Part:BBa_K2026000
LSSL, a composite part acts as genetic switch
LSSL(LoxP-B0015-B0015-LoxP) is a composite part acts as genetic switch. This part consist of two B0015(BBa_B0015) stop sequences which are flanked by LoxP sequences. LoxP sequence is a binding position for cre recombinase which binds to initial and last 13bp of LoxP to form a dimer. When Cre protein combined with Loxp sites, Cre could recombine DNA by deleting the sequence flanked by two Loxp sites.
This part use Cre-Lox system to function as a genetic switch which is similiar to the traditional LSL switch. However, we modified the stop sequence between the LoxPs in order to increase the stop efficiency of the switch. Furthermore, with the combination of genetic sensor such as pCpxP or pFadR promoter, this part could function as a critical component in signal amplification system.
This part is flanked by standard biobrick restriction sites.
Detailed information
The celebrated cre-loxp recombinase system, as many previous experiments and data suggests, is a widely used technology in DNA sequence modification. The system contains only 2 components – a recombinase Cre (stands for cause recombination) and the loxp site (stands for locus of crossing-over), no other phage or host components are necessary, in another word, the presence of cre is both necessary and sufficient to carry out the recombination. (Hoess et. Al, 1984) The mechanism was first discovered in the genome of bacteriophage P1. (Sternberg & Hamilton 1981) And its mature application was used in genetic engineering, even including some IGEM teams. (e.g. BBa_K1021005, BBa_K1179058, BBa_K1440000, IGEM parts) In our design, we focused on the appropriate arrangement of loxp sites thus achieve our goal – amplify the signal produced my inductive expression.
Design:
Based on the fact that ‘It has already been established that the loxP site exhibits directionality. When two sites on the same DNA molecule are in a directly repeated orientation, the DNA between the sites is excised after recombination’ (Hoess 1984) After several premature design, we designed the amplification plasmid PET28a-L-S-L-LacZW-Cre, once there’s Cre recombinase presented within the system, the two same oriented loxp sites flanking two B0015 terminators, would recombine thus results in deletion of the terminators thus enables the downstream expression. Furthermore, once the terminators have been removed while downstream LacZ reporter starts to express, the downstream Cre sequence would also express thus result in even higher level of Cre recombinase, which promotes removal of the terminators, amplify the signal with superb efficiency within a fairly short time.
References:
1. Hoess, Ronald H., and Ken Abremski. "Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP." Proceedings of the National Academy of Sciences 81.4 (1984): 1026-1029.
2. Sternberg, Nat, and Daniel Hamilton. "Bacteriophage P1 site-specific recombination: I. Recombination between loxP sites." Journal of molecular biology 150.4 (1981): 467-486.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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