Part:BBa_K1668003:Design

orfX (from Streptomyces avermitilis, increasing avermectin production)

The part orfX is a putative membrane-bound putative regulatory gene and its product is a putative membrane protein.

This gene sequence could not function in E.coli. If you would like to express frr in Streptomyces avermitilis, remember to add ermEp (BBa_K1668004) as its promoter.

Assembly Compatibility:

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

  Illegal NotI site found at 160
  Illegal NotI site found at 247
  Illegal NotI site found at 361
  Illegal NotI site found at 595
  Illegal NotI site found at 745
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 510
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 463
  Illegal AgeI site found at 264
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 441
  Illegal BsaI.rc site found at 195
  

Design Notes

PCR

The orfX 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 orfX1 and orfX2 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.
orfX1 (F, 5’-3’): GAATTCGCGGCCGCTTCTAGATGGTGAGCGCCT
orfX2 (R, 5’-3’): TGCAGCGGCCGCTACTAGTATTATTATCTGCGGTCC

Transformation and confirmation

After seamless assembly, standard plasmid pSB1C3 containing orfX 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_K1668003

Source

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

References

Hwang, Y. S., et al. (2003). "Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various Streptomyces avermitilis Strains." Appl Environ Microbiol 69(2): 1263-1269.