Team:Bielefeld-Germany/Results/Characterization

From 2010.igem.org

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(K389421, K389422, K389423: Sensitivity Tuner amlified Vir-test system)
(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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[[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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[[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.]]
=<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="http://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="http://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 [http://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Cultivation] and [http://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Measuring_of_mRFP Measurement]
Protocols for [http://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Cultivation] and [http://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="http://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="http://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016">Detailed information...</a></div></html>
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<center>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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=<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amplified Vir-test system=
=<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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'''PCR-Primer Design'''
 
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Primer forward activator phage P2:
 
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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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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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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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5`-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TTC TCC TCT TTC TCT AGT AAG TGG- 3`
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<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="http://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/Sen_Tuner">Detailed information...</a></div></html>
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'''Characterization tests'''
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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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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. ([http://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Protocols])
 
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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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'''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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=Mutated <partinfo>K389001</partinfo> (exemplary): Results of the directed mutagenesis=
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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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<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="http://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/Exemplary_results_DM">Detailed information...</a></div></html>
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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 [http://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Read_out_system Read out system]
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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.''']]
=References=
=References=

Latest revision as of 01:07, 28 October 2010

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Contents

BBa_K238008: 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 BBa_K238008 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 (BBa_K389001).

BBa_K238011: 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 BBa_K238011 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 (BBa_K389003).

BBa_P1010: 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 BBa_P1010, 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.

BBa_K389004: 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 BBa_K389004.
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 BBa_J23103 output
Limit of quantification (LOQ) 306 RLU ~ 0.7 % of BBa_J23103 output

BBa_K389011: VirA screening device

Detailed information...


Figure 1: Ratio of surviving colonies of E. coli EC100D carrying unmutated BBa_K389010 and BBa_K389011 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.


BBa_K389015: 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 BBa_K389015.
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

BBa_K389016: 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 BBa_K389016.
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

BBa_K389052: 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 BBa_C0012 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 BBa_C0012.

BBa_K389421, BBa_K389422, BBa_K389423: 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 BBa_K389421, BBa_K389422 and BBa_K389423, Standard deviation shown.


Figure 3: Vizualitation of induced (from left to right) BBa_K389421, BBa_K389422 and BBa_K389423 sensitivity tuner amplified vir-system.


Mutated BBa_K389001 (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