Part:BBa_K1433013

rT-rRFP-rRBS-attB-P-attP-RBS-lambda red-RBS-GFP-T-P-RBS-gp47-tag-T

This part is an important composition of Gene Socket. Gene Socket uses a plasmid, a circuit and a strain to construct circuits on chromosome by lambda-red and recombinase-derived bistable switch. This part is the helper plasmid, called SUPPORT DEVICE.

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 BBa_B0034, a strong RBS.
4. attB and attP sites: Recognition sites for Bxb1 gp35, Mycobacterium Phage Bxb1 DNA integrase.
5. Promoter: BBa_J23110, a middle-ground Bacterial constitutive promoter.
6. λ-red functional proteins : Exo, Beta and Gam.
7. GFP: BBa_E0040, green fluorescent protein derived from jellyfish Aequeora Victoria wild-type GFP.
8. Promoter: BBa_I0500, an inducible pBad/araC promoter
9. gp47: an excisionase in Mycobacterium phage Bxb1.
10. Tag: AAK tag, a distinct proteolysis tag on gp47 peptides.
11. Terminator: BBa_B0015, a strong double 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 Bxb1 gp35 and gp47 at certain ratio could change DNA from LR state to BP state.

Lambda red is a Lambda-phage-derived recombination system which contains three proteins: Exo, Beta and Gam. It can recombine dsDNA/ssDNA into different kinds of DNA molecules such as chromosome, bacteria artificial chromosome (BAC) or even multicopy plasmids, as long as each side of the donor dsDNA/ssDNA is flanked by 36-50bp homologous arms. The homologous arms also determine the accurate sites of recombination, which ensures that the donor fragment can be inserted into any sites by choosing and adding homologous arms using PCR. To get rid of PKD46, a helper plasmid to generate lambda red recombination, PKD46 functional unit (Exo, Beta and Gam) is introduced to this part. What’s more, there are three restrict enzyme sites of EcoRI and PstI. To improve the compatibility of this part and make the modules enable to be used in future assemblies, we removed these restrict enzyme sites by synonymous mutations.

SUPPORT DEVICE+SOCKET:
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SUPPORT DEVICE should be used with BBa_K1433018 (named SOCKET). SOCKET contains a BBa_J23110 promoter and two downstream homologous arms (homologous arms A and B) between two series unidirectional terminators. In the most downstream is a Bxb1 gp35 express unit BBa_K1433008. To allow Gene Socket to function normally, SUPPORT DEVICE is on plasmid and should be introduced to bacteria by transformation, SOCKET is a circuit segment introduced to bacteria by electrotransformation and should be recombined on chromosome by lambda red.

At first, gp35 is suppressed by two unidirectional terminators in series on SOCKET and gp47 on SUPPORT DEVICE is not induced, thus no reversion happening now. Only when lambda red functional proteins and GFP are expressed should green strain be observed. This state is called “green state”. To recombine genes on chromosome, users can link BBa_K1433009 or BBa_K1433010 to the downstream 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 arm A and add new homologous arms beside inserted genes. Then the inserted gene with homologous arms added by PCR can be introduced in the system by electrotransformation and recombined on SOCKET by lambda red functional proteins. Lambda-red-mediated homologous recombination can replace two terminators with exogenous sequences (inserted genes and BBa_K1433009 or BBa_K1433010), thus removing gp35 depression. Gp35 could mediate attBP sites change to attLR sites and the reverse of BBa_J23110 promoter as well, leading to the expression of RFP and turning off lambda red and GFP genes at the same time. Red strain can be observed, which is called “red state”. Red state indicates that the gene of interest has been inserted on SOCKET (SOCKET is on chromosome).

Additionally, Gene Socket can insert more than one genes. When pBAD promoter is induced, gp47 is expressed and heterologous expression of gp35 and gp47 could change DNA from LR state to BP state. AttL&R sites on SOCKET are reversed and the two reverse terminators become functional, suppressing gp35 again. BBa_J23110 Promoter on SUPPORT DEVICE then reverses, expressing lambda red proteins and GFP, thus the strain color turning from red to green. Gene Socket is back to its original state and the “green state” appears again. There is a tag on gp47, so gp47 will disappeared when we stop inducing for a while. To insert a second gene, repeating the referred procedure is enough. The only difference is the prepared homologous arms beside the inserted sequences 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 926
  Illegal NheI site found at 949
  Illegal NheI site found at 4991
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4930
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 873
  Illegal NgoMIV site found at 5025
  Illegal AgeI site found at 2429
  Illegal AgeI site found at 4765
  Illegal AgeI site found at 5793
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 893
  Illegal BsaI.rc site found at 976
  Illegal BsaI.rc site found at 3573
  Illegal SapI site found at 4747