Difference between revisions of "Part:BBa K3407019:Design"

 
 
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===Design Notes===
 
===Design Notes===
−
tetA/tetR  - RBD - YmdB - T7t
+
The gene was amplified by PCR from E.coli Bl21(DE3) genome as a template. YmdB amino acid sequence was taken from Uniprot (Uniprot ID: P0A8D6), it was introduced as a query in tBlastn from NCBI, against Escherichia coli BL21(DE3) genome and the resulting YmdB gene sequence (GenBank: AM946981.2) was used as a template for the design of the primers.
 +
Primers were designed to have a melting temperature of 59-60 ºC. Overhangs with BglBricks Prefix and Suffix were added, along with a His-Tag in the Reverse primer for a C-terminal-tagged protein. Designed to be cloned through Assembly. The primers used were the following:
  
 +
M3-037 (YmdB - Fw):
  
 +
5’ - gaattcaaaagatcttttaagaaggagatatacatATGAAAACGCGTATTCATGTTGTGC - 3’
 +
 +
M3-038  (YmdB - Rv):
 +
 +
5’- tttatttgatgcctggagatccttactcgagtttggatccttaGTGATGGTGGTGATGATGGTGGTGATGGTGACCTGTACTTCCTGTTTCATCTCCTTGTTGGGTAAGGAGTC - 3’
  
 
===Source===
 
===Source===
  
−
tetA/tetR  - RBD - YmdB - T7t
+
Plasmid used pBbA2k backbone from BglBricks. pBbA2k-RFP was a gift from Jay Keasling (Addgene plasmid # 35327 ; http://n2t.net/addgene:35327 ; RRID:Addgene_35327) <html><a href="#1">[1]</a></html>.
 +
Please refer to the Addgene page for more information about licences associated with the use of the plasmid.
  
 
===References===
 
===References===
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<a id="1" href="https://jbioleng.biomedcentral.com/articles/10.1186/1754-1611-5-12" target="_blank">
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BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, Prasad N, Lee SK, Keasling JD. J Biol Eng. 2011 Sep 20;5:12. 10.1186/1754-1611-5-12 PubMed 21933410</a>
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Latest revision as of 01:21, 28 October 2020


YmdB regulator of RNAseIII in E. coli with tetA/tetR promoters - RBD - T1 terminator


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 59
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 59
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 59
    Illegal BglII site found at 68
    Illegal BamHI site found at 673
    Illegal XhoI site found at 682
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 59
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 59
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The gene was amplified by PCR from E.coli Bl21(DE3) genome as a template. YmdB amino acid sequence was taken from Uniprot (Uniprot ID: P0A8D6), it was introduced as a query in tBlastn from NCBI, against Escherichia coli BL21(DE3) genome and the resulting YmdB gene sequence (GenBank: AM946981.2) was used as a template for the design of the primers. Primers were designed to have a melting temperature of 59-60 ºC. Overhangs with BglBricks Prefix and Suffix were added, along with a His-Tag in the Reverse primer for a C-terminal-tagged protein. Designed to be cloned through Assembly. The primers used were the following:

M3-037 (YmdB - Fw): 

5’ - gaattcaaaagatcttttaagaaggagatatacatATGAAAACGCGTATTCATGTTGTGC - 3’

M3-038 (YmdB - Rv): 

5’- tttatttgatgcctggagatccttactcgagtttggatccttaGTGATGGTGGTGATGATGGTGGTGATGGTGACCTGTACTTCCTGTTTCATCTCCTTGTTGGGTAAGGAGTC - 3’

Source

Plasmid used pBbA2k backbone from BglBricks. pBbA2k-RFP was a gift from Jay Keasling (Addgene plasmid # 35327 ; http://n2t.net/addgene:35327 ; RRID:Addgene_35327) [1]. Please refer to the Addgene page for more information about licences associated with the use of the plasmid.

References

Ordered List

  1. BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, Prasad N, Lee SK, Keasling JD. J Biol Eng. 2011 Sep 20;5:12. 10.1186/1754-1611-5-12 PubMed 21933410