Part:BBa_K1957004

HydA subunit of FeFe Hydrogenase

One of the three subunits that make up FeFe Hydrogenase in Shewanella oneidensis. Specifically this is the HydA subunit which is the membrane bound catalytic subunit. This unit is one out of the three subunits that can be used to make a the entire FeFe hydrogenase construct.

Figure 1: PCR Colony of FeFe Hydrogenase subunits. Lanes 6 and 7 have HydA gene insert, which is 1325bp. Ladder is Hyperladder Kb, sizes shown in bp

DNA of the colony on lane 6 and 7 were sent off for sequencing. After sequencing confirmation this was submitted to the iGEM registry. A Golden Gate cloning strategy was used with parts BBa_K1957001 & BBa_K1957002 to assemble the HydABC hydrogenase gene cluster into a single expression vector, with a C-terminal strep-tag on the B subunit. A Golden Gate cloning strategy was also used with parts BBa_K1957001 & BBa_K1957003 to assemble the HydABC gene cluster into a single pBAD expression vector with an N-terminal strep-tag on the B subunit.

Restriction site incompatibility, [1000], is with regards to MoClo or Golden Gate cloning. This restriction site was used in the described Golden Gate cloning experiments in order to assemble the gene cluster into a single expression vector. The Bsal restriction enzyme sites flank the coding sequence, and digestion with Bsal should not disrupt the protein coding sequence. For more details on our Golden Gate cloning strategy and instructions on how to assemble the HydABC gene cluster into a single expression vector please see the NRP-UEA 2016 iGEM teams wiki.

Figure 2: Sequencing data aligned with HydA gene insert using the software Benchling. (A) Screenshot of sequencing data at 28bp (B) Screenshot of sequencing data at 528bp

Sequence and Features

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