Coding

Part:BBa_K1957007:Design

Designed by: Nancy Teng   Group: iGEM16_NRP-UEA-Norwich   (2016-10-13)


HyaB C-terminally tagged subunit of NiFe Hydrogenase


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 258
    Illegal BamHI site found at 1455
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1043
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 1766


Design Notes

This is one of of the three NiFe Hydrogenase subunits. The entire NiFe construct can be made by ligating the remaining units into the pBAD expression vector via golden gate cloning. For the entire NiFe hydrogenase cluster the following BioBricks need to be ligated with Bba_K1957007: Bba_K1957005 with Bba_K1957006.

BBa_J04450, which contained pSB1C3, would not conjugate properly into S.oneidensis, most likely due to conflicts in the origin of replication. The conflict could result in low copy numbers of pSB1C3 in S.oneidensis resulting in little antibiotic resistance. pSB1C3 contains pUC19 derived pMB1 origin of replication. In a paper by Myers and Myers (1997) they observed that pMB1 did not replicate in Shewanella oneidensis MR-1, formerly known as Shewanella putrefaciens MR-1. To overcome this issue, the three subunits were ligated into the pBAD expression vector using Golden Gate cloning



Source

The source of the part will be its sequence which was retrieved from GenBank.


References

Myers C.R. and Myers J.M. (1997). 'Replication of plasmids with the p15A origin in Shewanella putrefaciens MR-1', Applied Microbiology, 24, pp.221-225