>
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Zairan Liu's Notes</font></font></font><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<a href="https://2010.igem.org/Team:Peking/Team/ZRLiu"><img src="https://static.igem.org/mediawiki/2010/a/af/Rr.jpg" width="40px" alt="goto her page"id="imggreen"> </a><br />
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I am responsible for the construction of metal binding peptide periplasm display module for Pb(II). This module aims at binding Pb(II) ions in the periplasmic space using the engineered anti-parallel coiled coil which is transported with the help of DsbA signal sequence. During the process several other intermediate plasmids are also constructed. Furthermore, I contribute to the characterization of Pc promoters of different intensity.<br />
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=='''Contents'''==<br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#July| July, 2010]]</span><br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#August| August, 2010]]</span><br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#September| September, 2010]]</span><br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#October| October, 2010]]</span><br />
<br />
<br />
==July==<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
|style="text-align:center"| Sat<br />
|style="text-align:center"| Sun<br />
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|style="text-align:center"| 2<br />
|style="text-align:center"| 3<br />
|- <br />
|style="text-align:center"| 4<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.5|5]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.6|6]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.7|7]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.8|8]]<br />
|style="text-align:center"| 9<br />
|style="text-align:center"| 10<br />
|- <br />
|style="text-align:center"| 11<br />
|style="text-align:center"| 12<br />
|style="text-align:center"| 13<br />
|style="text-align:center"| 14<br />
|style="text-align:center"| 15<br />
|style="text-align:center"| 16<br />
|style="text-align:center"| 17<br />
|- <br />
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|style="text-align:center"| 19<br />
|style="text-align:center"| 20<br />
|style="text-align:center"| 21<br />
|style="text-align:center"| 22<br />
|style="text-align:center"| 23<br />
|style="text-align:center"| 24<br />
|- <br />
|style="text-align:center"| 25<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.26|26]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.27|27]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.30|30]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.31|31]]<br />
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|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|}<br />
[<html><a href="#top">TOP</a></html>]<br />
===7.5===<br />
Purification of digested PCR product: merP, merT, and merC<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (merP / merT / merC digested with EcoRI and PstI): 7μL<br />
<br />
Vector (pSB1A2 backbone digested with EcoRI and PstI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
===7.6===<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
Digestion (20μL):<br />
<br />
pSB1A2: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
PCR to get merP, merT and merC fragments by Phusion (20μL)<br />
<br />
5*phusionHF buffer: 4μL<br />
<br />
2.5mM dNTPs: 1.6μL<br />
<br />
Polymerase: 0.2μL<br />
<br />
Primer_For:1μL<br />
<br />
Primer_Rev: 1μL<br />
<br />
Template: 0.5μL<br />
<br />
ddH2O: 11.7μL<br />
<br />
<br />
===7.7===<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
merT: 351bp<br />
<br />
merP: 256bp <br />
<br />
merC: 423bp<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (merP / merT / merC digested with EcoRI and PstI): 7μL<br />
<br />
Vector (pSB1A2 backbone digested with EcoRI and PstI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
===7.8===<br />
Pick up 3 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
[7.9-7.19 Field Practices @ Yantai & Beijing (^_<)]<br />
[7.20-7.21 Home @ Nanjing (#_#)]<br />
[7.22-7.24 World Exhibit @ Shanghai (*~*)Orz]<br />
[7.25 Home @ Nanjing (#_#)]<br />
<br />
===7.26===<br />
<br />
Design the PbrR Metal Binding Peptide (MBP) Construction<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by PFUEasyMix (20μL)<br />
<br />
EasyMix: 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Primer_For_N: 0.5μL<br />
<br />
Primer_Rev_N:0.5μL<br />
<br />
Template (pbrR): 0.5μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for MBP_STD<br />
<br />
EasyMix: 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Primer_For_C: 0.5μL<br />
<br />
Primer_Rev_C:0.5μL<br />
<br />
Template (pbrR): 0.5μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for MBP_STD<br />
<br />
Linker are designed into Primer_Rev_N and Primer_For_C<br />
<br />
<br />
===7.27===<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
N C<br />
<br />
Digestion:<br />
<br />
N terminal: EcoRI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+PstI+NEBuffer3<br />
<br />
Purification of digestion product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with EcoRI and BspEI): 3μL <br />
<br />
Insert2 (C terminal digested with BspEI and PstI): 3μL<br />
<br />
Vector (pSB1K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
<br />
===7.28===<br />
<br />
Transformation: OmniMAX<br />
<br />
Pick up 3 colonies from the agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
===7.29===<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR_MBP_STD (pSB1K3)<br />
<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by EasyPFU (50μL)<br />
<br />
10*EasyPFU buffer: 5μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_For_N’:1μL<br />
<br />
Primer_Rev_N’: 1μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
ddH2O: 36.8μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for MBP_COM<br />
<br />
10*EasyPFU buffer: 5μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_For_C’:1μL<br />
<br />
Primer_Rev_C’: 1μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
ddH2O: 36.8μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for MBP_COM<br />
<br />
Linker are designed into Primer_Rev_N’ and Primer_For_C’<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Digestion: <br />
<br />
N terminal: NdeI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+XhoI+NEBuffer3<br />
<br />
<br />
===7.30===<br />
Purification of digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with NdeI and BspEI): 3μL<br />
<br />
Insert2 (C terminal digested with BspEI and XhoI): 3μL<br />
<br />
Vector (pET21a backbone digested with NdeI and XhoI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===7.31===<br />
Pick up 3 colonies from the agar plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR_MBP_COM (pET21a)<br />
<br />
==August==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
|style="text-align:center"| Sat<br />
|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| 1<br />
|style="text-align:center"| 2<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.3|3]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.4|4]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.5|5]]<br />
|style="text-align:center"| 6<br />
|style="text-align:center"| 7<br />
|- <br />
|style="text-align:center"| 8<br />
|style="text-align:center"| 9<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.10|10]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.11|11]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.12|12]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.13|13]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.14|14]]<br />
|- <br />
|style="text-align:center"| 15<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.16|16]]<br />
|style="text-align:center"| 17<br />
|style="text-align:center"| 18<br />
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|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|}<br />
[<html><a href="#top">TOP</a></html>]<br />
===8.3===<br />
Design the DsbA-MBP Construction<br />
<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For_N: 2μL<br />
<br />
Primer_Rev_N:2μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for DsbA-MBP<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For_C: 2μL<br />
<br />
Primer_Rev_C:2μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for DsbA-MBP<br />
<br />
Linker are designed into Primer_Rev_N and Primer_For_C<br />
<br />
Electrophoresis to verify<br />
<br />
Purification of PCR product<br />
<br />
Digestion: <br />
<br />
N terminal: XbaI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+XhoI+NEBuffer3<br />
<br />
===8.4===<br />
Purification of digested product<br />
<br />
Digestion (20μL):<br />
<br />
pET39b: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
XhoI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with XbaI and BspEI): 3μL<br />
<br />
Insert2 (C terminal digested with BspEI and XhoI): 3μL<br />
<br />
Vector (pET39b backbone digested with SpeI and XhoI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===8.5===<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
NdeI: 1μL<br />
<br />
XhoI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
NdeI(1030) XhoI(174) MBP(abt 500bp)<br />
<br />
Correct band=1030-174+500=1356<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: DsbA-MBP (pET39b)<br />
<br />
<br />
===8.10===<br />
Design and the T7-RBS-DsbA-MBP Construction<br />
<br />
<br />
1st Round of Nest PCR by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For (T7-RBS-DsbA-F1): 2μL<br />
<br />
Primer_Rev (PbrR-MBP-C’-R):2μL<br />
<br />
Template (DsbA-MBP): 0.2μL<br />
<br />
=>RBS is prefixed to DsbA-MBP<br />
<br />
Electrophoresis<br />
<br />
<br />
<br />
FAILED!!!(TAT)<br />
<br />
<br />
REPEAT-PCR with Gradient<br />
<br />
Gradient: T=60℃ G=5<br />
<br />
Electrophoresis<br />
<br />
<br />
Gel extraction<br />
<br />
===8.11===<br />
2nd Round of Nest PCR by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For (T7-RBS-DsbA-F2): 2μL<br />
<br />
Primer_Rev (PbrR-MBP-C’-R):2μL<br />
<br />
Template (1st Round of Nest PCR product (RBS-DsbA-MBP)): 0.2μL<br />
<br />
=>T7 is prefixed to 1st Round of Nest PCR product (RBS-DsbA-MBP)<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
<br />
===8.12===<br />
Digestion (20μL):<br />
<br />
2nd Round of Nest PCR product (T7-RBS-DsbA-MBP): 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
SpeI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of digested product<br />
<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
SpeI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (T7-RBS-DsbA-MBP digested with EcoRI and SpeI): 6μL<br />
<br />
Vector (pSB1K3 backbone digested with EcoRI and SpeI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===8.13===<br />
Pick up 5 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
===8.14===<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be two bands; one is about 3000bp and the other is 600bp<br />
<br />
Verification: PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be one band, which is about 1200bp<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: DsbA-MBP (pET39b)<br />
<br />
<br />
===8.16===<br />
FAILED (T~T)<br />
<br />
Why?<br />
<br />
Because the reverse primer cannot specifically distinguish the C terminal from the N terminal of the MBP, new primer <br />
including the sequence of His-tag is designed in order to recognize the C terminal only<br />
<br />
<br />
Hand over this work to Donghai Liang<br />
<br />
[8.17-8.31] Physical Training<br />
<br />
==September==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
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|style="text-align:center"| Tue<br />
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|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.1|1]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.2|2]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.3|3]]<br />
|style="text-align:center"| 4<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.5|5]] <br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.6|6]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.7|7]]<br />
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|- <br />
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|style="text-align:center"|14<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.15|15]]<br />
|style="text-align:center"| 16<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.17|17]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.18|18]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.19|19]]<br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.20|20]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.21|21]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.22|22]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.23|23]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.24|24]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.25|25]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.26|26]]<br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.27|27]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.28|28]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.30|30]]<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|}<br />
[<html><a href="#top">TOP</a></html>]<br />
===9.1===<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Digestion (20μL):<br />
<br />
pbrR_MBP_STD (pSB1A2): 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (PbrR_MBP_STD digested with EcoRI and SpeI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and SpeI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
<br />
===9.2===<br />
<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be one band, which is about 500bp<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR-MBP (pSB1C3)<br />
<br />
<br />
<br />
===9.3===<br />
<br />
Construct the concentration of Hg(II) ions for induction<br />
<br />
Final Concentration/M Volume/μL Original Concentration/M<br />
<br />
1. 5*10-5 25 10-3<br />
<br />
2. 3*10-5 15 10-3<br />
<br />
3. 10-5 5 10-3<br />
<br />
4. 8*10-6 4 10-3<br />
<br />
5. 5*10-6 2.5 10-3<br />
<br />
6. 3*10-6 1.5 10-3<br />
<br />
7. 10-6 0.5 10-3<br />
<br />
8. 8*10-7 4 10-4<br />
<br />
9. 5*10-7 2.5 10-4<br />
<br />
10. 3*10-7 1.5 10-4<br />
<br />
11. 10-7 0.5 10-4<br />
<br />
12. 8*10-8 4 10-5<br />
<br />
13. 5*10-8 2.5 10-5<br />
<br />
14. 3*10-8 1.5 10-5<br />
<br />
15. 10-8 0.5 10-5<br />
<br />
16. 8*10-9 0.4 10-5<br />
<br />
17. 5*10-9 25 10-7<br />
<br />
18. 3*10-9 15 10-7<br />
<br />
19. 10-9 5 10-7<br />
<br />
20. 0 0 0<br />
<br />
<br />
<br />
===9.5===<br />
<br />
Learn the protocol for preparation of competent cells for transformation for bi-transformation<br />
<br />
Pick up one colony from each agar plate: J23109 and J23112<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===9.6===<br />
<br />
Re-activate the culture of J23109 and J23112 in fresh LB with ampicilin and kanamyclin<br />
<br />
Measure the value of OD600<br />
<br />
Induced with Hg(II) ions of different concentration for 2hrs<br />
<br />
Centrifugation at 5000r for 5min<br />
<br />
Resuspension with PBS<br />
<br />
Measure the value of OD600 and GFP with ELISA<br />
<br />
<br />
<br />
===9.7===<br />
<br />
Learn about Western Blot with Boxuan Zhao<br />
<br />
<br />
<br />
===9.15===<br />
<br />
Design the traffic light assay<br />
<br />
LacZ full length (RBS: B0034) BBa_I1732017 2_3K 3093bp pSB1A2<br />
<br />
LacZ α fragment BBa_I732006 1_23H 234bp pSB1AK3<br />
<br />
Plasmid1: 1_18I MerR (pSB3K3)<br />
<br />
Plasmid2: PmerT - LacZ full length / LacZ α fragment (pSB1C3)<br />
<br />
X-GAL assay<br />
<br />
<br />
<br />
Positive Transformation of 2_3K and 1_23H: TansMAX<br />
<br />
<br />
<br />
===9.17===<br />
<br />
Pick up 2 colonies from each agar plate: 2_3K and 1_23H<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Get the plasmids: 2_3K (LacZ full length_ pSB1A2)<br />
<br />
1_23H (LacZ α fragment_ pSB1AK3)<br />
<br />
<br />
<br />
===9.18===<br />
<br />
Digestion (20μL):<br />
<br />
LacZ full length: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
LacZα fragment: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
<br />
FAILED(T_T)<br />
<br />
Wrong enzyme used<br />
<br />
<br />
<br />
===9.19===<br />
<br />
Digestion (20μL):<br />
<br />
LacZ full length: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
LacZ α fragment: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
The band for LacZ full length is clear but not for LacZ α fragment<br />
<br />
Change method<br />
<br />
PCR from 1_23H by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for LacZ α fragment: 250bp<br />
<br />
<br />
<br />
===9.20===<br />
<br />
Digestion (20μL):<br />
<br />
RBS_pSB1A2: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer2): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Get the RBS_pSB1A2 backbone digested with SpeI and PstI<br />
<br />
Digestion (20μL):<br />
<br />
PmerT_pSB1C3: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer2): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Get the PmerT_pSB1C3 backbone digested with SpeI and PstI<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (RBS_pSB1A2 backbone digested with SpeI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (PmerT_pSB1C3 backbone digested with SpeI and PstI): 2μL<br />
<br />
Transformation of RBS-LacZ α fragment (pSB1A2)<br />
<br />
<br />
<br />
===9.21===<br />
<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
FAILED (>.<||)<br />
<br />
Forgot to digest the PCR product<br />
<br />
<br />
<br />
Digestion (20μL):<br />
<br />
1_23H PCR product: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of the digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (RBS_pSB1A2 backbone digested with SpeI and PstI): 2μL<br />
<br />
Transformation of RBS-LacZ α fragment (pSB1A2)<br />
<br />
Transformation of PmerT-LacZ full length (pSB1C3)<br />
<br />
<br />
<br />
===9.22===<br />
<br />
Pick up 6 colonies from each agar plate of RBS-LacZ α fragment (pSB1A2) and PmerT-LacZ full length (pSB1C3)<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-prep of PmerT-LacZ full length (pSB1C3)<br />
<br />
Digestion (20μL):<br />
<br />
PmerT-LacZ full length (pSB1C3): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 6 are of correct size<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: PmerT-LacZ full length (pSB1C3)<br />
<br />
<br />
<br />
Mini-prep of RBS-LacZ α fragment (pSB1A2)<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment (pSB1A2): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 4, 5, 6 are all of correct size<br />
<br />
<br />
<br />
===9.23===<br />
<br />
PCR to get RBS-LacZ α fragment by FastPFU (50μL)<br />
<br />
5*phusionHF buffer: 10μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.2μL<br />
<br />
ddH2O: 32μL<br />
<br />
Electrophoresis <br />
<br />
Excise the band of 250bp<br />
<br />
Gel Extraction<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (PmerT_pSB1C3 backbone digested with SpeI and PstI): 2μL<br />
<br />
<br />
<br />
===9.24===<br />
<br />
Transformation: Trans5α<br />
<br />
Pick up 6 colonies from the agar plate: PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
Grow the culture for overnight<br />
<br />
<br />
<br />
===9.25===<br />
<br />
Mini-prep of PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
Digestion (20μL):<br />
<br />
PmerT- RBS-LacZ α fragment (pSB1C3): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 4, 5, 6 are all of correct size<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
<br />
<br />
===9.26===<br />
<br />
Construct the mutant promoter P88 and P3 into the traffic light system<br />
<br />
<br />
<br />
===9.27===<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 1.5μL<br />
<br />
Primer_Rev_P88:1.5μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 4μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 1μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 1.5μL<br />
<br />
Primer_Rev_P3:1.5μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 4μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 1μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 3μL<br />
<br />
Primer_Rev_P88: 3μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 2μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 3μL<br />
<br />
Primer_Rev_P3: 3μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 2μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===9.28===<br />
<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
REPEAT<br />
<br />
<br />
<br />
===9.29===<br />
<br />
Still no recognizable colonies grew on the agar plates (TAT)<br />
<br />
Change the ratio of different components<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 15μL<br />
<br />
Primer_Rev_P88:15μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 3.5μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 1.5μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 10μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 15μL<br />
<br />
Primer_Rev_P3:15μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 3.5μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 1.5μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 10μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 7μL<br />
<br />
Primer_Rev_P88: 7μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1.7μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 3.3μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 7μL<br />
<br />
Primer_Rev_P3: 7μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1.7μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 3.3μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===9.30===<br />
<br />
Still no recognizable colonies grew on the agar plates (TT_TT)<br />
<br />
Hand over this work to Ying Sheng<br />
<br />
==October==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
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|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.13|13]]<br />
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|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.26|26]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.27|27]]<br />
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[[https://2010.igem.org/Team:Peking/Notebook/ZRLiu TOP]]<br />
===10.3===<br />
<br />
Prepare PARTS<br />
<br />
<br />
===10.4===<br />
<br />
Change backbone<br />
<br />
(1) T7-RBS-DsbA-MBP_pSB1K3=>T7-RBS-DsbA-MBP_pSB1C3<br />
<br />
(2) RBS-LacZ α fragment _pSB1A2=> RBS-LacZ α fragment _pSB1C3 <br />
<br />
<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
T7-RBS-DsbA-MBP_pSB1K3: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment _pSB1A2: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (T7-RBS-DsbA-MBP digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Mach-T1<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Mach-T1<br />
<br />
<br />
<br />
===10.5===<br />
<br />
Pick up 3 colonies from each agar plates<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: T7-RBS-DsbA-MBP (pSB1C3) and RBS-LacZ α fragment (pSB1C3)<br />
<br />
SUBMIT the plasmids<br />
<br />
Positive Transformation of T7-RBS-DsbA-MBP (pSB1C3) and RBS-LacZ α fragment (pSB1C3)<br />
<br />
<br />
<br />
===10.6===<br />
<br />
Pick up 1 colonies from each agar plates<br />
<br />
Grow the culture for 12hrs<br />
<br />
SUBMIT the culture preserved with glycerol<br />
<br />
<br />
<br />
Brief Characterization of PmerT-LacZ full length and PmerT-RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M<br />
<br />
(pictures see in PDF)<br />
<br />
<br />
<br />
===10.8===<br />
<br />
DNA sequencing shows that P88 / P3-LacZ α fragment (pSB1C3) are correct<br />
<br />
Positive Transformation<br />
<br />
Pick up one colony from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.9===<br />
<br />
Digestion (20μL):<br />
<br />
J23114_pSB3K3: 10μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
PCR from P88-LacZ α fragment_pSB1C3 by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for P88-RBS-LacZ α fragment: 350bp<br />
<br />
PCR from P3-LacZ α fragment_pSB1C3 by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for P3-RBS-LacZ α fragment: 350bp<br />
<br />
Digestion (20μL):<br />
<br />
P88-RBS-LacZ α fragment PCR product: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
P3-RBS-LacZ α fragment PCR product: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of the digested PCR product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P88-RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P3-RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===10.10===<br />
<br />
Pick up 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
Pick up one colony from plates: 1_18E and J23117<br />
<br />
Grow the culture for 12hrs<br />
<br />
<br />
<br />
<br />
<br />
===10.11===<br />
<br />
Fail in cultivating overnight culture<br />
<br />
PHAGE appeared??? (>…<)<br />
<br />
Pick up another 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
===10.12===<br />
<br />
Fail in cultivating overnight culture again (T_T)<br />
<br />
Pick up another 5 colonies from each plate<br />
<br />
Grow the culture in fresh LB with 1/10 kanamyclin for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
===10.13===<br />
<br />
Mini-prep<br />
<br />
Get the plasmids: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
P3-RBS-LacZα fragment_pSB3K3<br />
<br />
Re-activate the culture: 1_18E and J23117<br />
<br />
Make them competent cells for bi-transformation<br />
<br />
Transformation:<br />
<br />
Competent cell 1_18E: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
Competent cell J23117: P3-RBS-LacZα fragment_pSB3K3<br />
<br />
<br />
<br />
===10.14===<br />
<br />
Pick up 3 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.15===<br />
<br />
Brief Characterization of P88-RBS-LacZ α fragment and P3- RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M<br />
<br />
<br />
<br />
NO COLOR CHANGE (! _ !) <br />
<br />
<br />
<br />
[10.16-10.23] Prepare for GRE test<br />
<br />
<br />
<br />
===10.24===<br />
<br />
Pick up 6 colonies from each agar plate: J23109_pSB3K3<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.25===<br />
<br />
Mini-prep<br />
<br />
Get the plasmid: J23109_pSB3K3<br />
<br />
Digestion (20μL):<br />
<br />
J23109_pSB3K3: 10μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P88-RBS-LacZα fragment_pSB3K3): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P3-RBS-LacZα fragment_pSB3K3): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: TransT1-phage [14:00-0:00]<br />
<br />
Pick up one colony from plates: 1_18E and J23117 <br />
<br />
Grow the culture for 12hrs [19:30-7:30]<br />
<br />
<br />
<br />
===10.26===<br />
<br />
Pick up 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs [0:30-12:30]<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
Re-activate the culture of 1_18E and J23117 [7:30-11:30]<br />
<br />
Make them competent cells for bi-transformation [12:00-13:00]<br />
<br />
<br />
<br />
Mini-prep [12:30-13:30] Helped by Haoqian Zhang<br />
<br />
Get the plasmids P88-RBS-LacZα fragment_pSB3K3<br />
<br />
P3-RBS-LacZα fragment_pSB3K3<br />
<br />
<br />
<br />
Transformation: TransT1-phage [13:30-15:00]<br />
<br />
Competent cell 1_18E: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
Competent cell J23117: P3-RBS-LacZα fragment_pSB3K3<br />
<br />
[15:00-1:00]<br />
<br />
===10.27===<br />
<br />
Pick up 3 colonies on each agar plate<br />
<br />
Grow the culture for 12hrs [1:30-13:30]<br />
<br />
Re-activate the culture [13:30-15:30] <br />
<br />
<br />
<br />
Brief Characterization of PmerT-LacZ full length and PmerT-RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M [16:00-10:00]<br />
<br />
<br />
<br />
<br />
<br />
[<html><a href="#top">TOP</a></html>]<br />
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<a href="https://2010.igem.org/Team:Peking/Team/ZRLiu"><font color=#FFFFFF>==go to her page==</font></a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
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</html></div>Lgdeerhttp://2010.igem.org/Team:Peking/Notebook/ZRLiuTeam:Peking/Notebook/ZRLiu2010-10-27T10:31:50Z<p>Lgdeer: /* September */</p>
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__NOTOC__<br />
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<br><br><br />
<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Zairan Liu's Notes</font></font></font><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<a href="https://2010.igem.org/Team:Peking/Team/ZRLiu"><img src="https://static.igem.org/mediawiki/2010/a/af/Rr.jpg" width="40px" alt="goto her page"id="imggreen"> </a><br />
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I am responsible for the construction of metal binding peptide periplasm display module for Pb(II). This module aims at binding Pb(II) ions in the periplasmic space using the engineered anti-parallel coiled coil which is transported with the help of DsbA signal sequence. During the process several other intermediate plasmids are also constructed. Furthermore, I contribute to the characterization of Pc promoters of different intensity.<br />
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=='''Contents'''==<br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#July| July, 2010]]</span><br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#August| August, 2010]]</span><br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#September| September, 2010]]</span><br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#October| October, 2010]]</span><br />
<br />
<br />
==July==<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
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|style="text-align:center"| 2<br />
|style="text-align:center"| 3<br />
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|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.5|5]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.6|6]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.7|7]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.8|8]]<br />
|style="text-align:center"| 9<br />
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|style="text-align:center"| 23<br />
|style="text-align:center"| 24<br />
|- <br />
|style="text-align:center"| 25<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.26|26]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.27|27]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.30|30]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.31|31]]<br />
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[<html><a href="#top">TOP</a></html>]<br />
===7.5===<br />
Purification of digested PCR product: merP, merT, and merC<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (merP / merT / merC digested with EcoRI and PstI): 7μL<br />
<br />
Vector (pSB1A2 backbone digested with EcoRI and PstI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
===7.6===<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
Digestion (20μL):<br />
<br />
pSB1A2: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
PCR to get merP, merT and merC fragments by Phusion (20μL)<br />
<br />
5*phusionHF buffer: 4μL<br />
<br />
2.5mM dNTPs: 1.6μL<br />
<br />
Polymerase: 0.2μL<br />
<br />
Primer_For:1μL<br />
<br />
Primer_Rev: 1μL<br />
<br />
Template: 0.5μL<br />
<br />
ddH2O: 11.7μL<br />
<br />
<br />
===7.7===<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
merT: 351bp<br />
<br />
merP: 256bp <br />
<br />
merC: 423bp<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (merP / merT / merC digested with EcoRI and PstI): 7μL<br />
<br />
Vector (pSB1A2 backbone digested with EcoRI and PstI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
===7.8===<br />
Pick up 3 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
[7.9-7.19 Field Practices @ Yantai & Beijing (^_<)]<br />
[7.20-7.21 Home @ Nanjing (#_#)]<br />
[7.22-7.24 World Exhibit @ Shanghai (*~*)Orz]<br />
[7.25 Home @ Nanjing (#_#)]<br />
<br />
===7.26===<br />
<br />
Design the PbrR Metal Binding Peptide (MBP) Construction<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by PFUEasyMix (20μL)<br />
<br />
EasyMix: 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Primer_For_N: 0.5μL<br />
<br />
Primer_Rev_N:0.5μL<br />
<br />
Template (pbrR): 0.5μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for MBP_STD<br />
<br />
EasyMix: 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Primer_For_C: 0.5μL<br />
<br />
Primer_Rev_C:0.5μL<br />
<br />
Template (pbrR): 0.5μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for MBP_STD<br />
<br />
Linker are designed into Primer_Rev_N and Primer_For_C<br />
<br />
<br />
===7.27===<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
N C<br />
<br />
Digestion:<br />
<br />
N terminal: EcoRI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+PstI+NEBuffer3<br />
<br />
Purification of digestion product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with EcoRI and BspEI): 3μL <br />
<br />
Insert2 (C terminal digested with BspEI and PstI): 3μL<br />
<br />
Vector (pSB1K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
<br />
===7.28===<br />
<br />
Transformation: OmniMAX<br />
<br />
Pick up 3 colonies from the agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
===7.29===<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR_MBP_STD (pSB1K3)<br />
<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by EasyPFU (50μL)<br />
<br />
10*EasyPFU buffer: 5μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_For_N’:1μL<br />
<br />
Primer_Rev_N’: 1μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
ddH2O: 36.8μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for MBP_COM<br />
<br />
10*EasyPFU buffer: 5μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_For_C’:1μL<br />
<br />
Primer_Rev_C’: 1μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
ddH2O: 36.8μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for MBP_COM<br />
<br />
Linker are designed into Primer_Rev_N’ and Primer_For_C’<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Digestion: <br />
<br />
N terminal: NdeI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+XhoI+NEBuffer3<br />
<br />
<br />
===7.30===<br />
Purification of digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with NdeI and BspEI): 3μL<br />
<br />
Insert2 (C terminal digested with BspEI and XhoI): 3μL<br />
<br />
Vector (pET21a backbone digested with NdeI and XhoI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===7.31===<br />
Pick up 3 colonies from the agar plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR_MBP_COM (pET21a)<br />
<br />
==August==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
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|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| 1<br />
|style="text-align:center"| 2<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.3|3]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.4|4]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.5|5]]<br />
|style="text-align:center"| 6<br />
|style="text-align:center"| 7<br />
|- <br />
|style="text-align:center"| 8<br />
|style="text-align:center"| 9<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.10|10]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.11|11]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.12|12]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.13|13]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.14|14]]<br />
|- <br />
|style="text-align:center"| 15<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.16|16]]<br />
|style="text-align:center"| 17<br />
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|}<br />
[<html><a href="#top">TOP</a></html>]<br />
===8.3===<br />
Design the DsbA-MBP Construction<br />
<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For_N: 2μL<br />
<br />
Primer_Rev_N:2μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for DsbA-MBP<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For_C: 2μL<br />
<br />
Primer_Rev_C:2μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for DsbA-MBP<br />
<br />
Linker are designed into Primer_Rev_N and Primer_For_C<br />
<br />
Electrophoresis to verify<br />
<br />
Purification of PCR product<br />
<br />
Digestion: <br />
<br />
N terminal: XbaI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+XhoI+NEBuffer3<br />
<br />
===8.4===<br />
Purification of digested product<br />
<br />
Digestion (20μL):<br />
<br />
pET39b: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
XhoI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with XbaI and BspEI): 3μL<br />
<br />
Insert2 (C terminal digested with BspEI and XhoI): 3μL<br />
<br />
Vector (pET39b backbone digested with SpeI and XhoI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===8.5===<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
NdeI: 1μL<br />
<br />
XhoI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
NdeI(1030) XhoI(174) MBP(abt 500bp)<br />
<br />
Correct band=1030-174+500=1356<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: DsbA-MBP (pET39b)<br />
<br />
<br />
===8.10===<br />
Design and the T7-RBS-DsbA-MBP Construction<br />
<br />
<br />
1st Round of Nest PCR by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For (T7-RBS-DsbA-F1): 2μL<br />
<br />
Primer_Rev (PbrR-MBP-C’-R):2μL<br />
<br />
Template (DsbA-MBP): 0.2μL<br />
<br />
=>RBS is prefixed to DsbA-MBP<br />
<br />
Electrophoresis<br />
<br />
<br />
<br />
FAILED!!!(TAT)<br />
<br />
<br />
REPEAT-PCR with Gradient<br />
<br />
Gradient: T=60℃ G=5<br />
<br />
Electrophoresis<br />
<br />
<br />
Gel extraction<br />
<br />
===8.11===<br />
2nd Round of Nest PCR by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For (T7-RBS-DsbA-F2): 2μL<br />
<br />
Primer_Rev (PbrR-MBP-C’-R):2μL<br />
<br />
Template (1st Round of Nest PCR product (RBS-DsbA-MBP)): 0.2μL<br />
<br />
=>T7 is prefixed to 1st Round of Nest PCR product (RBS-DsbA-MBP)<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
<br />
===8.12===<br />
Digestion (20μL):<br />
<br />
2nd Round of Nest PCR product (T7-RBS-DsbA-MBP): 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
SpeI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of digested product<br />
<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
SpeI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (T7-RBS-DsbA-MBP digested with EcoRI and SpeI): 6μL<br />
<br />
Vector (pSB1K3 backbone digested with EcoRI and SpeI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===8.13===<br />
Pick up 5 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
===8.14===<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be two bands; one is about 3000bp and the other is 600bp<br />
<br />
Verification: PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be one band, which is about 1200bp<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: DsbA-MBP (pET39b)<br />
<br />
<br />
===8.16===<br />
FAILED (T~T)<br />
<br />
Why?<br />
<br />
Because the reverse primer cannot specifically distinguish the C terminal from the N terminal of the MBP, new primer <br />
including the sequence of His-tag is designed in order to recognize the C terminal only<br />
<br />
<br />
Hand over this work to Donghai Liang<br />
<br />
[8.17-8.31] Physical Training<br />
<br />
==September==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
|style="text-align:center"| Sat<br />
|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.1|1]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.2|2]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.3|3]]<br />
|style="text-align:center"| 4<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.5|5]] <br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.6|6]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.7|7]]<br />
|style="text-align:center"|8<br />
|style="text-align:center"| 9<br />
|style="text-align:center"| 10<br />
|style="text-align:center"| 11<br />
|style="text-align:center"| 12<br />
|- <br />
|style="text-align:center"| 13<br />
|style="text-align:center"|14<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.15|15]]<br />
|style="text-align:center"| 16<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.17|17]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.18|18]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.19|19]]<br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.20|20]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.21|21]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.22|22]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.23|23]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.24|24]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.25|25]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.26|26]]<br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.27|27]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.28|28]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.30|30]]<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|}<br />
[<html><a href="#top">TOP</a></html>]<br />
===9.1===<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Digestion (20μL):<br />
<br />
pbrR_MBP_STD (pSB1A2): 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (PbrR_MBP_STD digested with EcoRI and SpeI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and SpeI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
<br />
===9.2===<br />
<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be one band, which is about 500bp<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR-MBP (pSB1C3)<br />
<br />
<br />
<br />
===9.3===<br />
<br />
Construct the concentration of Hg(II) ions for induction<br />
<br />
Final Concentration/M Volume/μL Original Concentration/M<br />
<br />
1. 5*10-5 25 10-3<br />
<br />
2. 3*10-5 15 10-3<br />
<br />
3. 10-5 5 10-3<br />
<br />
4. 8*10-6 4 10-3<br />
<br />
5. 5*10-6 2.5 10-3<br />
<br />
6. 3*10-6 1.5 10-3<br />
<br />
7. 10-6 0.5 10-3<br />
<br />
8. 8*10-7 4 10-4<br />
<br />
9. 5*10-7 2.5 10-4<br />
<br />
10. 3*10-7 1.5 10-4<br />
<br />
11. 10-7 0.5 10-4<br />
<br />
12. 8*10-8 4 10-5<br />
<br />
13. 5*10-8 2.5 10-5<br />
<br />
14. 3*10-8 1.5 10-5<br />
<br />
15. 10-8 0.5 10-5<br />
<br />
16. 8*10-9 0.4 10-5<br />
<br />
17. 5*10-9 25 10-7<br />
<br />
18. 3*10-9 15 10-7<br />
<br />
19. 10-9 5 10-7<br />
<br />
20. 0 0 0<br />
<br />
<br />
<br />
===9.5===<br />
<br />
Learn the protocol for preparation of competent cells for transformation for bi-transformation<br />
<br />
Pick up one colony from each agar plate: J23109 and J23112<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===9.6===<br />
<br />
Re-activate the culture of J23109 and J23112 in fresh LB with ampicilin and kanamyclin<br />
<br />
Measure the value of OD600<br />
<br />
Induced with Hg(II) ions of different concentration for 2hrs<br />
<br />
Centrifugation at 5000r for 5min<br />
<br />
Resuspension with PBS<br />
<br />
Measure the value of OD600 and GFP with ELISA<br />
<br />
<br />
<br />
===9.7===<br />
<br />
Learn about Western Blot with Boxuan Zhao<br />
<br />
<br />
<br />
===9.15===<br />
<br />
Design the traffic light assay<br />
<br />
LacZ full length (RBS: B0034) BBa_I1732017 2_3K 3093bp pSB1A2<br />
<br />
LacZ α fragment BBa_I732006 1_23H 234bp pSB1AK3<br />
<br />
Plasmid1: 1_18I MerR (pSB3K3)<br />
<br />
Plasmid2: PmerT - LacZ full length / LacZ α fragment (pSB1C3)<br />
<br />
X-GAL assay<br />
<br />
<br />
<br />
Positive Transformation of 2_3K and 1_23H: TansMAX<br />
<br />
<br />
<br />
===9.17===<br />
<br />
Pick up 2 colonies from each agar plate: 2_3K and 1_23H<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Get the plasmids: 2_3K (LacZ full length_ pSB1A2)<br />
<br />
1_23H (LacZ α fragment_ pSB1AK3)<br />
<br />
<br />
<br />
===9.18===<br />
<br />
Digestion (20μL):<br />
<br />
LacZ full length: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
LacZα fragment: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
<br />
FAILED(T_T)<br />
<br />
Wrong enzyme used<br />
<br />
<br />
<br />
===9.19===<br />
<br />
Digestion (20μL):<br />
<br />
LacZ full length: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
LacZ α fragment: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
The band for LacZ full length is clear but not for LacZ α fragment<br />
<br />
Change method<br />
<br />
PCR from 1_23H by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for LacZ α fragment: 250bp<br />
<br />
<br />
<br />
===9.20===<br />
<br />
Digestion (20μL):<br />
<br />
RBS_pSB1A2: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer2): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Get the RBS_pSB1A2 backbone digested with SpeI and PstI<br />
<br />
Digestion (20μL):<br />
<br />
PmerT_pSB1C3: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer2): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Get the PmerT_pSB1C3 backbone digested with SpeI and PstI<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (RBS_pSB1A2 backbone digested with SpeI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (PmerT_pSB1C3 backbone digested with SpeI and PstI): 2μL<br />
<br />
Transformation of RBS-LacZ α fragment (pSB1A2)<br />
<br />
<br />
<br />
===9.21===<br />
<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
FAILED (>.<||)<br />
<br />
Forgot to digest the PCR product<br />
<br />
<br />
<br />
Digestion (20μL):<br />
<br />
1_23H PCR product: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of the digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (RBS_pSB1A2 backbone digested with SpeI and PstI): 2μL<br />
<br />
Transformation of RBS-LacZ α fragment (pSB1A2)<br />
<br />
Transformation of PmerT-LacZ full length (pSB1C3)<br />
<br />
<br />
<br />
===9.22===<br />
<br />
Pick up 6 colonies from each agar plate of RBS-LacZ α fragment (pSB1A2) and PmerT-LacZ full length (pSB1C3)<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-prep of PmerT-LacZ full length (pSB1C3)<br />
<br />
Digestion (20μL):<br />
<br />
PmerT-LacZ full length (pSB1C3): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 6 are of correct size<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: PmerT-LacZ full length (pSB1C3)<br />
<br />
<br />
<br />
Mini-prep of RBS-LacZ α fragment (pSB1A2)<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment (pSB1A2): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 4, 5, 6 are all of correct size<br />
<br />
<br />
<br />
===9.23===<br />
<br />
PCR to get RBS-LacZ α fragment by FastPFU (50μL)<br />
<br />
5*phusionHF buffer: 10μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.2μL<br />
<br />
ddH2O: 32μL<br />
<br />
Electrophoresis <br />
<br />
Excise the band of 250bp<br />
<br />
Gel Extraction<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (PmerT_pSB1C3 backbone digested with SpeI and PstI): 2μL<br />
<br />
<br />
<br />
===9.24===<br />
<br />
Transformation: Trans5α<br />
<br />
Pick up 6 colonies from the agar plate: PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
Grow the culture for overnight<br />
<br />
<br />
<br />
===9.25===<br />
<br />
Mini-prep of PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
Digestion (20μL):<br />
<br />
PmerT- RBS-LacZ α fragment (pSB1C3): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 4, 5, 6 are all of correct size<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
<br />
<br />
===9.26===<br />
<br />
Construct the mutant promoter P88 and P3 into the traffic light system<br />
<br />
<br />
<br />
===9.27===<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 1.5μL<br />
<br />
Primer_Rev_P88:1.5μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 4μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 1μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 1.5μL<br />
<br />
Primer_Rev_P3:1.5μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 4μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 1μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 3μL<br />
<br />
Primer_Rev_P88: 3μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 2μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 3μL<br />
<br />
Primer_Rev_P3: 3μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 2μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===9.28===<br />
<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
REPEAT<br />
<br />
<br />
<br />
===9.29===<br />
<br />
Still no recognizable colonies grew on the agar plates (TAT)<br />
<br />
Change the ratio of different components<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 15μL<br />
<br />
Primer_Rev_P88:15μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 3.5μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 1.5μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 10μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 15μL<br />
<br />
Primer_Rev_P3:15μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 3.5μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 1.5μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 10μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 7μL<br />
<br />
Primer_Rev_P88: 7μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1.7μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 3.3μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 7μL<br />
<br />
Primer_Rev_P3: 7μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1.7μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 3.3μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===9.30===<br />
<br />
Still no recognizable colonies grew on the agar plates (TT_TT)<br />
<br />
Hand over this work to Ying Sheng<br />
<br />
==October==<br />
<br />
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|style="text-align:center"|7<br />
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[[https://2010.igem.org/Team:Peking/Notebook/ZRLiu TOP]]<br />
===10.3===<br />
<br />
Prepare PARTS<br />
<br />
<br />
===10.4===<br />
<br />
Change backbone<br />
<br />
(1) T7-RBS-DsbA-MBP_pSB1K3=>T7-RBS-DsbA-MBP_pSB1C3<br />
<br />
(2) RBS-LacZ α fragment _pSB1A2=> RBS-LacZ α fragment _pSB1C3 <br />
<br />
<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
T7-RBS-DsbA-MBP_pSB1K3: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment _pSB1A2: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (T7-RBS-DsbA-MBP digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Mach-T1<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Mach-T1<br />
<br />
<br />
<br />
===10.5===<br />
<br />
Pick up 3 colonies from each agar plates<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: T7-RBS-DsbA-MBP (pSB1C3) and RBS-LacZ α fragment (pSB1C3)<br />
<br />
SUBMIT the plasmids<br />
<br />
Positive Transformation of T7-RBS-DsbA-MBP (pSB1C3) and RBS-LacZ α fragment (pSB1C3)<br />
<br />
<br />
<br />
===10.6===<br />
<br />
Pick up 1 colonies from each agar plates<br />
<br />
Grow the culture for 12hrs<br />
<br />
SUBMIT the culture preserved with glycerol<br />
<br />
<br />
<br />
Brief Characterization of PmerT-LacZ full length and PmerT-RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M<br />
<br />
(pictures see in PDF)<br />
<br />
<br />
<br />
===10.8===<br />
<br />
DNA sequencing shows that P88 / P3-LacZ α fragment (pSB1C3) are correct<br />
<br />
Positive Transformation<br />
<br />
Pick up one colony from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.9===<br />
<br />
Digestion (20μL):<br />
<br />
J23114_pSB3K3: 10μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
PCR from P88-LacZ α fragment_pSB1C3 by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for P88-RBS-LacZ α fragment: 350bp<br />
<br />
PCR from P3-LacZ α fragment_pSB1C3 by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for P3-RBS-LacZ α fragment: 350bp<br />
<br />
Digestion (20μL):<br />
<br />
P88-RBS-LacZ α fragment PCR product: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
P3-RBS-LacZ α fragment PCR product: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of the digested PCR product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P88-RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P3-RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===10.10===<br />
<br />
Pick up 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
Pick up one colony from plates: 1_18E and J23117<br />
<br />
Grow the culture for 12hrs<br />
<br />
<br />
<br />
<br />
<br />
===10.11===<br />
<br />
Fail in cultivating overnight culture<br />
<br />
PHAGE appeared??? (>…<)<br />
<br />
Pick up another 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
===10.12===<br />
<br />
Fail in cultivating overnight culture again (T_T)<br />
<br />
Pick up another 5 colonies from each plate<br />
<br />
Grow the culture in fresh LB with 1/10 kanamyclin for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
===10.13===<br />
<br />
Mini-prep<br />
<br />
Get the plasmids: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
P3-RBS-LacZα fragment_pSB3K3<br />
<br />
Re-activate the culture: 1_18E and J23117<br />
<br />
Make them competent cells for bi-transformation<br />
<br />
Transformation:<br />
<br />
Competent cell 1_18E: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
Competent cell J23117: P3-RBS-LacZα fragment_pSB3K3<br />
<br />
<br />
<br />
===10.14===<br />
<br />
Pick up 3 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.15===<br />
<br />
Brief Characterization of P88-RBS-LacZ α fragment and P3- RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M<br />
<br />
<br />
<br />
NO COLOR CHANGE (! _ !) <br />
<br />
<br />
<br />
[10.16-10.23] Prepare for GRE test<br />
<br />
<br />
<br />
===10.24===<br />
<br />
Pick up 6 colonies from each agar plate: J23109_pSB3K3<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.25===<br />
<br />
Mini-prep<br />
<br />
Get the plasmid: J23109_pSB3K3<br />
<br />
Digestion (20μL):<br />
<br />
J23109_pSB3K3: 10μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P88-RBS-LacZα fragment_pSB3K3): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P3-RBS-LacZα fragment_pSB3K3): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: TransT1-phage [14:00-0:00]<br />
<br />
Pick up one colony from plates: 1_18E and J23117 <br />
<br />
Grow the culture for 12hrs [19:30-7:30]<br />
<br />
<br />
<br />
===10.26===<br />
<br />
Pick up 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs [0:30-12:30]<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
Re-activate the culture of 1_18E and J23117 [7:30-11:30]<br />
<br />
Make them competent cells for bi-transformation [12:00-13:00]<br />
<br />
<br />
<br />
Mini-prep [12:30-13:30] Helped by Haoqian Zhang<br />
<br />
Get the plasmids P88-RBS-LacZα fragment_pSB3K3<br />
<br />
P3-RBS-LacZα fragment_pSB3K3<br />
<br />
<br />
<br />
Transformation: TransT1-phage [13:30-15:00]<br />
<br />
Competent cell 1_18E: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
Competent cell J23117: P3-RBS-LacZα fragment_pSB3K3<br />
<br />
[15:00-1:00]<br />
<br />
===10.27===<br />
<br />
Pick up 3 colonies on each agar plate<br />
<br />
Grow the culture for 12hrs [1:30-13:30]<br />
<br />
Re-activate the culture [13:30-15:30] <br />
<br />
<br />
<br />
Brief Characterization of PmerT-LacZ full length and PmerT-RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M [16:00-10:00]<br />
<br />
<br />
<br />
<br />
<br />
[[https://2010.igem.org/Team:Peking/Notebook/ZRLiu TOP]]<br />
<br />
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<a href="https://2010.igem.org/Team:Peking/Team/ZRLiu"><font color=#FFFFFF>==go to her page==</font></a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
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</html></div>Lgdeerhttp://2010.igem.org/Team:Peking/Notebook/ZRLiuTeam:Peking/Notebook/ZRLiu2010-10-27T10:31:19Z<p>Lgdeer: /* August */</p>
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__NOTOC__<br />
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Zairan Liu's Notes</font></font></font><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<a href="https://2010.igem.org/Team:Peking/Team/ZRLiu"><img src="https://static.igem.org/mediawiki/2010/a/af/Rr.jpg" width="40px" alt="goto her page"id="imggreen"> </a><br />
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I am responsible for the construction of metal binding peptide periplasm display module for Pb(II). This module aims at binding Pb(II) ions in the periplasmic space using the engineered anti-parallel coiled coil which is transported with the help of DsbA signal sequence. During the process several other intermediate plasmids are also constructed. Furthermore, I contribute to the characterization of Pc promoters of different intensity.<br />
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=='''Contents'''==<br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#July| July, 2010]]</span><br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#August| August, 2010]]</span><br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#September| September, 2010]]</span><br />
<br />
* <span style="font-size:4mm;">[[Team:Peking/Notebook/ZRLiu#October| October, 2010]]</span><br />
<br />
<br />
==July==<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
|style="text-align:center"| Sat<br />
|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| 1<br />
|style="text-align:center"| 2<br />
|style="text-align:center"| 3<br />
|- <br />
|style="text-align:center"| 4<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.5|5]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.6|6]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.7|7]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.8|8]]<br />
|style="text-align:center"| 9<br />
|style="text-align:center"| 10<br />
|- <br />
|style="text-align:center"| 11<br />
|style="text-align:center"| 12<br />
|style="text-align:center"| 13<br />
|style="text-align:center"| 14<br />
|style="text-align:center"| 15<br />
|style="text-align:center"| 16<br />
|style="text-align:center"| 17<br />
|- <br />
|style="text-align:center"| 18<br />
|style="text-align:center"| 19<br />
|style="text-align:center"| 20<br />
|style="text-align:center"| 21<br />
|style="text-align:center"| 22<br />
|style="text-align:center"| 23<br />
|style="text-align:center"| 24<br />
|- <br />
|style="text-align:center"| 25<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.26|26]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.27|27]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.30|30]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#7.31|31]]<br />
|- <br />
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[<html><a href="#top">TOP</a></html>]<br />
===7.5===<br />
Purification of digested PCR product: merP, merT, and merC<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (merP / merT / merC digested with EcoRI and PstI): 7μL<br />
<br />
Vector (pSB1A2 backbone digested with EcoRI and PstI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
===7.6===<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
Digestion (20μL):<br />
<br />
pSB1A2: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
PCR to get merP, merT and merC fragments by Phusion (20μL)<br />
<br />
5*phusionHF buffer: 4μL<br />
<br />
2.5mM dNTPs: 1.6μL<br />
<br />
Polymerase: 0.2μL<br />
<br />
Primer_For:1μL<br />
<br />
Primer_Rev: 1μL<br />
<br />
Template: 0.5μL<br />
<br />
ddH2O: 11.7μL<br />
<br />
<br />
===7.7===<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
merT: 351bp<br />
<br />
merP: 256bp <br />
<br />
merC: 423bp<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (merP / merT / merC digested with EcoRI and PstI): 7μL<br />
<br />
Vector (pSB1A2 backbone digested with EcoRI and PstI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
===7.8===<br />
Pick up 3 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
[7.9-7.19 Field Practices @ Yantai & Beijing (^_<)]<br />
[7.20-7.21 Home @ Nanjing (#_#)]<br />
[7.22-7.24 World Exhibit @ Shanghai (*~*)Orz]<br />
[7.25 Home @ Nanjing (#_#)]<br />
<br />
===7.26===<br />
<br />
Design the PbrR Metal Binding Peptide (MBP) Construction<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by PFUEasyMix (20μL)<br />
<br />
EasyMix: 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Primer_For_N: 0.5μL<br />
<br />
Primer_Rev_N:0.5μL<br />
<br />
Template (pbrR): 0.5μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for MBP_STD<br />
<br />
EasyMix: 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Primer_For_C: 0.5μL<br />
<br />
Primer_Rev_C:0.5μL<br />
<br />
Template (pbrR): 0.5μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for MBP_STD<br />
<br />
Linker are designed into Primer_Rev_N and Primer_For_C<br />
<br />
<br />
===7.27===<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
N C<br />
<br />
Digestion:<br />
<br />
N terminal: EcoRI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+PstI+NEBuffer3<br />
<br />
Purification of digestion product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with EcoRI and BspEI): 3μL <br />
<br />
Insert2 (C terminal digested with BspEI and PstI): 3μL<br />
<br />
Vector (pSB1K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
<br />
===7.28===<br />
<br />
Transformation: OmniMAX<br />
<br />
Pick up 3 colonies from the agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
===7.29===<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR_MBP_STD (pSB1K3)<br />
<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by EasyPFU (50μL)<br />
<br />
10*EasyPFU buffer: 5μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_For_N’:1μL<br />
<br />
Primer_Rev_N’: 1μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
ddH2O: 36.8μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for MBP_COM<br />
<br />
10*EasyPFU buffer: 5μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_For_C’:1μL<br />
<br />
Primer_Rev_C’: 1μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
ddH2O: 36.8μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for MBP_COM<br />
<br />
Linker are designed into Primer_Rev_N’ and Primer_For_C’<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Digestion: <br />
<br />
N terminal: NdeI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+XhoI+NEBuffer3<br />
<br />
<br />
===7.30===<br />
Purification of digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with NdeI and BspEI): 3μL<br />
<br />
Insert2 (C terminal digested with BspEI and XhoI): 3μL<br />
<br />
Vector (pET21a backbone digested with NdeI and XhoI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===7.31===<br />
Pick up 3 colonies from the agar plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR_MBP_COM (pET21a)<br />
<br />
==August==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
|style="text-align:center"| Sat<br />
|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| 1<br />
|style="text-align:center"| 2<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.3|3]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.4|4]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.5|5]]<br />
|style="text-align:center"| 6<br />
|style="text-align:center"| 7<br />
|- <br />
|style="text-align:center"| 8<br />
|style="text-align:center"| 9<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.10|10]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.11|11]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.12|12]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.13|13]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.14|14]]<br />
|- <br />
|style="text-align:center"| 15<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#8.16|16]]<br />
|style="text-align:center"| 17<br />
|style="text-align:center"| 18<br />
|style="text-align:center"| 19<br />
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|style="text-align:center"| 21<br />
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|style="text-align:center"|24<br />
|style="text-align:center"| 25<br />
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|style="text-align:center"| 28<br />
|- <br />
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|style="text-align:center"| 31<br />
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|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|}<br />
[<html><a href="#top">TOP</a></html>]<br />
===8.3===<br />
Design the DsbA-MBP Construction<br />
<br />
<br />
PCR from pbrR to get the N terminal and C terminal of MBP by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For_N: 2μL<br />
<br />
Primer_Rev_N:2μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
=>Get the metal binding domain of pbrR as the N terminal for DsbA-MBP<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For_C: 2μL<br />
<br />
Primer_Rev_C:2μL<br />
<br />
Template (pbrR): 0.2μL<br />
<br />
=>Get the metal binding domain of pbrR as the C terminal for DsbA-MBP<br />
<br />
Linker are designed into Primer_Rev_N and Primer_For_C<br />
<br />
Electrophoresis to verify<br />
<br />
Purification of PCR product<br />
<br />
Digestion: <br />
<br />
N terminal: XbaI+BspEI+NEBuffer3<br />
<br />
C terminal: BspEI+XhoI+NEBuffer3<br />
<br />
===8.4===<br />
Purification of digested product<br />
<br />
Digestion (20μL):<br />
<br />
pET39b: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
XhoI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert1 (N terminal digested with XbaI and BspEI): 3μL<br />
<br />
Insert2 (C terminal digested with BspEI and XhoI): 3μL<br />
<br />
Vector (pET39b backbone digested with SpeI and XhoI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===8.5===<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
NdeI: 1μL<br />
<br />
XhoI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
NdeI(1030) XhoI(174) MBP(abt 500bp)<br />
<br />
Correct band=1030-174+500=1356<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: DsbA-MBP (pET39b)<br />
<br />
<br />
===8.10===<br />
Design and the T7-RBS-DsbA-MBP Construction<br />
<br />
<br />
1st Round of Nest PCR by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For (T7-RBS-DsbA-F1): 2μL<br />
<br />
Primer_Rev (PbrR-MBP-C’-R):2μL<br />
<br />
Template (DsbA-MBP): 0.2μL<br />
<br />
=>RBS is prefixed to DsbA-MBP<br />
<br />
Electrophoresis<br />
<br />
<br />
<br />
FAILED!!!(TAT)<br />
<br />
<br />
REPEAT-PCR with Gradient<br />
<br />
Gradient: T=60℃ G=5<br />
<br />
Electrophoresis<br />
<br />
<br />
Gel extraction<br />
<br />
===8.11===<br />
2nd Round of Nest PCR by EasyPFU SuperMix (50μL)<br />
<br />
2*EasyPFU SuperMix: 25μL<br />
<br />
ddH2O: 20.8μL<br />
<br />
Primer_For (T7-RBS-DsbA-F2): 2μL<br />
<br />
Primer_Rev (PbrR-MBP-C’-R):2μL<br />
<br />
Template (1st Round of Nest PCR product (RBS-DsbA-MBP)): 0.2μL<br />
<br />
=>T7 is prefixed to 1st Round of Nest PCR product (RBS-DsbA-MBP)<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
<br />
===8.12===<br />
Digestion (20μL):<br />
<br />
2nd Round of Nest PCR product (T7-RBS-DsbA-MBP): 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
SpeI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of digested product<br />
<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
SpeI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (T7-RBS-DsbA-MBP digested with EcoRI and SpeI): 6μL<br />
<br />
Vector (pSB1K3 backbone digested with EcoRI and SpeI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
===8.13===<br />
Pick up 5 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
===8.14===<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be two bands; one is about 3000bp and the other is 600bp<br />
<br />
Verification: PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be one band, which is about 1200bp<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: DsbA-MBP (pET39b)<br />
<br />
<br />
===8.16===<br />
FAILED (T~T)<br />
<br />
Why?<br />
<br />
Because the reverse primer cannot specifically distinguish the C terminal from the N terminal of the MBP, new primer <br />
including the sequence of His-tag is designed in order to recognize the C terminal only<br />
<br />
<br />
Hand over this work to Donghai Liang<br />
<br />
[8.17-8.31] Physical Training<br />
<br />
==September==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
|style="text-align:center"| Sat<br />
|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.1|1]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.2|2]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.3|3]]<br />
|style="text-align:center"| 4<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.5|5]] <br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.6|6]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.7|7]]<br />
|style="text-align:center"|8<br />
|style="text-align:center"| 9<br />
|style="text-align:center"| 10<br />
|style="text-align:center"| 11<br />
|style="text-align:center"| 12<br />
|- <br />
|style="text-align:center"| 13<br />
|style="text-align:center"|14<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.15|15]]<br />
|style="text-align:center"| 16<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.17|17]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.18|18]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.19|19]]<br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.20|20]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.21|21]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.22|22]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.23|23]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.24|24]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.25|25]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.26|26]]<br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.27|27]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#9.28|28]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.29|29]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#9.30|30]]<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|}<br />
[[https://2010.igem.org/Team:Peking/Notebook/ZRLiu TOP]]<br />
===9.1===<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Digestion (20μL):<br />
<br />
pbrR_MBP_STD (pSB1A2): 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (PbrR_MBP_STD digested with EcoRI and SpeI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and SpeI): 2μL<br />
<br />
Transformation: OmniMAX<br />
<br />
<br />
<br />
===9.2===<br />
<br />
Mini-Prep<br />
<br />
Verification: Digestion (20μL):<br />
<br />
Plasmid: 5μL<br />
<br />
EcoRI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (EcoRI Buffer): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
There should be one band, which is about 500bp<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: pbrR-MBP (pSB1C3)<br />
<br />
<br />
<br />
===9.3===<br />
<br />
Construct the concentration of Hg(II) ions for induction<br />
<br />
Final Concentration/M Volume/μL Original Concentration/M<br />
<br />
1. 5*10-5 25 10-3<br />
<br />
2. 3*10-5 15 10-3<br />
<br />
3. 10-5 5 10-3<br />
<br />
4. 8*10-6 4 10-3<br />
<br />
5. 5*10-6 2.5 10-3<br />
<br />
6. 3*10-6 1.5 10-3<br />
<br />
7. 10-6 0.5 10-3<br />
<br />
8. 8*10-7 4 10-4<br />
<br />
9. 5*10-7 2.5 10-4<br />
<br />
10. 3*10-7 1.5 10-4<br />
<br />
11. 10-7 0.5 10-4<br />
<br />
12. 8*10-8 4 10-5<br />
<br />
13. 5*10-8 2.5 10-5<br />
<br />
14. 3*10-8 1.5 10-5<br />
<br />
15. 10-8 0.5 10-5<br />
<br />
16. 8*10-9 0.4 10-5<br />
<br />
17. 5*10-9 25 10-7<br />
<br />
18. 3*10-9 15 10-7<br />
<br />
19. 10-9 5 10-7<br />
<br />
20. 0 0 0<br />
<br />
<br />
<br />
===9.5===<br />
<br />
Learn the protocol for preparation of competent cells for transformation for bi-transformation<br />
<br />
Pick up one colony from each agar plate: J23109 and J23112<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===9.6===<br />
<br />
Re-activate the culture of J23109 and J23112 in fresh LB with ampicilin and kanamyclin<br />
<br />
Measure the value of OD600<br />
<br />
Induced with Hg(II) ions of different concentration for 2hrs<br />
<br />
Centrifugation at 5000r for 5min<br />
<br />
Resuspension with PBS<br />
<br />
Measure the value of OD600 and GFP with ELISA<br />
<br />
<br />
<br />
===9.7===<br />
<br />
Learn about Western Blot with Boxuan Zhao<br />
<br />
<br />
<br />
===9.15===<br />
<br />
Design the traffic light assay<br />
<br />
LacZ full length (RBS: B0034) BBa_I1732017 2_3K 3093bp pSB1A2<br />
<br />
LacZ α fragment BBa_I732006 1_23H 234bp pSB1AK3<br />
<br />
Plasmid1: 1_18I MerR (pSB3K3)<br />
<br />
Plasmid2: PmerT - LacZ full length / LacZ α fragment (pSB1C3)<br />
<br />
X-GAL assay<br />
<br />
<br />
<br />
Positive Transformation of 2_3K and 1_23H: TansMAX<br />
<br />
<br />
<br />
===9.17===<br />
<br />
Pick up 2 colonies from each agar plate: 2_3K and 1_23H<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Get the plasmids: 2_3K (LacZ full length_ pSB1A2)<br />
<br />
1_23H (LacZ α fragment_ pSB1AK3)<br />
<br />
<br />
<br />
===9.18===<br />
<br />
Digestion (20μL):<br />
<br />
LacZ full length: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
LacZα fragment: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
<br />
<br />
FAILED(T_T)<br />
<br />
Wrong enzyme used<br />
<br />
<br />
<br />
===9.19===<br />
<br />
Digestion (20μL):<br />
<br />
LacZ full length: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
LacZ α fragment: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
The band for LacZ full length is clear but not for LacZ α fragment<br />
<br />
Change method<br />
<br />
PCR from 1_23H by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for LacZ α fragment: 250bp<br />
<br />
<br />
<br />
===9.20===<br />
<br />
Digestion (20μL):<br />
<br />
RBS_pSB1A2: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer2): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Get the RBS_pSB1A2 backbone digested with SpeI and PstI<br />
<br />
Digestion (20μL):<br />
<br />
PmerT_pSB1C3: 5μL<br />
<br />
SpeI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer2): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
Get the PmerT_pSB1C3 backbone digested with SpeI and PstI<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (RBS_pSB1A2 backbone digested with SpeI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (PmerT_pSB1C3 backbone digested with SpeI and PstI): 2μL<br />
<br />
Transformation of RBS-LacZ α fragment (pSB1A2)<br />
<br />
<br />
<br />
===9.21===<br />
<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
FAILED (>.<||)<br />
<br />
Forgot to digest the PCR product<br />
<br />
<br />
<br />
Digestion (20μL):<br />
<br />
1_23H PCR product: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of the digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (RBS_pSB1A2 backbone digested with SpeI and PstI): 2μL<br />
<br />
Transformation of RBS-LacZ α fragment (pSB1A2)<br />
<br />
Transformation of PmerT-LacZ full length (pSB1C3)<br />
<br />
<br />
<br />
===9.22===<br />
<br />
Pick up 6 colonies from each agar plate of RBS-LacZ α fragment (pSB1A2) and PmerT-LacZ full length (pSB1C3)<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-prep of PmerT-LacZ full length (pSB1C3)<br />
<br />
Digestion (20μL):<br />
<br />
PmerT-LacZ full length (pSB1C3): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 6 are of correct size<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: PmerT-LacZ full length (pSB1C3)<br />
<br />
<br />
<br />
Mini-prep of RBS-LacZ α fragment (pSB1A2)<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment (pSB1A2): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 4, 5, 6 are all of correct size<br />
<br />
<br />
<br />
===9.23===<br />
<br />
PCR to get RBS-LacZ α fragment by FastPFU (50μL)<br />
<br />
5*phusionHF buffer: 10μL<br />
<br />
2.5mM dNTPs: 5μL<br />
<br />
Polymerase: 1μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.2μL<br />
<br />
ddH2O: 32μL<br />
<br />
Electrophoresis <br />
<br />
Excise the band of 250bp<br />
<br />
Gel Extraction<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment: 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of digested product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (LacZ α fragment digested with XbaI and PstI): 6μL<br />
<br />
Vector (PmerT_pSB1C3 backbone digested with SpeI and PstI): 2μL<br />
<br />
<br />
<br />
===9.24===<br />
<br />
Transformation: Trans5α<br />
<br />
Pick up 6 colonies from the agar plate: PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
Grow the culture for overnight<br />
<br />
<br />
<br />
===9.25===<br />
<br />
Mini-prep of PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
Digestion (20μL):<br />
<br />
PmerT- RBS-LacZ α fragment (pSB1C3): 5μL<br />
<br />
XbaI: 1μL<br />
<br />
PstI: 1μL<br />
<br />
Buffer (NEBuffer3): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis to verify<br />
<br />
Band No.1, 2, 3, 4, 5, 6 are all of correct size<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: PmerT- RBS-LacZ α fragment (pSB1C3)<br />
<br />
<br />
<br />
===9.26===<br />
<br />
Construct the mutant promoter P88 and P3 into the traffic light system<br />
<br />
<br />
<br />
===9.27===<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 1.5μL<br />
<br />
Primer_Rev_P88:1.5μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 4μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 1μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 1.5μL<br />
<br />
Primer_Rev_P3:1.5μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 4μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 1μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 1μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 3μL<br />
<br />
Primer_Rev_P88: 3μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 2μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 3μL<br />
<br />
Primer_Rev_P3: 3μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 2μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===9.28===<br />
<br />
No recognizable colonies grew on the agar plates (T_T)<br />
<br />
REPEAT<br />
<br />
<br />
<br />
===9.29===<br />
<br />
Still no recognizable colonies grew on the agar plates (TAT)<br />
<br />
Change the ratio of different components<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 15μL<br />
<br />
Primer_Rev_P88:15μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 3.5μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 1.5μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 10μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 15μL<br />
<br />
Primer_Rev_P3:15μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 3.5μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 1.5μL<br />
<br />
Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 10μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P88: 7μL<br />
<br />
Primer_Rev_P88: 7μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1.7μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 3.3μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
Annealing to form the promoter from designed primers (95℃ 5min):<br />
<br />
Primer_For_P3: 7μL<br />
<br />
Primer_Rev_P3: 7μL<br />
<br />
Phosphorylation (37℃ 30min):<br />
<br />
[ADD]<br />
<br />
PNK: 1μL<br />
<br />
Ligase buffer: 1.7μL<br />
<br />
ddH2O: 1μL<br />
<br />
Ligation:<br />
<br />
[ADD]<br />
<br />
Ligase: 5μL<br />
<br />
Ligase buffer: 3.3μL<br />
<br />
Insert: (LacZ full length digested with XbaI and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 10μL<br />
<br />
ddH2O: 9μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===9.30===<br />
<br />
Still no recognizable colonies grew on the agar plates (TT_TT)<br />
<br />
Hand over this work to Ying Sheng<br />
<br />
==October==<br />
<br />
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"<br />
|- <br />
|style="text-align:center"| Mon<br />
|style="text-align:center"| Tue<br />
|style="text-align:center"| Wed<br />
|style="text-align:center"| Thu<br />
|style="text-align:center"| Fri<br />
|style="text-align:center"| Sat<br />
|style="text-align:center"| Sun<br />
|- <br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"|-<br />
|style="text-align:center"|-<br />
|style="text-align:center"|1<br />
|style="text-align:center"|2<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.3|3]] <br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.4|4]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.5|5]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.6|6]]<br />
|style="text-align:center"|7<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.8|8]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.9|9]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.10|10]]<br />
|- <br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.11|11]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.12|12]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.13|13]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.14|14]]<br />
|style="text-align:center"| 15<br />
|style="text-align:center"|16<br />
|style="text-align:center"| 17<br />
|- <br />
|style="text-align:center"| 18<br />
|style="text-align:center"| 19<br />
|style="text-align:center"|20<br />
|style="text-align:center"|21<br />
|style="text-align:center"|22<br />
|style="text-align:center"|23<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.24|24]]<br />
|- <br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.25|25]]<br />
|style="text-align:center"|[[Team:Peking/Notebook/ZRLiu#10.26|26]]<br />
|style="text-align:center"| [[Team:Peking/Notebook/ZRLiu#10.27|27]]<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|style="text-align:center"| -<br />
|}<br />
[[https://2010.igem.org/Team:Peking/Notebook/ZRLiu TOP]]<br />
===10.3===<br />
<br />
Prepare PARTS<br />
<br />
<br />
===10.4===<br />
<br />
Change backbone<br />
<br />
(1) T7-RBS-DsbA-MBP_pSB1K3=>T7-RBS-DsbA-MBP_pSB1C3<br />
<br />
(2) RBS-LacZ α fragment _pSB1A2=> RBS-LacZ α fragment _pSB1C3 <br />
<br />
<br />
<br />
Digestion (20μL):<br />
<br />
pSB1C3: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
T7-RBS-DsbA-MBP_pSB1K3: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
RBS-LacZ α fragment _pSB1A2: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (T7-RBS-DsbA-MBP digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Mach-T1<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Mach-T1<br />
<br />
<br />
<br />
===10.5===<br />
<br />
Pick up 3 colonies from each agar plates<br />
<br />
Grow the culture for 12hrs<br />
<br />
Mini-Prep<br />
<br />
Verification<br />
<br />
DNA Sequencing<br />
<br />
To get the plasmid: T7-RBS-DsbA-MBP (pSB1C3) and RBS-LacZ α fragment (pSB1C3)<br />
<br />
SUBMIT the plasmids<br />
<br />
Positive Transformation of T7-RBS-DsbA-MBP (pSB1C3) and RBS-LacZ α fragment (pSB1C3)<br />
<br />
<br />
<br />
===10.6===<br />
<br />
Pick up 1 colonies from each agar plates<br />
<br />
Grow the culture for 12hrs<br />
<br />
SUBMIT the culture preserved with glycerol<br />
<br />
<br />
<br />
Brief Characterization of PmerT-LacZ full length and PmerT-RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M<br />
<br />
(pictures see in PDF)<br />
<br />
<br />
<br />
===10.8===<br />
<br />
DNA sequencing shows that P88 / P3-LacZ α fragment (pSB1C3) are correct<br />
<br />
Positive Transformation<br />
<br />
Pick up one colony from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.9===<br />
<br />
Digestion (20μL):<br />
<br />
J23114_pSB3K3: 10μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
PCR from P88-LacZ α fragment_pSB1C3 by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for P88-RBS-LacZ α fragment: 350bp<br />
<br />
PCR from P3-LacZ α fragment_pSB1C3 by EasyPFU SuperMix (20μL)<br />
<br />
2*EasyPFU SuperMix: 10μL<br />
<br />
ddH2O: 7μL<br />
<br />
Primer_Univ_For: 1.5μL<br />
<br />
Primer_Univ_Rev:1.5μL<br />
<br />
Template: 0.3μL<br />
<br />
Electrophoresis<br />
<br />
Gel Extraction<br />
<br />
There should be one band for P3-RBS-LacZ α fragment: 350bp<br />
<br />
Digestion (20μL):<br />
<br />
P88-RBS-LacZ α fragment PCR product: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Digestion (20μL):<br />
<br />
P3-RBS-LacZ α fragment PCR product: 5μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Purification of the digested PCR product<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P88-RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P3-RBS-LacZ α fragment digested with EcoRI-HF and PstI): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: Trans5α<br />
<br />
<br />
<br />
===10.10===<br />
<br />
Pick up 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
Pick up one colony from plates: 1_18E and J23117<br />
<br />
Grow the culture for 12hrs<br />
<br />
<br />
<br />
<br />
<br />
===10.11===<br />
<br />
Fail in cultivating overnight culture<br />
<br />
PHAGE appeared??? (>…<)<br />
<br />
Pick up another 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
===10.12===<br />
<br />
Fail in cultivating overnight culture again (T_T)<br />
<br />
Pick up another 5 colonies from each plate<br />
<br />
Grow the culture in fresh LB with 1/10 kanamyclin for 12hrs<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
===10.13===<br />
<br />
Mini-prep<br />
<br />
Get the plasmids: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
P3-RBS-LacZα fragment_pSB3K3<br />
<br />
Re-activate the culture: 1_18E and J23117<br />
<br />
Make them competent cells for bi-transformation<br />
<br />
Transformation:<br />
<br />
Competent cell 1_18E: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
Competent cell J23117: P3-RBS-LacZα fragment_pSB3K3<br />
<br />
<br />
<br />
===10.14===<br />
<br />
Pick up 3 colonies from each agar plate<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.15===<br />
<br />
Brief Characterization of P88-RBS-LacZ α fragment and P3- RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M<br />
<br />
<br />
<br />
NO COLOR CHANGE (! _ !) <br />
<br />
<br />
<br />
[10.16-10.23] Prepare for GRE test<br />
<br />
<br />
<br />
===10.24===<br />
<br />
Pick up 6 colonies from each agar plate: J23109_pSB3K3<br />
<br />
Grow the culture overnight<br />
<br />
<br />
<br />
===10.25===<br />
<br />
Mini-prep<br />
<br />
Get the plasmid: J23109_pSB3K3<br />
<br />
Digestion (20μL):<br />
<br />
J23109_pSB3K3: 10μL<br />
<br />
EcoRI-HF: 0.5μL<br />
<br />
PstI: 0.5μL<br />
<br />
Buffer (NEBuffer4): 2μL<br />
<br />
ddH2O: 12μL<br />
<br />
Electrophoresis<br />
<br />
Gel extraction<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P88-RBS-LacZα fragment_pSB3K3): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Ligation (10μL):<br />
<br />
Ligase: 1μL<br />
<br />
Ligase buffer: 1μL<br />
<br />
Insert (P3-RBS-LacZα fragment_pSB3K3): 6μL<br />
<br />
Vector (pSB3K3 backbone digested with EcoRI and PstI): 2μL<br />
<br />
Transformation: TransT1-phage [14:00-0:00]<br />
<br />
Pick up one colony from plates: 1_18E and J23117 <br />
<br />
Grow the culture for 12hrs [19:30-7:30]<br />
<br />
<br />
<br />
===10.26===<br />
<br />
Pick up 6 colonies from each plate<br />
<br />
Grow the culture for 12hrs [0:30-12:30]<br />
<br />
Colony PCR by EasyTaq SuperMix<br />
<br />
Template: 0.2μL<br />
<br />
Primer_Univ_For: 1μL<br />
<br />
Primer_Univ_Rev: 1μL<br />
<br />
2*EasyTaq SuperMix: 5μL<br />
<br />
ddH2O:3μL<br />
<br />
Electrophoresis to verify<br />
<br />
<br />
<br />
Re-activate the culture of 1_18E and J23117 [7:30-11:30]<br />
<br />
Make them competent cells for bi-transformation [12:00-13:00]<br />
<br />
<br />
<br />
Mini-prep [12:30-13:30] Helped by Haoqian Zhang<br />
<br />
Get the plasmids P88-RBS-LacZα fragment_pSB3K3<br />
<br />
P3-RBS-LacZα fragment_pSB3K3<br />
<br />
<br />
<br />
Transformation: TransT1-phage [13:30-15:00]<br />
<br />
Competent cell 1_18E: P88-RBS-LacZα fragment_pSB3K3<br />
<br />
Competent cell J23117: P3-RBS-LacZα fragment_pSB3K3<br />
<br />
[15:00-1:00]<br />
<br />
===10.27===<br />
<br />
Pick up 3 colonies on each agar plate<br />
<br />
Grow the culture for 12hrs [1:30-13:30]<br />
<br />
Re-activate the culture [13:30-15:30] <br />
<br />
<br />
<br />
Brief Characterization of PmerT-LacZ full length and PmerT-RBS-LacZ α fragment<br />
<br />
Re-activate the culture to OD600 0.4~0.6<br />
<br />
Centrifugation (5000r 5min)<br />
<br />
Resuspension with ddH2O (0.01M Xgal added)<br />
<br />
Induced by Hg(II) ions of different concentrations from 10-3M to 10-7M [16:00-10:00]<br />
<br />
<br />
<br />
<br />
<br />
[[https://2010.igem.org/Team:Peking/Notebook/ZRLiu TOP]]<br />
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<a href="https://2010.igem.org/Team:Peking/Team/ZRLiu"><font color=#FFFFFF>==go to her page==</font></a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
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