Part:BBa_K1433018

P-A-T-T-B-B0034-gp35-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, which is called SOCKET, is a circuit need to be recombined on chromosome by a helper plasmid BBa_K1433013, called SUPPORT DEVICE.

Composition:

1. Promoter: BBa_J23110, a middle-ground Bacterial constitutive promoter.
2. Homologous arms: homologous arms A and B.
3. Terminator: BBa_B0015, a strong double terminator.
4. RBS: BBa_B0034, a strong RBS.
5. gp35: a serine integrase in Mycobacterium phage Bxb1.

SUPPORT DEVICE+SOCKET:

SOCKET should be used with BBa_K1433013 (named SUPPORT DEVICE). 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 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 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 gene. 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. 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.

SET+SOCKET:
Co-transformation of this part and BBa_K1433011 (SET) can be used to test the background expression of gp35. SOCKET contains two BBa_B0015 terminators between two homologous arms, which can suppress the expression of gp35. The co-transformed bacteria should be pure green. Indeed, there are a few bacteria presenting red or mix of green and red because of background leakage. 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, thus removing gp35 depression and changing the color from green to red. Measuring this leakage of gp35 is important in recombination experiment because the real reverse ability of gp35 should be under no interference of background leakage.

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 592
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 866
  Illegal XhoI site found at 953
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1505
  Illegal NgoMIV site found at 1592
  Illegal AgeI site found at 642
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1700