Team:Bielefeld-Germany/Results/Characterization

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(Tested BioBricks)
(K389421, K389422, K389423: Sensitivity Tuner amplified Vir-test system)
 
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     <li><a href="/Team:Bielefeld-Germany/Results">Results</a></li>
     <li><a href="/Team:Bielefeld-Germany/Results">Results</a></li>
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     <li><a href="/Team:Bielefeld-Germany/Results/Tests">Tested</a></li>
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     <li><a href="/Team:Bielefeld-Germany/Results/Characterization">Characterization</a></li>
     <li><a href="/Team:Bielefeld-Germany/Results/Used">Used</a></li>
     <li><a href="/Team:Bielefeld-Germany/Results/Used">Used</a></li>
     <li><a href="/Team:Bielefeld-Germany/Results/Submitted">Submitted</a></li>
     <li><a href="/Team:Bielefeld-Germany/Results/Submitted">Submitted</a></li>
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     <li><a href="/Team:Bielefeld-Germany/Results/Unfinished">Unfinished</a></li>
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     <li><a href="/Team:Bielefeld-Germany/Results/Sequencing">Sequencing</a></li>
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-
=Tested BioBricks=
 
-
We tested the following BioBricks:
 
-
===<partinfo>K238008</partinfo>: ''virA''===
 
-
We made a restriction analysis and sequenced parts of this BioBrick. There were problems - more information within a short time.
 
 +
=<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===
+
=<partinfo>BBa_K238011</partinfo>: ''vir''-promoter=
-
We made a restriction analysis and sequenced parts of this BioBrick.
+
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=
-
===<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.  
-
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.  
+
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).
-
<center>
+
<center>Table 1: Results of the transformation of the cell-death gene ''ccdB'', BioBrick <partinfo>P1010</partinfo>, into different strains of ''E. coli''.  
-
Table 1: Results of the transformation of the cell-death gene ''ccdB'', BioBrick <partinfo>P1010</partinfo>, into different strains of ''E. coli''.  
+
{|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"| 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?
-
!style="border-style: solid; border-width: 1px"| Gyrase genotype [2,3]
+
!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])
|-
|-
|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.
 +
=<partinfo>K389004</partinfo>: Luciferase from pGL4.10[luc2]=
 +
[[Team:Bielefeld-Germany/Results/Characterization/K389004#mRFP vs. luciferase as reporter gene | For a comparison between mRFP and luciferase as reporter genes click here. ]]
-
===<partinfo>K389011</partinfo>: VirA screening device===
+
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:
-
[[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.]]
+
-
The ratio of surviving colonies ϕS was calculated like
+
<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>
-
[[Image:IGEM-Bielefeld-formel-LD50.jpg|100px|center]]
 
-
with the number of colony forming units CFU, the concentration of kanamycin on the considered plate KanX and no kanamycin on the plate Kan0.
+
<center>Table 2: Parameters for <partinfo>K389004</partinfo>.  
 +
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
 +
|-
 +
!style="border-style: solid; border-width: 1px"| Experiment
 +
!style="border-style: solid; border-width: 1px"| Result
 +
|-
 +
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Accumulation of luciferase | Behaviour during cultivation]]
 +
|style="border-style: solid; border-width: 1px"|
 +
* production is growth dependent
 +
* degradation in stationary growth phase
 +
|-
 +
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Kinetic of luciferin conversion | Kinetic of luciferin conversion]]
 +
|style="border-style: solid; border-width: 1px"| max. output between 20 - 40 s
 +
|-
 +
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of detection (LOD)]]
 +
|style="border-style: solid; border-width: 1px"| 162 RLU ~ 0.3 % of <partinfo>J23103</partinfo> output
 +
|-
 +
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of quantification (LOQ)]]
 +
|style="border-style: solid; border-width: 1px"| 306 RLU ~ 0.7 % of <partinfo>J23103</partinfo> output
 +
|}
 +
</center>
-
===<partinfo>K389012</partinfo>: VirA reporter system with luciferase===
+
=<partinfo>K389011</partinfo>: VirA screening device=
-
coming more soon
+
-
===<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase===
 
-
Control of BioBrick quality by capillar gel elektrophoresis (GCE)
+
<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>
-
[[Image:Bielelefeld_CGE_K389015_1.jpg|500px|center|thumb|STANDARD CGE ANALYSIS
 
-
SAMPLE ID: K389015 
 
-
Run: 10/22/2010 8:00:07 AM
 
-
REPORT: 10/25/2010 10:01:30 AM
 
-
PLASMIDFACTORY GMBH & CO KG
 
-
]]
 
-
===<partinfo>K389016</partinfo>: VirA/G reporter device with mRFP===
 
-
Control of BioBrick quality by capillar gel elektrophoresis (GCE)
+
[[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.]]
-
[[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/K389422/K389423</partinfo>: Sensitivity Tuner amlified Vir-test system===
+
=<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:
-
By PCR-Primer designed 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 I746370, I746380  and I746390 so that standard assembly would be possible. Assembling of PCR-products took place by Silver Assembly.
+
<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>
-
=Sequenced BioBricks=
 
 +
<center>Table 3: Parameters for <partinfo>K389015</partinfo>.
 +
{|{{Table}}
 +
!Experiment
 +
!Characteristic
 +
!Value
 +
|-
 +
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Transfer function | 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
 +
|-
 +
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Growth functions and Luciferase expression for BBa_K389015 | Doubling time / h]]
 +
|without plasmid
 +
|1.98
 +
|-
 +
|carrying K389015
 +
|2.24
 +
|-
 +
|carrying K389015 with 400 µM acetosyringone
 +
|2.67
 +
|-
 +
|rowspan="2"|Response time
 +
|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Response time | exponential phase]]
 +
|>1 h
 +
|-
 +
|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data Analysis | begin of cultivation]]
 +
|max. induction at OD<sub>600</sub> = 1 +/- 0.5
 +
|-
 +
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Plasmid conformation analysis | Conformation analysis]]
 +
|ratio ccc monomer / %
 +
|91
 +
|-
 +
|ratio ccc dimer / %
 +
|3.7
 +
|-
 +
|ratio oc forms / %
 +
|5.3
 +
|-
 +
|}
 +
</center>
-
===Own BioBricks===
+
=<partinfo>K389016</partinfo>: VirA/G reporter device with mRFP=
-
The sequencing of the following of our own BioBricks was succesful and lead to the expected results:  
+
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]
-
* <partinfo>K389001</partinfo> (not fully completed)
+
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:
-
* <partinfo>K389002</partinfo>
+
<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>
-
* <partinfo>K389003</partinfo>
 
-
* <partinfo>K389004</partinfo>
+
<center>Table 4: Parameters for <partinfo>K389016</partinfo>.
 +
{|{{Table}}
 +
!Experiment
 +
!Characteristic
 +
!Value
 +
|-
 +
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Transfer function of BBa_K389016 | 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
 +
|-
 +
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Growth functions and mRFP expression for BBa_K389016 | 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
 +
|-
 +
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Plasmid conformation analysis | Conformation analysis]]
 +
|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>
-
* <partinfo>K389005</partinfo>
+
=<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>.
-
* <partinfo>K389010</partinfo> (not fully completed)
+
=<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amplified Vir-test system=
-
* <partinfo>K389011</partinfo> (not fully completed)
 
-
* <partinfo>K389012</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>K389013</partinfo> (not fully completed)
 
-
* <partinfo>K389015</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>K389016</partinfo> (not fully completed)
 
-
* <partinfo>K389050</partinfo>
+
[[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.''']]
-
===Other BioBricks===
+
=Mutated <partinfo>K389001</partinfo> (exemplary): Results of the directed mutagenesis=
-
* <partinfo>K238008</partinfo> sequencing gave negative results - infos in registry are not correct!
 
-
* <partinfo>K238011</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>
 +
 +
[[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.
+
 
 +
 
 +
*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

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:

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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

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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

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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