Team:Bielefeld-Germany/Results/Characterization

From 2010.igem.org

(Difference between revisions)
(K389015: VirA/G reporter device with luciferase)
(K389421, K389422, K389423: Sensitivity Tuner amplified Vir-test system)
 
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=<partinfo>BBa_K238011</partinfo>: ''vir''-promoter=
=<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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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>P1010</partinfo>: ''ccdB''-gene=
=<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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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).
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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!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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[[Team:Bielefeld-Germany/Results/Characterization/K389004#mRFP vs. luciferase as reporter gene | For a comparison between mRFP and luciferase as reporter genes click here. ]]
[[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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Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. 2 or click here:  
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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:  
<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>
<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=
=<partinfo>K389011</partinfo>: VirA screening device=
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[[Image:Bielefeld_LD50_Graph2.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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<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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[[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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=<partinfo>K389012</partinfo>: VirA reporter system with luciferase=
 
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coming more soon
 
=<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase=
=<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase=
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Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. X or click here:  
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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:  
<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>
<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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<center>Table X: Parameters for <partinfo>K389015</partinfo>.  
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<center>Table 3: Parameters for <partinfo>K389015</partinfo>.  
{|{{Table}}
{|{{Table}}
!Experiment
!Experiment
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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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Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. X or click here:  
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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:  
<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>
<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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<center>
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Table X: Parameters for <partinfo>K389016</partinfo>.  
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<center>Table 4: Parameters for <partinfo>K389016</partinfo>.  
{|{{Table}}
{|{{Table}}
!Experiment
!Experiment
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</center>
</center>
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=<partinfo>K389052</partinfo>: Tightly regulated ''lac'' operon with mRFP readout=
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=<partinfo>K389052</partinfo>: tightly regulated ''lac'' operon with mRFP readout=
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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>.
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>.
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=<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amlified Vir-test system=
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=<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amplified Vir-test system=
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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.
 
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'''Accomplishment'''
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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/Sen_Tuner">Detailed information...</a></div></html>
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'''PCR-Primer Design'''
 
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Primer forward activator phage P2:
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[[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.''']]
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5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GTT TCA TTG TCC TTT ATG CC-3`
 
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Primer forward activator phage PSP3:
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[[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.''']]
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5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GAT GCA CTG CCC GTT ATG- 3`
 
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Primer forward activator phage phi R73:
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=Mutated <partinfo>K389001</partinfo> (exemplary): Results of the directed mutagenesis=
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5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GCG CTG CCC TTT CTG-3`
 
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Primer backward Promotor PF from phage P2:
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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/Exemplary_results_DM">Detailed information...</a></div></html>
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5`-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TTC TCC TCT TTC TCT AGT AAG TGG- 3`
 
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[[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.''']]
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'''Characterization tests'''
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=References=
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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.
 
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'''Results'''
 
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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.
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*Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.
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[[Image:ST Tuner.png|600px|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.''']]
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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.
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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.
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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.
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For further theory click [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Read_out_system Read out system]
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=References=
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[1] http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).
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[2] http://openwetware.org/wiki/E._coli_genotypes, E. coli genotypes (seen on 10.10.10).
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*http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).
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[3] Metcalf, W.W. ''et al.'' (1994) Gene 138, 1.
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*http://openwetware.org/wiki/E._coli_genotypes, E. coli genotypes (seen on 10.10.10).
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[4] Stadler J, Lemmens R, Nyhammar T 2004, ''Plasmid DNA purification'', The J. of Gene Medicine,Vol.6, pp.54–S66
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*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.
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[5] Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.
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*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 (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
(Metcalf et al., 1994; 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.
  • Stadler J, Lemmens R, Nyhammar T 2004, Plasmid DNA purification, The J. of Gene Medicine,Vol.6, pp.54–S66