Part:BBa_K525224

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.