Coding

Part:BBa_K1957004:Design

Designed by: Nancy Teng   Group: iGEM16_NRP-UEA-Norwich   (2016-10-06)
Revision as of 20:42, 8 October 2016 by Nteng (Talk | contribs)


HydA subunit of FeFe Hydrogenase


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 1
    Illegal BsaI.rc site found at 1261
    Illegal SapI site found at 1219


Design Notes

This is one of of the three FeFe Hydrogenase subunits. To make the entire FeFe Hydrogenase construct the following BioBricks need to be ligated with BBa_K1957004: BBa_K1957001 and BBa_K1957002 or BBa_K1957001 and BBa_K1957003. pSB1C3 would not conjugate properly into S.oneidensis, thus the three subunits were ligated using golden gate cloning into the pBAD expression vector.

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 Shewanellaputrefaciens MR-1', Applied Microbiology, 24, pp.221-225