Coding
cspB

Part:BBa_K525223

Designed by: Anna Drong   Group: iGEM11_Bielefeld-Germany   (2011-09-12)
Revision as of 21:08, 21 September 2011 by JSchwarzhans (Talk | contribs)

S-layer cspB from Corynebacterium halotolerans with lipid anchor, PT7 and RBS

S-layer cspB from Corynebacterium halotolerans with TAT-Sequence and lipid anchor, PT7 and RBS

Important parameters

Experiment Characteristic Result
Expression (E. coli) Localisation Cell membrane
Compatibility E. coli KRX
Induction of expression L-rhamnose for induction of T7 polymerase
Specific growth rate (un-/induced) 0.248 h-1 / 0.098 h-1
Doubling time (un-/induced) 2.79 h / 7.07 h

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1102
    Illegal XhoI site found at 558
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 225
    Illegal NgoMIV site found at 1314
    Illegal NgoMIV site found at 1425
    Illegal AgeI site found at 216
    Illegal AgeI site found at 457
    Illegal AgeI site found at 504
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 906
    Illegal BsaI.rc site found at 213
    Illegal BsaI.rc site found at 591
    Illegal BsaI.rc site found at 993


Expression in E. coli

The CspB gen was fused with a monomeric RFP (BBa_E1010) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.

The CspB|mRFP fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase by supplementation of 0,1 % L-rhamnose using the [http://2011.igem.org/Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_S-layer_genes_in_E._coli autinduction protocol] from promega.

Figure 1: Growthcurve of E. coli KRX expressing the fusion protein of CspB and mRFP with and without induction. A curve depicting KRX wildtype is shown for comparsion.
Figure 2: RFU to OD600 ratio of E. coli KRX expressing the fusion protein of CspB and mRFP with and without induction. A curve depicting KRX wildtype is shown for comparsion.
[edit]
Categories
//chassis/prokaryote/ecoli
//proteindomain/internal
//rnap/bacteriophage/t7
Parameters
biologyS-layer
originCorynebacterium halotolerans