Part:BBa_K2609026

Lambda Red Recombinases under T7 expression system

This part generates the three Lambda RED recombinases - exo, gam, beta - when expressed in a T7 expression strain like E. coli BL21(DE3).

Biology

The genes exo, gam and beta are a part of Bacteriophage Lambda's genome and is used in recomibination of bacteriophage DNA. The gam protein binds to the RecBCD nuclease of the host, thus protecting linear viral DNA from degradation. The protein exo is a 5' to 3' exonuclease which exposes the ends of linear dsDNA. The protein beta promotes single strand annealing and hence promotes homologous recombination. It is also important in rollin circle DNA replication which comes late in the infective cycle of the lambda phage.

Usage

IISc-Bangalore 2018

For their Phage Assisted Imune Recruitment (PAIR) the IISc 2018 iGEM Team used this part to recombine the Bacteriophage T4 genome to produce a lysis deficient phage. This was done using the parts BBa_K2609008 and BBa_K2609009 which have flanking sequences homologous to the flanking regions of Endolysin gene (e gene) on the T4 genome. A recombinant T4 Endolysin (BBa_K2609017) was used in conjuction with this part to screen for recombinants. Following successful recombination, their lysis deficient phage would trigger it's host to secrete mcp-1 (BBa_K2609000).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1943
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1460
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Mosberg, J. A., Lajoie, M. J., & Church, G. M. (2010). Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate. Genetics, 186(3), 791–799. http://doi.org/10.1534/genetics.110.120782

[2]Sharan S K, Thomason L C, Kuznetsov S G, et al. Recombineering: a homologous recombination-based method of genetic engineering[J]. Nature protocols, 2009, 4(2): 206-223.

[3]Datsenko K A, Wanner B L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proceedings of the National Academy of Sciences, 2000, 97(12): 6640-6645.