Team:Bielefeld-Germany/Results/Characterization

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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