Difference between revisions of "Part:BBa K525224"
Line 3: Line 3:
  
 
S-layer cspB from Corynebacterium halotolerans with PT7 and RBS
 
S-layer cspB from Corynebacterium halotolerans with PT7 and RBS
 +
 +
===Important parameters===
 +
<center>
 +
{|{{Table}}
 +
!Experiment
 +
!Characteristic
 +
!Result
 +
|-
 +
|rowspan="5"|[[Part:BBa_K525224#Expression_in_E._coli | 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)
 +
|0.251 h<sup>-1</sup> / 0.157 h<sup>-1</sup>
 +
|-
 +
|Doubling time (un-/induced)
 +
|2.76 h / 4.42 h
 +
|-
 +
|}
 +
</center>
  
 
After processing:  
 
After processing:  

Revision as of 21:10, 21 September 2011

S-layer cspB from Corynebacterium halotolerans with TAT-sequence, PT7 and RBS

S-layer cspB from Corynebacterium halotolerans with 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) 0.251 h-1 / 0.157 h-1
Doubling time (un-/induced) 2.76 h / 4.42 h

After processing:

Molecular weight: 50.6 kDa

Theoretical pI: 4.25


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 440
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 440
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1191
    Illegal XhoI site found at 647
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 440
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 440
    Illegal NgoMIV site found at 314
    Illegal NgoMIV site found at 1403
    Illegal AgeI site found at 89
    Illegal AgeI site found at 305
    Illegal AgeI site found at 546
    Illegal AgeI site found at 593
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 995
    Illegal BsaI.rc site found at 302
    Illegal BsaI.rc site found at 680
    Illegal BsaI.rc site found at 1082


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.