Team:Bielefeld-Germany/Results/Characterization

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=Characterized BioBricks=
 
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We submitted the following BioBricks:
 
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<groupparts>iGEM010 Bielefeld-Germany</groupparts>
 
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=<partinfo>K238008</partinfo>: ''virA''=
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We wanted to use this part in our project, but could only obtain unexpected/faulty restriction patterns. Finally we chose to sequence the part, hoping to find the cause for the maintained restriction patterns. Unfortunately we could not approve the sequence of <partinfo>BBa_K238008</partinfo> deposited in the parts registry so that we chose to design our own VirA BioBrick. I strongly recommend using our VirA since it has been approved by multiple means, e.g. restriction patterns and sequencing (<partinfo>K389001</partinfo>).
 +
=<partinfo>BBa_K238011</partinfo>: ''vir''-promoter=
 +
We wanted to use this part in our project, but could only obtain unexpected/faulty restriction patterns. Finally we chose to sequence the part, hoping to find the cause for the maintained restriction patterns. Unfortunately we could not approve the sequence of <partinfo>BBa_K238011</partinfo> deposited in the parts registry so that we chose to design our own VirA BioBrick. I strongly recommend using our VirA since it has been approved by multiple means, e.g. restriction patterns and sequencing (<partinfo>K389003</partinfo>).
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===<partinfo>K238008</partinfo>: ''virA''===
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=<partinfo>P1010</partinfo>: ''ccdB''-gene=
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We made a restriction analysis and sequenced parts of this BioBrick. There were problems - more information within a short time.
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The ''ccdB'' gene targets the gyrase of ''Escherichia coli'' and is lethal for all ''E. coli'' strains without the gyrase mutation gyrA462 ([http://openwetware.org/wiki/CcdB Openwetware]). The ''ccdB'' BioBrick is used for the 3A-assembly as a positive selection marker.  
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===<partinfo>BBa_K238011</partinfo>: ''vir''-promoter===
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We made a restriction analysis and sequenced parts of this BioBrick.
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-
 
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===<partinfo>P1010</partinfo>: ''ccdB''-gene===
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The ''ccdB'' gene targets the gyrase of ''Escherichia coli'' and is lethal for all ''E. coli'' strains without the gyrase mutation gyrA462 [1]. The ''ccdB'' BioBrick is used for the 3A-assembly as a positive selection marker.  
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We transformed this BioBrick into ''E. coli'' JM109, DH5α, TOP10, XL1-Blue, EC100D and DB3.1. ''E. coli'' JM109, XL1-Blue and DH5α seem to be ''ccdB'' resistant because there were as much colonies after P1010 transformation as observed with DB3.1. The P1010 works as expected in ''E. coli'' TOP10, EC100D (no colonies after transformation) and DB3.1 (many colonies after transformation).
We transformed this BioBrick into ''E. coli'' JM109, DH5α, TOP10, XL1-Blue, EC100D and DB3.1. ''E. coli'' JM109, XL1-Blue and DH5α seem to be ''ccdB'' resistant because there were as much colonies after P1010 transformation as observed with DB3.1. The P1010 works as expected in ''E. coli'' TOP10, EC100D (no colonies after transformation) and DB3.1 (many colonies after transformation).
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<center>
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<center>Table 1: Results of the transformation of the cell-death gene ''ccdB'', BioBrick <partinfo>P1010</partinfo>, into different strains of ''E. coli''.  
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Table 1: Results of the transformation of the cell-death gene ''ccdB'', BioBrick <partinfo>P1010</partinfo>, into different strains of ''E. coli''.  
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{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
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!style="border-style: solid; border-width: 1px"| Resistant to ''ccdB''?
!style="border-style: solid; border-width: 1px"| Resistant to ''ccdB''?
!style="border-style: solid; border-width: 1px"| Expected result?
!style="border-style: solid; border-width: 1px"| Expected result?
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!style="border-style: solid; border-width: 1px"| Gyrase genotype [2,3]
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!style="border-style: solid; border-width: 1px"| Gyrase genotype <br> ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a Metcalf ''et al.'', 1994]; [http://openwetware.org/wiki/E._coli_genotypes Openwetware])
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|style="border-style: solid; border-width: 1px"| DB3.1
|style="border-style: solid; border-width: 1px"| DB3.1
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It seems that not only the gyrase mutation gyrA462 is causing a ''ccdB'' resistance. Also the gyrase mutation gyrA96 gives ''E. coli'' a ''ccdB'' resistance. This should be kept in mind when assembling BioBricks with the 3A assembly.
It seems that not only the gyrase mutation gyrA462 is causing a ''ccdB'' resistance. Also the gyrase mutation gyrA96 gives ''E. coli'' a ''ccdB'' resistance. This should be kept in mind when assembling BioBricks with the 3A assembly.
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=<partinfo>K389004</partinfo>: Luciferase from pGL4.10[luc2]=
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[[Team:Bielefeld-Germany/Results/Characterization/K389004#mRFP vs. luciferase as reporter gene | For a comparison between mRFP and luciferase as reporter genes click here. ]]
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===<partinfo>K389004</partinfo>: Luciferase from pGL4.10[luc2]===
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Some important parameters determined by the characterization experiments are shown in tab. 2. For more information concerning these experiments click on the corresponding link in tab. 2 or click here:
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<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389004">Detailed information...</a></div></html>
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===<partinfo>K389011</partinfo>: VirA screening device===
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<center>Table 2: Parameters for <partinfo>K389004</partinfo>.  
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[[Image:IGEM-Bielefeld-LD50.jpg|600px|thumb|center|Ratio of surviving colonies of ''E. coli'' EC100D carrying unmutated <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo> plated on PA agar plates with chloramphenicol, ampicillin and different concentrations of kanamycin. Comparison between cells that were induced with acetosyringone with cells that were not induced.]]
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The ratio of surviving colonies ϕS was calculated like
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[[Image:IGEM-Bielefeld-formel-LD50.jpg|100px|center]]
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with the number of colony forming units CFU, the concentration of kanamycin on the considered plate KanX and no kanamycin on the plate Kan0.
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===<partinfo>K389012</partinfo>: VirA reporter system with luciferase===
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coming more soon
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===<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase===
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==== Growth functions and Luciferase expression for <partinfo>K389015</partinfo>====
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To characterize this part we performed several cultivations with different concentrations of [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] as inducer and measured the luminescence emitted by the luciferasereaction with Luciferin ([[#Protocols|Protocol]]). We used ''Escherichia coli'' DB3.1 carrying the pSB1C3::K389015 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stocksolution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that ''E. coli'' carrying the pSB1C3::K389015 plasmid growths nearly linear.
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[[Image:K389015growth.jpg|600px|thumb|center|'''Fig. 1: Growth Curves for ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.''']]
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The specific growth rates µ and doubling times t<sub>d</sub> are calculated with the OD<sub>600</sub> and following formulas:
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[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div>
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[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div>
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<center>
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Table 1: Averaged specific growth rates and doubling times for cultivations of  ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.
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{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
|-
|-
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!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1
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!style="border-style: solid; border-width: 1px"| Experiment
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!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup>
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!style="border-style: solid; border-width: 1px"| Result
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!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h
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|-
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|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Accumulation of luciferase | Behaviour during cultivation]]
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|style="border-style: solid; border-width: 1px"|
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* production is growth dependent
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* degradation in stationary growth phase
|-
|-
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|style="border-style: solid; border-width: 1px"| without plasmid
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|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Kinetic of luciferin conversion | Kinetic of luciferin conversion]]
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|style="border-style: solid; border-width: 1px"| 0.35
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|style="border-style: solid; border-width: 1px"| max. output between 20 - 40 s
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|style="border-style: solid; border-width: 1px"| 1.98
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|-
|-
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|style="border-style: solid; border-width: 1px"| carrying K389015
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|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of detection (LOD)]]
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|style="border-style: solid; border-width: 1px"| 0.31
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|style="border-style: solid; border-width: 1px"| 162 RLU ~ 0.3 % of <partinfo>J23103</partinfo> output
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|style="border-style: solid; border-width: 1px"| 2.24
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|-
|-
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|style="border-style: solid; border-width: 1px"| carrying K389015 with 400 µM acetosyringone
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|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of quantification (LOQ)]]
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|style="border-style: solid; border-width: 1px"| 0.26
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|style="border-style: solid; border-width: 1px"| 306 RLU ~ 0.7 % of <partinfo>J23103</partinfo> output
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|style="border-style: solid; border-width: 1px"| 2.67
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|}
|}
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</center>
</center>
 +
=<partinfo>K389011</partinfo>: VirA screening device=
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Exemplary induction curves with the luminescence normalized to OD<sub>600</sub> are shown in Fig. 2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed [[#Data_Analysis | data analysis]] and [[#Transfer_function | transfer function]] is described below.
 
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[[Image:K389015induction.jpg|600px|thumb|center|'''Fig. 2: Induction curves for ''E. coli'' DB3.1 carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol. The relative luminescence units (RLU) are normalized to OD<sub>600</sub> and plotted against the cultivation time in h''']]
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<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389011">Detailed information...</a></div></html>
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====Transfer function====
 
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The data for the transfer function was measured and analyzed as [[Part:BBa_K389015:Experience#Data Analysis | described below]]. The data was fitted with a dose response function of the form
 
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[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div>
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[[Image:Bielefeld_LD50_Graph2.jpg|600px|thumb|center|Figure 1: Ratio of surviving colonies of ''E. coli'' EC100D carrying unmutated <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo> plated on PA agar plates with chloramphenicol, ampicillin and different concentrations of kanamycin. Comparison between cells that were induced with acetosyringone with cells that were not induced.]]
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with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 3 shows the measured [[Part:BBa_K389015:Experience#Data analysis | ratio ɸ<sub>I</sub>]] between induced (i) and uninduced (u) relative luminescence units (RLU) per OD<sub>600</sub> plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R<sup>2</sup> = 0.98.
 
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=<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase=
 +
Some important parameters determined by the characterization experiments are shown in tab. 3. For more information concerning these experiments click on the corresponding link in tab. 3 or click here:
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[[Image:Bielefeld_Fit_Luc_final.jpg|600px|thumb|center|'''Fig. 3: Transfer function for the part <partinfo>K389015</partinfo> (R<sup>2</sup> = 0.98).''']]
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<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389015">Detailed information...</a></div></html>
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The important data from the transfer function is summarized in table 1:
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<center>Table 3: Parameters for <partinfo>K389015</partinfo>.  
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{|{{Table}}
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<center>
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!Experiment
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Table 2: Data from the transfer function for the part <partinfo>K389015</partinfo>.
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!Characteristic
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!Value
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{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
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|-
|-
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!style="border-style: solid; border-width: 1px"| Parameter
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|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Transfer function | Transfer Function]]
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!style="border-style: solid; border-width: 1px"| Value
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|Maximum induction level
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|2.2 fold
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|style="border-style: solid; border-width: 1px"| Hill coefficient
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|Maximum induction level reached
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|style="border-style: solid; border-width: 1px"| 1.092
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|200 µM acetosyringone
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|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]
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|Hill coefficient
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|style="border-style: solid; border-width: 1px"| 31.6 µM
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|1.09
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|-
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|style="border-style: solid; border-width: 1px"| Top asymptote
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|Switch Point
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|style="border-style: solid; border-width: 1px"| 2.16
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|31.6 µM acetosyringone
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|}
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|-
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|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Growth functions and Luciferase expression for BBa_K389015 | Doubling time / h]]
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</center>
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|without plasmid
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|1.98
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|-
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The fully induced VirA/G signaling system with luciferase read-out has a 2.2 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 32 µM, so this is the concentration at which the device output is 50% of the maximum output.
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|carrying K389015
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|2.24
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====Response time====
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|-
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|carrying K389015 with 400 µM acetosyringone
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The system needs at least one hour to show a measurable reaction to an induction with acetosyringone. In the following illustration the reaction of the system to induction with 200 µM acetosyringone in the exponential growth phase is shown. For a good separation of the induced system from the uninduced system at least two hours are needed.  
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|2.67
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|-
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[[Image:Bielefeld_Induktionsverhalten_luc.jpg|600px|center|thumb|'''Fig. 4: Cultivation of <partinfo>K389015</partinfo> in ''Escherichia coli'' DB3.1. Induction with 200 µM acetosyringone during the exponential growth phase.''']]
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|rowspan="2"|Response time
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|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Response time | exponential phase]]
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====Data Analysis====
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|>1 h
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Because the luciferase accumulation is very different in different cultivations, the uninduced negative control was used as internal standard. To show the behaviour of the VirA/G signaling system when induced, the ratio ɸ<sub>I</sub> between induced (i) and uninduced (u) relative luminescence units (RLU) per OD<sub>600</sub> is calculated:
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|-
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|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data Analysis | begin of cultivation]]
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|max. induction at OD<sub>600</sub> = 1 +/- 0.5
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[[Image:Bielefeld_Quotient_RLU.jpg|200px|center]] <div align="right">(4)</div>
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[[Part:BBa_K389015:Experience#Response time | As seen above]], at least one hour is needed to separate the induced luminescence signal from the uninduced, so ɸ<sub>I</sub> > 1. Within a cultivation ɸ<sub>I</sub> is rising during the first hours and is decreasing after it reached a maximum at OD<sub>600</sub> ~ 1. This is shown in figure 3:
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[[Image:Bielefeld_Luc_fit_ratio_to_OD.jpg|650px|thumb|center|'''Fig. 5A: Typical development of ɸ<sub>I</sub> plotted to OD<sub>600</sub> of a cultivation with <partinfo>K389015</partinfo> in ''E. coli'' DB3.1 with polynomial fit (5th order, R<sup>2</sup> = 0.75, induced with 200 µM acetosyringone). Fig. 5B: Another typical development of ɸ<sub>I</sub> plotted to OD<sub>600</sub> of a cultivation with <partinfo>K389015</partinfo> in ''E. coli'' DB3.1 with polynomial fit (5th order, R<sup>2</sup> = 0.87, induced with 400 µM acetosyringone).''']]
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To measure the ratio of increasing promoter activity by inducing the system ɸ<sub>I</sub> samples for analyzation should be taken at OD<sub>600</sub> = 1 +/- 0.5. The highest ɸ<sub>I</sub> in this range of the cultivation is taken for the calculation of the [[Part:BBa_K389015:Experience#Transfer function | transfer function]].
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====Plasmid conformation analysis====
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A plasmid conformation analysis for the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 6 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]). 
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[[Image:Bielefeld_CGE_K389015.jpg|600px|thumb|center|'''Fig. 6: Chromatogram of the CGE of the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).''']]
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<center>
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Table 3: Data from the CGE of the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).
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{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
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|-
|-
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!style="border-style: solid; border-width: 1px"| Conformation
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|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Plasmid conformation analysis | Conformation analysis]]
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!style="border-style: solid; border-width: 1px"| Ratio / %
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|ratio ccc monomer / %
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|91
|-
|-
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|style="border-style: solid; border-width: 1px"| ccc monomer
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|ratio ccc dimer / %
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|style="border-style: solid; border-width: 1px"| 91
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|3.7
|-
|-
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|style="border-style: solid; border-width: 1px"| ccc dimer
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|ratio oc forms / %
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|style="border-style: solid; border-width: 1px"| 3.7
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|5.3
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|-
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|style="border-style: solid; border-width: 1px"| oc
 
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|style="border-style: solid; border-width: 1px"| 5.3
 
|}
|}
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</center>
</center>
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===<partinfo>K389016</partinfo>: VirA/G reporter device with mRFP===
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=<partinfo>K389016</partinfo>: VirA/G reporter device with mRFP=
Protocols for [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Cultivation] and [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Measuring_of_mRFP Measurement]
Protocols for [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Cultivation] and [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Measuring_of_mRFP Measurement]
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==== Growth functions and mRFP expression for <partinfo>K389016</partinfo>====
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Some important parameters determined by the characterization experiments are shown in tab. 4. For more information concerning these experiments click on the corresponding link in tab. 4 or click here:
-
To characterize this part we performed several cultivations with different concentrations of acetosyringone as inducer and measured the fluorescence emitted by mRFP ([[#Protocols|Protocol]]). We used Escherichia Coli DB3.1 carrying the pSB1C3::K389016 plasmid. Even without inducer the bacteria, carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growthrates (we used a stocksolution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that E.coli carrying the pSB1C3::K389016 plasmid growths nearly linear.  
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<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016">Detailed information...</a></div></html>
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[[Image:K389016growth.jpg|600px|thumb|center|'''Fig. 1: Growth Curves for ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.''']]
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<center>Table 4: Parameters for <partinfo>K389016</partinfo>.  
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{|{{Table}}
-
 
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!Experiment
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The specific growth rates µ and doubling times td are calculated with the OD600 and following formulas:
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!Characteristic
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!Value
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[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div>
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-
 
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[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div>
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-
 
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<center>
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-
Table 1: Averaged specific growth rates and doubling times for cultivations of  ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.
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{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
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|-
|-
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!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1
+
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Transfer function of BBa_K389016 | Transfer Function]]
-
!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup>
+
|Maximum induction level
-
!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h
+
|2.6 fold
|-
|-
-
|style="border-style: solid; border-width: 1px"| without plasmid
+
|Maximum induction level reached
-
|style="border-style: solid; border-width: 1px"| 0.35
+
|150 µM acetosyringone
-
|style="border-style: solid; border-width: 1px"| 1.98
+
|-
|-
-
|style="border-style: solid; border-width: 1px"| carrying K389016
+
|Hill coefficient
-
|style="border-style: solid; border-width: 1px"| 0.27
+
|1.67
-
|style="border-style: solid; border-width: 1px"| 2.57
+
|-
|-
-
|style="border-style: solid; border-width: 1px"| carrying K389016 with 150 µM acetosyringone
+
|Switch Point
-
|style="border-style: solid; border-width: 1px"| 0.25
+
|26.5 µM acetosyringone
-
|style="border-style: solid; border-width: 1px"| 2.77
+
|-
|-
-
|style="border-style: solid; border-width: 1px"| carrying K389016 with 1000 µM acetosyringone
+
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Growth functions and mRFP expression for BBa_K389016 | Doubling time / h]]
-
|style="border-style: solid; border-width: 1px"| 0.23
+
|without plasmid
-
|style="border-style: solid; border-width: 1px"| 3.01
+
|1.98
-
|}
+
-
 
+
-
</center>
+
-
 
+
-
 
+
-
Exemplary induction curves with the fluorescence normalized to OD600 are shown in Fig.2. We observed a basal Transcription, but the Induction with acetosyringone is undoubtedly. The detailed [[#Data analysis for BBa_K389016 | data analysis]] and [[#Transfer function of BBa_K389016 | transfer function]] is described below.
+
-
 
+
-
 
+
-
[[Image:K389016induction.jpg|600px|thumb|center|'''Fig. 2: Induction Curves for ''E. coli'' DB3.1 carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol. The relative fluorescence units are normalized to OD600 and plotted against the ime in h''']]
+
-
 
+
-
 
+
-
====Transfer function of <partinfo>K389016</partinfo>====
+
-
The data for the transfer function was measured and analyzed as [[Part:BBa_K389016:Experience#Data analysis for BBa_K389016 | described below]]. The data was fitted with a dose response function of the form
+
-
 
+
-
 
+
-
[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div>
+
-
 
+
-
 
+
-
with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 1 shows the measured normalized specific production rates q<sub>P,n</sub> (eq. 8) plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R<sup>2</sup> = 0.99.
+
-
 
+
-
 
+
-
[[Image:Bielefeld_Final_RFP_fit.jpg|600px|thumb|center|'''Fig. 1: Transfer function for the part <partinfo>K389016</partinfo> (R<sup>2</sup> = 0.99).''']]
+
-
 
+
-
 
+
-
The important data from the transfer function is summarized in table 1:
+
-
 
+
-
<center>
+
-
Table 1: Data from the transfer function for the part <partinfo>K389016</partinfo>.
+
-
 
+
-
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
+
|-
|-
-
!style="border-style: solid; border-width: 1px"| Parameter
+
|carrying K389016
-
!style="border-style: solid; border-width: 1px"| Value
+
|2.57
|-
|-
-
|style="border-style: solid; border-width: 1px"| Hill coefficient
+
|carrying K389016 with 150 µM acetosyringone
-
|style="border-style: solid; border-width: 1px"| 1.673
+
|2.77
|-
|-
-
|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]
+
|carrying K389016 with 1000 µM acetosyringone
-
|style="border-style: solid; border-width: 1px"| 26.5 µM
+
|3.01
|-
|-
-
|style="border-style: solid; border-width: 1px"| Top asymptote
+
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Plasmid conformation analysis | Conformation analysis]]
-
|style="border-style: solid; border-width: 1px"| 2.62
+
|ratio ccc monomer / %
 +
|91.2
 +
|-
 +
|ratio ccc dimer / %
 +
|3.2
 +
|-
 +
|ratio oc forms / %
 +
|5.6
 +
|-
 +
|rowspan="5"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Different possible inducers | Inducers]]
 +
|Induction by
 +
|Acetosyringone
 +
|-
 +
|rowspan="4"|No Induction by
 +
|Capsaicin
 +
|-
 +
|Dopamine
 +
|-
 +
|Homovanillic acid
 +
|-
 +
|3-Methoxytyramine
|}
|}
-
 
</center>
</center>
-
So the fully induced VirA/G signaling system has a 2.6 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 25 µM, so this is the concentration at which the device output is 50% of the maximum output.
+
=<partinfo>K389052</partinfo>: Tightly regulated ''lac'' operon with mRFP readout=
-
 
+
This construct was plated for plasmid isolation in a ''lacI<sup>q</sup>'' negative ''E. coli'' strain after assembly - and we have never seen such red plates when working with constructs with mRFP downstream of a promoter. This ''lac'' operon definitely shows a very high basal transcription, so it is not tightly repressed. It seems that the ''lacI'' repressor <partinfo>BBa_C0012</partinfo> is not suitable for this purpose due to its LVA degradation tag or it does not work properly. Another indicator for this assumption is the experience page of <partinfo>C0012</partinfo>.
-
====Data analysis for <partinfo>K389016</partinfo>====
+
-
The data analysis is made in three steps. First step is the processing of the fluorescence raw data gained by the fluorescence plate reader for every sample:  
+
-
 
+
-
 
+
-
[[Image:Bielefeld_RFU_corrected.jpg|250px|center]] <div align="right">(4)</div>
+
-
 
+
-
 
+
-
In the second step the RFU<sub>corrected</sub> of every sample is plotted against the cultivation time it was drawn. The data is fitted by an exponential fit of the following style:
+
-
 
+
-
 
+
-
[[Image:Bielefeld_Expfit.jpg|100px|center]] <div align="right">(5)</div>
+
-
 
+
-
 
+
-
The accumulation of mRFP in the cells is always exponential. A typical fitted product accumulation curve is shown below:
+
-
 
+
-
[[Image:Bielefeld_ExpFit_auf_RFU.jpg|500px|thumb|center|'''Fig. 2: Exponential fit on the measured RFU plotted against cultivation time of a cultivation of <partinfo>K389016</partinfo> in ''Escherichia coli'' DB3.1 in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol and 150 µM acetosyringone. ''']]
+
-
 
+
-
The product accumulation in a cultivation can be described as:
+
-
 
+
-
 
+
-
[[Image:Bielefeld_Produktbildung.jpg|100px|center]] <div align="right">(6)</div>
+
-
 
+
-
 
+
-
with the amount of product P, the cell count X and the specific production rate q<sub>P</sub>.
+
-
 
+
-
RFU is commensurate to the concentration of mRFP (P) and the OD<sub>600</sub> is commensurate to the cell count (X) ([[Part:BBa_F2620:Experience/Endy/Data_analysis | Canton and Labno, 2004]]):
+
-
 
+
-
 
+
-
[[Image:Bielefeld_RFUpropP.jpg|100px|center]] <div align="right">(7)</div>
+
-
 
+
-
[[Image:Bielefeld_ODpropX.jpg|80px|center]] <div align="right">(8)</div>
+
-
 
+
-
 
+
-
With these assumptions it is possible to calculate the specific production rate of mRFP q<sub>P</sub> in the third step: the specific production rate for every sample of a cultivation is calculated by the derivation of the exponential fit line which describes the accumulation of product in the culture (dRFU/dt) and the measured OD<sub>600</sub> data:
+
-
 
+
-
 
+
-
[[Image:Bielefeld_specific_production_rate.jpg|150px|center]] <div align="right">(9)</div>
+
-
 
+
-
 
+
-
The specific production rates q<sub>P</sub> of all samples of all cultivations made with a specific inductor concentration c are averaged and normalized against the specific production rate of the uninduced system q<sub>P,0</sub>:
+
-
 
+
-
 
+
-
[[Image:Bielefeld_QPN.jpg|100px|center]] <div align="right">(10)</div>
+
-
 
+
-
 
+
-
This normalized specific production rate we calculated is commensurate to relative promotor units (RPU) which is commensurate to PoPS (polymerase per seconds) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]; [http://partsregistry.org/Part:BBa_J23101:Experience Pasotti ''et al.'', 2009]):
+
-
 
+
-
 
+
-
[[Image:Bielefeld_QPNRPUPoPS.jpg|150px|center]] <div align="right">(11)</div>
+
-
 
+
-
 
+
-
==== Control of BioBrick quality by capillar gel electrophoresis (GCE) ====
+
-
 
+
-
The representative tested BioBrick shows ccc-type of over 91 %, meaning pDNA quality is optimal.
+
-
 
+
-
[[Image:Bielefeld_CGE_K289016_3.jpg|500px|center|thumb|Standard CGE Analysis
+
-
Sample ID: K389016
+
-
Lauf: 10/22/2010 12:55:04 PM
+
-
Report: 10/25/2010 10:03:46 AM
+
-
PlasmidFactory GmbH & Co KG
+
-
]]
+
-
 
+
-
===<partinfo>K389052</partinfo>: tightly regulated ''lac'' operon with mRFP readout===
+
-
Fails...more soon.
+
-
 
+
-
===<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amlified Vir-test system===
+
-
 
+
-
By self designed PCR-Primer we excluded terminal GFP and the initial promoter pBAD/araC, for replacing our own VirB promotor and reporter gene luc (luciferase). Primers were designed for sensitivity tuner [http://partsregistry.org/Part:BBa_I746370 I746370], [http://partsregistry.org/Part:BBa_I746380 I746380]  and [http://partsregistry.org/Part:BBa_I746390 I746390] so that standard assembly would be possible. Assembling of PCR-products took place by Silver Assembly.
+
-
 
+
-
'''Accomplishment'''
+
-
 
+
-
'''PCR-Primer Design'''
+
-
 
+
-
Primer forward activator phage P2:
+
-
 
+
-
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GTT TCA TTG TCC TTT ATG CC-3`
+
-
 
+
-
Primer forward activator phage PSP3:
+
-
 
+
-
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GAT GCA CTG CCC GTT ATG- 3`
+
-
 
+
-
Primer forward activator phage phi R73:
+
-
 
+
-
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GCG CTG CCC TTT CTG-3`
+
-
 
+
-
Primer backward Promotor PF from phage P2:
+
-
 
+
-
5`-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TTC TCC TCT TTC TCT AGT AAG TGG- 3`
+
-
 
+
-
 
+
-
'''Characterization tests'''
+
-
 
+
-
Cultivation was done by induction with Acetosyringone at 50 µM. Controls were not induced Sensitivity Tuner devices as well as induced and not induced nativ system ([http://partsregistry.org/Part:BBa_K389015 K389015]; without tuning elements). Induction was done upon inoculation.  Measuring point for amplification factor calculation was OD 1.0.
+
-
 
+
-
 
+
-
'''Results'''
+
-
 
+
-
Three sensitivity tuned Vir-Gen sensing systems were obtained: [http://partsregistry.org/Part:BBa_K389421 K389421], [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] distinguishing by the amplification level of luc transcription.
+
-
 
+
-
[[Image:Bielefeld amp factor.png|400px|thumb|center| Figure 1: Amplification factor of induced, 50 µM Acetosyringone (red) and not induced (green) modified Sensitivity Tuner K389421, K389422 and K389423, Standard deviation shown.]]
+
-
 
+
-
The amplification factor was received by apply [http://partsregistry.org/Part:BBa_K389015 K389015] as reference. Amplification calculation was done by normalizing relative luminescence units emitted from luciferase per OD.
+
-
Output-signal amplification is in the induced contructs (red) [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] 100 and respectively 200 fold higher than in not induced controls (green). An exception is K389422 were induced and not indiced system revealed analog results. Corresponding to data of iGEM Team, Cambridge 2009, K389423 (originated from [http://partsregistry.org/Part:BBa_I746390 I746390]) shows the highest amplification rate of all tested Sensitivity Tuners. Our results indicate to higher amplification rate of [http://partsregistry.org/Part:BBa_K389421 K389421] than [http://partsregistry.org/Part:BBa_K389422 K389422] of 100 fold under induced conditions. The controls also show high basal transcription rates.
+
-
 
+
-
Because there is small difference in induced and not induced system visible and basal transcription rates are high, we assume that the sensitivity tuning constructs are not well applicable for luciferase measurements.
+
-
 
+
-
For further theory click [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Read_out_system Read out system]
+
-
 
+
-
=Sequenced BioBricks=
+
-
 
+
-
 
+
-
===Own BioBricks===
+
-
 
+
-
The sequencing of the following of our own BioBricks was succesful and lead to the expected results:
+
-
 
+
-
* <partinfo>K389001</partinfo> (not fully completed)
+
-
 
+
-
* <partinfo>K389002</partinfo>
+
-
 
+
-
* <partinfo>K389003</partinfo>
+
-
 
+
-
* <partinfo>K389004</partinfo>
+
-
* <partinfo>K389005</partinfo>
+
=<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amplified Vir-test system=
-
* <partinfo>K389010</partinfo> (not fully completed)
 
-
* <partinfo>K389011</partinfo> (not fully completed)
+
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/Sen_Tuner">Detailed information...</a></div></html>
-
* <partinfo>K389012</partinfo> (not fully completed)
 
-
* <partinfo>K389013</partinfo> (not fully completed)
+
[[Image:ST Tuner.png|600px|thumb|center| '''Figure 2: Amplification factor of induced, 50 µM Acetosyringone (red) and not induced (green) modified Sensitivity Tuner <partinfo>K389421</partinfo>, <partinfo>K389422</partinfo> and <partinfo>K389423</partinfo>, Standard deviation shown.''']]
-
* <partinfo>K389015</partinfo> (not fully completed)
 
-
* <partinfo>K389016</partinfo> (not fully completed)
+
[[Image:Bielefeld_luc.jpg|600px|thumb|center| '''Figure 3: Vizualitation of induced (from left to right) <partinfo>K389421</partinfo>, <partinfo>K389422</partinfo> and <partinfo>K389423</partinfo> sensitivity tuner amplified vir-system.''']]
-
* <partinfo>K389050</partinfo>
 
 +
=Mutated <partinfo>K389001</partinfo> (exemplary): Results of the directed mutagenesis=
-
===Other BioBricks===
 
-
* <partinfo>K238008</partinfo> sequencing gave negative results - infos in registry are not correct!
+
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/Exemplary_results_DM">Detailed information...</a></div></html>
-
* <partinfo>K238011</partinfo> sequencing gave negative results - infos in registry are not correct!
 
 +
[[Image:Bielefeld_results_screening.jpg|600px|thumb|center| '''Figure 4: Luciferase production rates of the exemplary clone “21” are shown under conditions of induction with acetosyringone (21-A), capsaicin (21-C) and in uninduced state (21-U). The right bar indicates the production rate of the native system without acetosyringone (212-U), all the values in the figure had been normalized to.''']]
=References=
=References=
-
[1] http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).
 
-
[2] http://openwetware.org/wiki/E._coli_genotypes, E. coli genotypes (seen on 10.10.10).
 
-
[3] Metcalf, W.W. ''et al.'' (1994) Gene 138, 1.
 
-
[4] Stadler J, Lemmens R, Nyhammar T 2004, ''Plasmid DNA purification'', The J. of Gene Medicine,Vol.6, pp.54–S66
 
-
[5] Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.
+
*Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.
 +
*http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).
-
Canton B  and Labno A (2004) [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data processing of Part BBa_F2620].  
+
*http://openwetware.org/wiki/E._coli_genotypes, E. coli genotypes (seen on 10.10.10).
-
Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], ''J Theor Biol'' 257(3):419-429.  
+
*Metcalf, WW ''et al.'' (1994) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a ''Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kλ origin plasmids at different copy numbers''], Gene 138(1):1-7.
-
Pasotti L, Zucca S, Del Fabbro E (2009) Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118, http://partsregistry.org/Part:BBa_J23101:Experience.
+
*Stadler J, Lemmens R, Nyhammar T 2004, ''Plasmid DNA purification'', The J. of Gene Medicine,Vol.6, pp.54–S66

Latest revision as of 01:07, 28 October 2010

http://igem-bielefeld.de/img/banner_lab.png


Contents

<partinfo>K238008</partinfo>: virA

We wanted to use this part in our project, but could only obtain unexpected/faulty restriction patterns. Finally we chose to sequence the part, hoping to find the cause for the maintained restriction patterns. Unfortunately we could not approve the sequence of <partinfo>BBa_K238008</partinfo> deposited in the parts registry so that we chose to design our own VirA BioBrick. I strongly recommend using our VirA since it has been approved by multiple means, e.g. restriction patterns and sequencing (<partinfo>K389001</partinfo>).

<partinfo>BBa_K238011</partinfo>: vir-promoter

We wanted to use this part in our project, but could only obtain unexpected/faulty restriction patterns. Finally we chose to sequence the part, hoping to find the cause for the maintained restriction patterns. Unfortunately we could not approve the sequence of <partinfo>BBa_K238011</partinfo> deposited in the parts registry so that we chose to design our own VirA BioBrick. I strongly recommend using our VirA since it has been approved by multiple means, e.g. restriction patterns and sequencing (<partinfo>K389003</partinfo>).

<partinfo>P1010</partinfo>: ccdB-gene

The ccdB gene targets the gyrase of Escherichia coli and is lethal for all E. coli strains without the gyrase mutation gyrA462 ([http://openwetware.org/wiki/CcdB Openwetware]). The ccdB BioBrick is used for the 3A-assembly as a positive selection marker. We transformed this BioBrick into E. coli JM109, DH5α, TOP10, XL1-Blue, EC100D and DB3.1. E. coli JM109, XL1-Blue and DH5α seem to be ccdB resistant because there were as much colonies after P1010 transformation as observed with DB3.1. The P1010 works as expected in E. coli TOP10, EC100D (no colonies after transformation) and DB3.1 (many colonies after transformation).


Table 1: Results of the transformation of the cell-death gene ccdB, BioBrick <partinfo>P1010</partinfo>, into different strains of E. coli.
E. coli strain Resistant to ccdB? Expected result? Gyrase genotype
([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a Metcalf et al., 1994]; [http://openwetware.org/wiki/E._coli_genotypes Openwetware])
DB3.1 yes yes gyrA462
DH5α yes no gyrA96
EC100D no yes WT
JM109 yes no gyrA96
TOP10 no yes WT
XL1-Blue yes no gyrA96


It seems that not only the gyrase mutation gyrA462 is causing a ccdB resistance. Also the gyrase mutation gyrA96 gives E. coli a ccdB resistance. This should be kept in mind when assembling BioBricks with the 3A assembly.

<partinfo>K389004</partinfo>: Luciferase from pGL4.10[luc2]

For a comparison between mRFP and luciferase as reporter genes click here.

Some important parameters determined by the characterization experiments are shown in tab. 2. For more information concerning these experiments click on the corresponding link in tab. 2 or click here:

Detailed information...


Table 2: Parameters for <partinfo>K389004</partinfo>.
Experiment Result
Behaviour during cultivation
  • production is growth dependent
  • degradation in stationary growth phase
Kinetic of luciferin conversion max. output between 20 - 40 s
Limit of detection (LOD) 162 RLU ~ 0.3 % of <partinfo>J23103</partinfo> output
Limit of quantification (LOQ) 306 RLU ~ 0.7 % of <partinfo>J23103</partinfo> output

<partinfo>K389011</partinfo>: VirA screening device

Detailed information...


Figure 1: Ratio of surviving colonies of E. coli EC100D carrying unmutated <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo> plated on PA agar plates with chloramphenicol, ampicillin and different concentrations of kanamycin. Comparison between cells that were induced with acetosyringone with cells that were not induced.


<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase

Some important parameters determined by the characterization experiments are shown in tab. 3. For more information concerning these experiments click on the corresponding link in tab. 3 or click here:

Detailed information...


Table 3: Parameters for <partinfo>K389015</partinfo>.
Experiment Characteristic Value
Transfer Function Maximum induction level 2.2 fold
Maximum induction level reached 200 µM acetosyringone
Hill coefficient 1.09
Switch Point 31.6 µM acetosyringone
Doubling time / h without plasmid 1.98
carrying K389015 2.24
carrying K389015 with 400 µM acetosyringone 2.67
Response time Induction: exponential phase >1 h
Induction: begin of cultivation max. induction at OD600 = 1 +/- 0.5
Conformation analysis ratio ccc monomer / % 91
ratio ccc dimer / % 3.7
ratio oc forms / % 5.3

<partinfo>K389016</partinfo>: VirA/G reporter device with mRFP

Protocols for Cultivation and Measurement

Some important parameters determined by the characterization experiments are shown in tab. 4. For more information concerning these experiments click on the corresponding link in tab. 4 or click here:

Detailed information...


Table 4: Parameters for <partinfo>K389016</partinfo>.
Experiment Characteristic Value
Transfer Function Maximum induction level 2.6 fold
Maximum induction level reached 150 µM acetosyringone
Hill coefficient 1.67
Switch Point 26.5 µM acetosyringone
Doubling time / h without plasmid 1.98
carrying K389016 2.57
carrying K389016 with 150 µM acetosyringone 2.77
carrying K389016 with 1000 µM acetosyringone 3.01
Conformation analysis ratio ccc monomer / % 91.2
ratio ccc dimer / % 3.2
ratio oc forms / % 5.6
Inducers Induction by Acetosyringone
No Induction by Capsaicin
Dopamine
Homovanillic acid
3-Methoxytyramine

<partinfo>K389052</partinfo>: Tightly regulated lac operon with mRFP readout

This construct was plated for plasmid isolation in a lacIq negative E. coli strain after assembly - and we have never seen such red plates when working with constructs with mRFP downstream of a promoter. This lac operon definitely shows a very high basal transcription, so it is not tightly repressed. It seems that the lacI repressor <partinfo>BBa_C0012</partinfo> is not suitable for this purpose due to its LVA degradation tag or it does not work properly. Another indicator for this assumption is the experience page of <partinfo>C0012</partinfo>.

<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amplified Vir-test system

Detailed information...


Figure 2: Amplification factor of induced, 50 µM Acetosyringone (red) and not induced (green) modified Sensitivity Tuner <partinfo>K389421</partinfo>, <partinfo>K389422</partinfo> and <partinfo>K389423</partinfo>, Standard deviation shown.


Figure 3: Vizualitation of induced (from left to right) <partinfo>K389421</partinfo>, <partinfo>K389422</partinfo> and <partinfo>K389423</partinfo> sensitivity tuner amplified vir-system.


Mutated <partinfo>K389001</partinfo> (exemplary): Results of the directed mutagenesis

Detailed information...


Figure 4: Luciferase production rates of the exemplary clone “21” are shown under conditions of induction with acetosyringone (21-A), capsaicin (21-C) and in uninduced state (21-U). The right bar indicates the production rate of the native system without acetosyringone (212-U), all the values in the figure had been normalized to.

References

  • Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.
  • http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).
  • http://openwetware.org/wiki/E._coli_genotypes, E. coli genotypes (seen on 10.10.10).
  • Metcalf, WW et al. (1994) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kλ origin plasmids at different copy numbers], Gene 138(1):1-7.
  • Stadler J, Lemmens R, Nyhammar T 2004, Plasmid DNA purification, The J. of Gene Medicine,Vol.6, pp.54–S66
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