Part:BBa_K1668001:Design

metK (from Streptomyces avermitilis, increasing avermectin production)

The part metK is the coding sequence of S-adenosylmethionine synthetase in Streptomyces avermitilis. It was found to stimulate the production of avermectins, one kind of pesticide.
This gene sequence could not function in E.coli. If you would like to express metK in Streptomyces avermitilis, remember to add ermEp (BBa_K1668004) as its promoter.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 376
  Illegal BsaI.rc site found at 304
  Illegal BsaI.rc site found at 595
  

Source

The metK gene was amplified by PCR with genomic DNA extracted from S. avermitilis ATCC31267 strain as template. We commercially purchased this strain.

Design Notes

PCR

The metK gene was amplified by PCR with genomic DNA extracted from S. avermitilis ATCC31267 strain as template. We commercially purchased this strain. By PCR with primers metK1 and metK2 shown below, we added the standard prefix and suffix at both ends of the metK sequence.

Seamless assembly

We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol. By this way, prefix sequence, metK, and suffix sequence can be ligated seamlessly.
metK1 (F, 5’-3’): GAATTCGCGGCCGCTTCTAGATGTTCGGCTACGC
metK2 (R, 5’-3’): TGCAGCGGCCGCTACTAGTATTATTACAGCCCCACA

Transformation and confirmation

After seamless assembly, standard plasmid pSB1C3 containing metK gene was transformed into E.coli DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2/VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.

Plasmid map

Fig.3 the plasmid map of BBa_K1668001

References

Zhao, X., et al. (2013). "Overexpression of metK shows different effects on avermectin production in various Streptomyces avermitilis strains." World J Microbiol Biotechnol 29(10): 1869-1875.
http://www.uniprot.org/uniprot/Q827Q0