Team:DTU-Denmark/BBrick Characterisation

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<title>Welcome to the DTU iGEM wiki!</title>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Basics">Basics</a></li><br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems">Regulatory Systems</a></li><br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch">The Switch</a></li><br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL">Synthetic Promoter Library</a></li><br>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/BBrick_Characterisation">Results</a></li><br>
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<h3>The RNA Polymerase (RNAP)</h3>
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<h1>Introduction</h1>
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<p align="justify">In synthetic biology when creating the minimal cell  XXX factors have been identified as essential for normal function of RNAP, both regarding normal elongation but also in terms of normal termination function. (REFFF) of these XXX have been identified to take part in regulatory function. While the rest is core subunits. Of interest for regulation in terms of termination Anti-termination will be highlighted:</p>
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<p align="justify">In order to optimize the lab work, we split up the work so that we could have two lab teams working in parallel to design different parts of the switch. We split up the lab work so we had:<br>
<ul>
<ul>
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<li>NusA:</li>
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<li>Team 1: <b>The Repressor - Anti-Repressor Team</b></li>
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<li>NusG:</li>
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<li>Team 2: <b>The Terminator - Anti-Terminator Team</b></li>
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<li>NusE:</li>
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<li>NusB:</li>
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</ul>
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<p align="justify">Due to the construction of the RNAP of many subcomponents and systems, the function of the RNAP can be regulated by only adding or changing one or a few factors. This is the mechanism in the different termination and anti-termination functions described below.</p><br>
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The Repressor (Repressor - Anti-Repressor) Team is responsible for assembling the construct illustrated below in Figure 1:<br>
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<p align="justify">Figure and table containing normal transcription and normal termination and table with sub-part names and explanation.</p><br>
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<p align="center"><img scr=""></img></p>
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<h3>Termination</h3>
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The Anti-Terminator (Terminator - Anti-Terminator) Team is responsible for assembling the construct illustrated in Figure 2:<br>
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<p align="justify">(terminator introduction)</p>
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<p align="center"><img scr=""></img></p>
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<p align="justify">Termination can fall into one of two categories:</p>
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</p>
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<ul>
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<h1>Repressor Group</h1>
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<li>Intrinsic Termination</li>
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<p align="justify">The repressor group will be assembling the constructs step-by-step:</p>
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<li>Factor-dependent Termination</li>
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<h3>Step 1</h3>
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</ul>
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<p align="justify">The construction of a plasmid containing the divergent promoters is the first step, the effect of this will be the uninhibited expression of GFP as illustrated by the green colonies observed in Figure 4.</p>
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<h4>Intrinsic Termination</h4>
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<p align="center"><img scr=""></img></p>
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<p align="justify">Intrinsic Termination can be found to occur at defined template sequences, usually a region of hyphenated inverted sequence symmetry followed by a run of T residues. Termination through intrinsic terminators is stimulated by additional factors, e.g. NusA. Termination occurs due to the stem-loop structure formed by the base-pairing of mRNA with itself caused by inverted sequence symmetry, followed by the run of T residues. The NusA protein causes the RNA-p complex to temporarily stall at the stem-loop structure, when this is followed by a poly-A tail, the RNA-DNA duplex is destabilized. This causes the RNA-p to dissociate from the DNA, thereby terminating transcription. Termination function step-by-step.</p>
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<font size="1.5">
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<h4>Factor dependent Termination</h4>
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<p align="justify"><b>Figure 3</b>: The initial plasmid constructed is illustrated. The divergent promoters have been inserted into a plasmid and transformed into the electro-competent <i>E.coli</i> cells.</p>
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<p align="justify">Factor-dependent Termination occurs due to events that are not directly related to transcription, such as the release of ribosomes from nascent transcript or DNA damage. One such host termination factor is Rho, which acts on many sites along the bacterial chromosome.
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<p align="center"><img scr=""></img></p>
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(( ??MFD, is a host termination factor that is responsible for releasing RNA-p stalled at sites of UV-induced DNA lesions. ??))
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<p align="justify"><b>Figure 3</b>: The success of the plasmid construction and transformation is illustrated by the fluorescent green colonies seen on the LB-agar plates.</p>
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rho-dependent termination is characterized by not having a specific hairpin structure involved in the termination.  
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</font>
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The termination thus happens  whendue to XXXXXX, and what have been found of the termination site any commen sequences or consensus ?????????????????
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<h3>Step 2</h3>
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the function of rho dependent termination, have been shown to be affected by XXXX.
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<p align="center"><img src="https://static.igem.org/mediawiki/2010/e/ef/DTU_BB_Repressor1.png" width="570px"  align="center"> </img></p>
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The rho binding sites on the mRNA, have been identified from XXbp to XXXbp of stream of the termination site.
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<font size="1.5">
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Rho-termination is thus an example of the more complex termination regulation that is not fully understood and can be very difficult to define and use for  engineering purposes. Thus for a more defined anti-termination system the lambda N-protein system and the interaction with the nut-site in the phage genome ????? is a more defined system. Recent research have found out that the Rho termination..? </p>
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<p align="justify"><b>Figure 4</b>: The construction of a plasmid containing the Repressor protein (GogR or GtgR) expressed from the pRM promoter is shown. The continually expressed repressor protein will then inhibit the pR promoter and no GFP will be expressed.</p>
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</font>
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<h3>Phages</h3>
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<h3>Step 3</h3>
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<p align="justify">What do they need to be succesfull? How stable have the function been proved to be. Examples of known phage regulation systems. Lambda, p21, P22, gifsy1,2,3,. What does the genomes contain of proteins, mRNA, binding sequences, ANJA??? Identification of mechanisms in regulation, compare with the Chinese paper (hong kong) using cI protein and UV-radiation.
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<font size="1.5">
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Link from phages to anti-terminator and/or Repressor function in phages (next sections).</p>
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<p align="center"><img src="https://static.igem.org/mediawiki/2010/7/7c/DTU_BB_Repressor2.png" width="570px"  align="center"> </img></p>
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<h3>Fluorescence Proteins</h3>
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<p align="justify"><b>Figure 5</b>: The independent plasmid is constructed is shown. This plasmid contains the gene encoding the anti-repressor is found downstream of the promoter induced by arabinose, pBAD.</p>
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<p align="justify">as we departed in the idea from the terminator screening plasmids described in the partsregistry.org (REFFF) we had a primary focus on fluorescence proteins as our reporter systems. Further we wanted to have high quality data, with a high resolution. We descided on the in-house expertice on using a continous microfermentor system that can measure two fluorescence proteins continuously (biolector) and a flow cytometer, also capable of measuring at two different wave length. </p>
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<h3>Results Simulation</h3>
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<font size="1.5">
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<p align="center"><img src="https://static.igem.org/mediawiki/2010/3/34/DTU_BB_Repressor3_graph.png" width="570px"  align="center"> </img></p>
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<p align="justify"><b>Figure 7</b>: The graphs illustrated are a simulation of the expected results from <b>Construct 2</b> and <b>Construct 3</b>. The expected results from <b>Construct 2</b> would be a baseline expression of GFP, as the promoter would continually be repressed. With <b>Construct 3</b>, there would be a baseline expression of GFP until the pBAD is induced. At this point, the anti-repressor is expressed and binds to the repressor preventing its activity. This leads to an increase in the expression of GFP as illustrated by the red curve.</p>
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</font>
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<p align="center"><img src="https://static.igem.org/mediawiki/2010/a/a0/DTU_BB_AntiT1.png" width="570px"  align="center"> </img></p>
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<p align="center"><img src="https://static.igem.org/mediawiki/2010/7/7b/DTU_BB_AntiT2.png" width="570px" align="center"> </img></p>
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<font size="1.5">
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<p align="center"><img src="https://static.igem.org/mediawiki/2010/8/84/DTU_BB_AntiT3_graph.png" width="570px"  align="center"> </img></p>
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<p align="justify"><b>Figure 7</b>: The graphs illustrated are a simulation of the expected results from <b>Construct 2</b> and <b>Construct 3</b>. The expected results from <b>Construct 2</b> would be a baseline expression of GFP, as the promoter would continually be repressed. With <b>Construct 3</b>, there would be a baseline expression of GFP until the pBAD is induced. At this point, the anti-repressor is expressed and binds to the repressor preventing its activity. This leads to an increase in the expression of GFP as illustrated by the red curve.</p>
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= Characterizing BBricks as parts of the switch =
 
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'''(Materials and Methods)''' section<br>
 
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Mainly the hard control of the switch is due to a double regulation system build on a both terminator-anti-terminator and repressor anti-repressor regulation. It was out of the scope of this project to construct the entire theoretical developed switch, and characterize the fully constructed switch. Have focused on characterizing the two regulatory systems individually. This was done in order to investigate if the responses were satisfactory to use in a future complete switch construction. (????  By getting the regulatory mechanism of the subparts we further, by modeling, could conclude constraints for successful function of the system and other subparts.
 
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In this section we describe the design of and the experimental setup used to characterize the subparts of the system and our bio-bricks.
 
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== Anti-terminator function ==
 
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(experimental work)
 
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==== selecting subparts ====
 
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Why were these subparts chosen ?<br>
 
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AIM
 
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==== Design and experimental setup ====
 
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'''presentation - Figure of setup and explanation'''
 
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==== Materials and methods ====
 
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HOW ? what plasmids and why, what measurering method and why?
 
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refer to the notebook page with protocols - and actual info from lab.
 
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'''mRNA-stability'''
 
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when introducing non-coding sequences problems will acour  with rna-degredation of RNAP is not attracted to the area, to fast degredation, unwanted steam loops. (Reference to the terminator screening plasmids for BB)
 
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'''Synthetic promoter library (SPL)'''<br>
 
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How does it work, examples, what have it been used to characterize?
 
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how do you construct it? Figures and illustrations to explain.
 
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Figures to explain our use? And example on our specific design primer sequences illustration on the double stranded DNA, with BB - prefix suffix.
 
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==== Results ====
 
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comments to the results and reference to the BB pages with info and results.
 
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== Repressor function ==
 
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(experimental work)
 
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==== selecting supparts ====
 
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Why were these supparts chosen ?<br>
 
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AIM
 
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==== Design and experimental setup ====
 
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'''presentation - Figure of setup and explanation'''
 
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==== Materials and methods ====
 
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HOW ? what plasmids and why, what measurering method and why?
 
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refer to the notebook page with protocols - and actual info from lab.
 
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==== Results ====
 
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comments to the results and reference to the BB pages with info and results.
 

Latest revision as of 17:27, 20 October 2010

Welcome to the DTU iGEM wiki!

Home The Team The Project Parts submitted Modelling Notebook Blog

Introduction

In order to optimize the lab work, we split up the work so that we could have two lab teams working in parallel to design different parts of the switch. We split up the lab work so we had:

  • Team 1: The Repressor - Anti-Repressor Team
  • Team 2: The Terminator - Anti-Terminator Team
The Repressor (Repressor - Anti-Repressor) Team is responsible for assembling the construct illustrated below in Figure 1:

The Anti-Terminator (Terminator - Anti-Terminator) Team is responsible for assembling the construct illustrated in Figure 2:

Repressor Group

The repressor group will be assembling the constructs step-by-step:

Step 1

The construction of a plasmid containing the divergent promoters is the first step, the effect of this will be the uninhibited expression of GFP as illustrated by the green colonies observed in Figure 4.

Figure 3: The initial plasmid constructed is illustrated. The divergent promoters have been inserted into a plasmid and transformed into the electro-competent E.coli cells.

Figure 3: The success of the plasmid construction and transformation is illustrated by the fluorescent green colonies seen on the LB-agar plates.

Step 2

Figure 4: The construction of a plasmid containing the Repressor protein (GogR or GtgR) expressed from the pRM promoter is shown. The continually expressed repressor protein will then inhibit the pR promoter and no GFP will be expressed.

Step 3

Figure 5: The independent plasmid is constructed is shown. This plasmid contains the gene encoding the anti-repressor is found downstream of the promoter induced by arabinose, pBAD.

Results Simulation

Figure 7: The graphs illustrated are a simulation of the expected results from Construct 2 and Construct 3. The expected results from Construct 2 would be a baseline expression of GFP, as the promoter would continually be repressed. With Construct 3, there would be a baseline expression of GFP until the pBAD is induced. At this point, the anti-repressor is expressed and binds to the repressor preventing its activity. This leads to an increase in the expression of GFP as illustrated by the red curve.

Figure 7: The graphs illustrated are a simulation of the expected results from Construct 2 and Construct 3. The expected results from Construct 2 would be a baseline expression of GFP, as the promoter would continually be repressed. With Construct 3, there would be a baseline expression of GFP until the pBAD is induced. At this point, the anti-repressor is expressed and binds to the repressor preventing its activity. This leads to an increase in the expression of GFP as illustrated by the red curve.

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