Team:Bielefeld-Germany/Results/Characterization

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Contents

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>BBa_K238011</partinfo>: vir-promoter

We made a restriction analysis and sequenced parts of this BioBrick.


<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 [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).


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 [2,3]
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>K389011</partinfo>: VirA screening device

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

IGEM-Bielefeld-formel-LD50.jpg

with the number of colony forming units CFU, the concentration of kanamycin on the considered plate KanX and no kanamycin on the plate Kan0.


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

Fails...more soon.


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>K389010</partinfo> (not fully completed)
  • <partinfo>K389011</partinfo> (not fully completed)
  • <partinfo>K389012</partinfo> (not fully completed)
  • <partinfo>K389013</partinfo> (not fully completed)
  • <partinfo>K389015</partinfo> (not fully completed)
  • <partinfo>K389016</partinfo> (not fully completed)
  • <partinfo>K389050</partinfo>


Other BioBricks

  • <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!


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.

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