Part:BBa_K1433021

Promoter-B0034-gp35-T

This part, which is also called Int-B0034, is a circuit designed to express Bxb1 gp35. Bxb1 gp35 is a serine integrase of Mycobacterium phage Bxb1. This integrase can exclusively catalyze site-specific recombination between attB and attP, the attachment sites on phage chromosome and host chromosome. This recombination results in reverse of the sequence between attB and attP, changing the two sites to attL and attR at the meantime.

Int-B0031 is a tool to provide functional protein gp35 to work with other circuits which contain attB and attP sites. Gp35 can reverse the sequence between the two sites and change the two sites to attL and attR, which make it possible to realize more difficult function by using it.

Composition:

1. Promoter: BBa_J23110, a middle-ground bacterial constitutive promoter.
2. RBS: BBa_B0034, a strong RBS.
3. gp35: a serine integrase in Mycobacterium phage Bxb1.
4. Terminator: BBa_B0015, a strong double terminator.

This part has sister parts BBa_K1433015 and BBa_K1433014, whose nickname is INT-6N and INT-B0031. The only difference among these parts is RBS. Different from INT-B0031, Int-6N contains a RBS named 6N. 6N is stronger than B0031 (Bonnet, et al. 2012). Different RBSs can results in different expression quantities of gp35, thus causing different reverse effects. As experimental data shows, the effect of 6N is better than B0031. Therefore some circuits containing gp35 like BBa_K1433017 and BBa_K1433018 which we construct later using 6N only.

INT+SET:
BBa_K1433011 is designed to test the function of INT-RBS circuit. There are two reporter genes on BBa_K1433011, GFP and RFP. At first, the promoter between attB and attP sites is in forward direction and can promote the expression of downstream GFP gene to make bacteria green. When the co-transform of this part and INT-RBS occurs, gp35 will express and then reverse the promoter, which will promote the expression of upstream RFP gene. The change of color of bacterial colony or solution can be easily observed. Bxb1 gp47 is an excisionase in Mycobacterium phage Bxb1. Different expression quantity of Bxb1 integrase gp35 and excisionase gp47 could transform DNA from LR state to BP state, the gene between which could turn to previous direction, too. BBa_K1433012 is designed to accompany INT-RBS to test the function from LR state to BP state.

INT+RESET:
BBa_K1433012 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. Before inducing pBAD promoter by arabinose，co-transformation of INT-RBS and BBa_K1433012 can results in red strains (In this state, only the RFP gene at the downstream of the promoter is expressed. At the meantime, 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, resulting in different levels of expression of gp35 and gp47 and changing DNA from LR state to BP state. The change of color of strains from red to green can be easily observed.

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 239
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 513
  Illegal XhoI site found at 600
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1152
  Illegal NgoMIV site found at 1239
  Illegal AgeI site found at 289
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1347