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>K389012</partinfo>: VirA reporter system with luciferase

coming more soon


<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase

Control of BioBrick quality by capillar gel electrophoresis (GCE)

The quality of plasmid-conformation is crucial for following works such as transformation. A high content of covalent closed circular (ccc-type) pDNA indicates to high pDNA quality ([http://onlinelibrary.wiley.com/doi/10.1002/jgm.512/full Stadler et al., 2004]), ([http://www.eppendorf.com/script/cms-newspic.php?id=4966&col=DOWNLOADFILE Behrens B, Eppendorf AG]).

The representative tested BioBrick shows ccc-type of over 90 %, meaning pDNA quality is optimal.

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 electrophoresis (GCE)

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</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amlified Vir-test system

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.

Accomplishment

PCR-Primer Design

Primer forward activator phage P2:

5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GTT TCA TTG TCC TTT ATG CC-3`

Primer forward activator phage PSP3:

5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GAT GCA CTG CCC GTT ATG- 3`

Primer forward activator phage phi R73:

5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GCG CTG CCC TTT CTG-3`

Primer backward Promotor PF from phage P2:

5`-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TTC TCC TCT TTC TCT AGT AAG TGG- 3`


Characterization tests

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.

Results

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.

Space Illustration


The amplification factor was received by apply [http://partsregistry.org/Part:BBa_K389015 K389015] as reference. Amplification calculation was done by normalizing relative light units emmitted from luciferase per OD. 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.

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.

For further theory click Read out system

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.

[4] Stadler J, Lemmens R, Nyhammar T, 2004, Plasmid DNA purification, The J. of Gene Medicine,Vol.6, pp.54–S66

[5] Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.

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