Part:BBa_K907005

Dual phase protein generator(mRFP default). mRFP and GFP

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<Part Description>
This part is derivative of BBa_K907003, twins with BBa_K907005

BBa_E1010(mRFP, reversed) and BBa_0040(wtGFP) are attached to both ends of BBa_K907003 by overlapping extension PCR. This part normally generates mRFP in E.coli(strain MG1655). When BBa_K907000(Bxb1 integrase) inverts the promoter orientation, it starts to generate GFP.

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<Part Demonstration>

Figure 1. Experimental results of BBa_K907005.

Two ep-tubes designated as BBa_K907005 are containing centrifuged E.coli MG1655 cells possessing BBa_K907005. pTrcHis2A vector containing BBa_K907000, controlled by Trc promoter, transformed into MG1655-BBa_K907005. The double transformed E.coli MG1655 cells showed pink color rather than intense red color of MG1655-BBa_K907005. Gathered cells are not showing perfect yellowish green color because initial state color of mRFP was too strong. With this result, we can say that our designed parts(BBa_K907000, BBa_K907003 and BBa_K907005) are working as we expected. The cells were cultured in 100 mL LB media(1% Cm,AP each) and gathered incubating 12 hours more after induction with 1mM of IPTG. Culture condition was maintained at 37'C and 220 rpm.
Trc promoter, however, has basal level expression of Bxb1 gp35, Mycobacterium Phage Bxb1 DNA integrase, the double transformants were available to change its color without IPTG induction. To diminish the effect of basal transcription level, we performed several optimizations.
Further information are available at [http://2012.igem.org/Team:KAIST_Korea KAIST_iGEM_2012 Wiki]

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<Related Parts>
BBa_K907000 - Mycobacterium Phage Bxb1 integrase
BBa_K907001 - Mycobacterium Phage Bxb1 excisionase
BBa_K907002 - Binary Signal Generator, RBS(reverse) - attB - Promoter - attP - RBS
BBa_K907003 - Binary Signal Generator, Promoter Reversed, RBS(reverse) - attB - Promoter(reverse) - attP - RBS
BBa_K907004 - Dual Phase Protein Generator(GFP default). mRFP and GFP

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Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 750
  Illegal NheI site found at 773
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 838
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 703
  Illegal AgeI site found at 10
  Illegal AgeI site found at 122
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 723
  Illegal BsaI.rc site found at 806
  Illegal BsaI.rc site found at 1499