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).

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]

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).

Characterisation

IISc-Bangalore 2018

Expression

The part was transformed into E. coli BL21(DE3) since it contains the T7 polymerase under a lac promoter. It was induced with IPTG in mid log phase and the cell pellet was loaded onto an SDS PAGE. The following are the exact conditions:

Following growth and induction, 1mL of the culture was pelleted down and resuspended in 100μL of dH₂O and mixed with 100μL of SDS sample buffer. 10μL of this was loaded onto a 12% acrylamide SDS PAGE.

• Initial Growth Temperature: 37°C
• Growth Medium: LB
• Chloramphenicol Concentration: 35μg/mL
• Induction OD600: 0.6
• IPTG Concentration: 500μM
• Growth Temperature Following Induction: 37°C
• Growth Time Following Induction: 3hrs

Bands were observed slightly below 32kDa ladder band and at the 25kDa ladder band. These two correspond to the bet protein (29.7 kDa) and the exo protein (25.9 kDa). The other protein - gam (16.3 kDa) - cannot be seen on the gel. This could be because the part has only one RBS sequence for all three proteins. It could be either that the third protein is being produced in very small amounts or not at all.

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.

[4]https://www.uniprot.org/uniprot/P03697

[5]https://www.uniprot.org/uniprot/P03698

[6]https://www.uniprot.org/uniprot/P03702