Part:BBa_K907000

Mycobacterium Phage Bxb1 gp35, DNA integrase

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This part is a protein coding part which encodes Bxb1 gp35, which is a DNA integrase of Mycobacterium phage Bxb1.
The Bxb1 integrase is a DNA recombinase, more precisely a member of serine integrase family. It recognizes specific sequences, called attB and attP, and then integrates, inverts, or excises dsDNA depending on the orientation of recognition sequences. We used this integrase to invert specific sequence in plasmid. The protein is well expressed in E.coli(strain MG1655) When it inverts DNA sequence, the attB and attP sequences are changed into attL and attR, as other DNA recombinases do. Another protein called Bxb1 gp47(BBa_K0907002) binds to integrase-DNA complex and this complex flips inverted DNA back into original sequence by regenerating attB and attP sequences.

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<Part Demonstration>

Figure 1. Experimental results of BBa_K907000.

Two ep-tubes designated as BBa_K907004 are containing centrifuged E.coli MG1655 cells possessing BBa_K907004. pTrcHis2A vector containing BBa_K907000, controlled by Trc promoter, transformed into MG1655-BBa_K907004. The double transformed E.coli MG1655 cells showed color red rather than yellowish green color of MG1655-BBa_K907004. 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. This results demonstrate that this part BBa_K907000 is 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. Even though the basal expression level is very low, the double transformants were available to change its color without IPTG induction. Thus we presume that BBa_K907000 is very efficient in bacterial cells. 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_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
BBa_K907005 - Dual Phase Protein Generator(mRFP 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 192
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 466
  Illegal XhoI site found at 553
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1105
  Illegal NgoMIV site found at 1192
  Illegal AgeI site found at 242
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1300