Part:BBa_K1433016

P-C-attL-rT-rT-attR-B-B0031-gp35-T

Bxb1 gp47 is an excisionase and Bxb1 gp35 is a serine integrase of Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination and mediate DNA inversion from BP state to LR state. Expression of gp35 and gp47 at different ratio could change DNA from LR state back to BP state.

This part, also called REINT, is designed to test whether gp35 with gp47 can reverse sequence between attL and attR sites and change it to attB&P sites. What’s more, this circuit is used to test the terminating effect of two B0015 terminators on gp35.

Composition:

1. Promoter: BBa_J23110, a middle-ground Bacterial constitutive promoter.
2. Homologous arms: homologous arms C and B.
3. Terminator (reverse): BBa_B0015, a strong double terminator.
4. attL and attR sites: Recognition sites for Bxb1 gp35 and gp47, Mycobacterium Phage Bxb1 DNA integrase and excisionase.
5. RBS: BBa_B0031, a weak RBS.
6. gp35: a serine integrase in Mycobacterium phage Bxb1.
7. Terminator: BBa_B0015, a strong double terminator.

REINT+RESET:
Due to the tag on gp47, the degradation rate of gp47 is faster than that of gp35, so there would be some gp35 remaining changing some attL&R sites back to attB&P sites. Measuring this leakage is important for evaluating promoter reverse effects in the next turn.

Co-transformation of REINT and BBa_K1433013(RESET) can enable the test of the reverse function of gp35 and gp47. Before arabinose is added to induce pBAD promoter，strain shows red color (In this situation, only downstream RFP is expressed; the expressed gp35 which can only act on attB and attP sites cannot turn attL and attR to its previous state, which results in the maintenance of GFP expression). When pBAD promoter is induced, gp47 is expressed. The different expression quantities of gp35 and gp47 could change DNA from LR state to BP state. After reversion, these two reversed B0015 become functional and terminate the expression of gp35. With tag and without induction of pBAD, gp47 disappears. Then the color of strains will change from red to green, which can be easily observed. However, two B0015 terminators may not suppress gp35 totally. After some time not degraded or newly expressed gp35 may lead to a few attB&P sites reversing back to attL&R sites, resulting in some strains turning red. Measuring this leakage is significant for evaluating promoter reverse effects in the next turn.

Besides, REINT+RESET system can also achieve some function of Gene Socket like inserting genes continuously. Users can choose BBa_K1433009 or BBa_K1433010, linking it to the end of inserted gene by ligase (these parts contains two reverse B0015 terminators between attL and attR sites; the former should be chosen if the inserted gene has no promoter, while the latter one should be chosen if the inserted gene has a promoter). Our software GS-BOX can give primers to add homologous armC and add new homologous arms beside inserted genes. After this preparation, the gene could recombine to REINT by lambda red. Lambda red is a Lambda-phage-derived recombination system, which can recombine dsDNA/ssDNA into different kinds of DNA molecules as long as each side of the donor dsDNA/ssDNA is flanked by 36-50bp homologous arms. Lambda-red-mediated homologous recombination can replace two terminators with exogenous sequences (inserted genes together with BBa_K1433009 or BBa_K1433010), thus removing gp35 depression. The expression of gp35 can reverse attB&P sites and J23110 promoter to RFP, changing the strain color from green to red. Observed red color means that the gene insertion is successful. To insert a second gene, re-inducing BBa_I0500 pBAD promoter and repeating the referred procedure are enough. The only difference is that the prepared homologous arms beside the inserted gene should be new homologous arms.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 643
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 917
  Illegal XhoI site found at 1004
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 79
  Illegal NgoMIV site found at 1556
  Illegal NgoMIV site found at 1643
  Illegal AgeI site found at 693
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 99
  Illegal BsaI.rc site found at 357
  Illegal BsaI.rc site found at 1751