Part:BBa_K1433012

Terminator-RFP-RBS-attL-Promotor-attR-RBS-GFP-Terminator-Promotor-Xis-Terminator

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 is a circuit used to test the function of gp35 and gp47 at certain ratio which is to change DNA from LR state to BP state, which we call RESET. As high gp35 to gp47 ratio is important in this reversion, too much expression of gp47 may make this system ineffective. To prevent the possible low ratio, an AAK proteolysis tag is added to decrease the amount of gp47 peptides.

Composition:

1. Terminator (reverse): BBa_B0015, a strong double terminator.
2. RFP (reverse): BBa_J06504, a monomeric RFP optimized for bacteria.
3. RBS: BBa_B0034, a strong RBS.
4. attL and attR sites: Recognition sites for Bxb1 gp35 and gp47, Mycobacterium Phage Bxb1 DNA integrase and excisionase.
5. Promoter: BBa_J23110, a middle-ground Bacterial constitutive promoter.
6. GFP: BBa_E0040, green fluorescent protein derived from jellyfish Aequeora Victoria wild-type GFP.
7. Promoter: BBa_I0500, an inducible pBad/araC promoter
8. gp47: an excisionase in Mycobacterium phage Bxb1.
9. Tag: AAK tag, a distinct proteolysis tag on gp47 peptides.
10. Terminator: BBa_B0015, a strong double terminator.

This part contains a reverse promoter between attL and attR sites. There are two reporter genes, GFP and RFP in upstream and downstream of this promoter respectively. Besides, there is a gp47 gene controlled by an inducible pBAD promoter in upstream. Strain contains this parts should be red because of the expression of downstream RFP gene.

INT+RESET
BBa_K1433014 or BBa_K1433015 is designed to express Bxb1 gp35, which we call INT. As gp47 cannot reverse LR state to BP state alone, this part should be used with gp35-production circuit. Co-transformation of INT and 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. Then the color of strains will change from red to green, which can be easily observed.

REINT+RESET
There is a tag on gp47 while no one on gp35, causing the degradation of gp47 become faster than gp35. Therefore, after some time a few attB&P sites may reverse back to attL&R sites, resulting in some strains turning red. After the reversion, the strain produces GFP continuously and stably which indicates that the current LR state. Besides, to make gene socket come true, we designed another circuit to cooperate with this part: BBa_K1433016 or BBa_K1433017, which we call REINT. The only difference of these two parts is RBS. Instead of the BBa_J23110 promoter in INT, REINT has two reverse B0015 terminators between attL and attR sites, where two terminators are put to ensure the completeness of termination. What’s more, there are two homologous arms flanking attLR sites, called homologous armC and armB. When pBAD promoter is induced, gp47 is expressed and expression of gp35 and gp47 at certain ratio could change DNA from LR state to BP state, turning the strain color from red to green. Different from INT, after reversion, these two reversed BBa_B0015 become functional and terminate the expression of gp35. With tag and without induction of pBAD, gp47 disappears. The strain shows green color which indicates that DNA is in LR state.

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 BBa_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 andBBa_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 920
  Illegal NheI site found at 943
  Illegal NheI site found at 3087
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3026
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 873
  Illegal NgoMIV site found at 3121
  Illegal AgeI site found at 2861
  Illegal AgeI site found at 3889
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 893
  Illegal BsaI.rc site found at 976
  Illegal BsaI.rc site found at 1669
  Illegal SapI site found at 2843