Difference between revisions of "Part:BBa K2609026"
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| − | This part generates the three Lambda RED recombinases - exo, gam, beta - when expressed in a T7 expression strain like E. coli BL21(DE3). | + | This part generates the three Lambda RED recombinases - exo, gam, beta - when expressed in a T7 expression strain like E. coli BL21(DE3). |
| − | + | <h4>Sequence and Features</h4> | |
| + | <partinfo>BBa_K2609026 SequenceAndFeatures</partinfo> | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
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| − | 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. | + | 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. |
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===References=== | ===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 | [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 | ||
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[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. | [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. | ||
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| + | [4]https://www.uniprot.org/uniprot/P03697 | ||
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| + | [5]https://www.uniprot.org/uniprot/P03698 | ||
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| + | [6]https://www.uniprot.org/uniprot/P03702 | ||
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
Latest revision as of 22:56, 17 October 2018
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1943
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1460
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
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:
- 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