Part:BBa_K1957008

HyaB N-terminally tagged subunit of NiFe Hydrogenase

One of the three subunits that make up NiFe Hydrogenase in Shewanella oneidensis. Specifically this is the HyaB subunit, which is the membrane-bound subunit. This subunit has a strep-II tag on its CN-terminal for downstream protein assays. This gene encodes one out of the three subunits that can be used to make the entire NiFe hydrogenase construct.

Figure 1: PCR Colony of NiFe Hydrogenase subunits. Lanes 8 to 10 have HyaB N-terminus gene insert, which is 1830bp. Ladder is Hyperladder Kb, sizes shown in bp

Colonies from lanes 9 and 10 were sent off for sequencing. After sequencing data came back and confirmed the correct gene insert, the DNA from the colony of lane 10 was submitted into the registry.

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 HyaCBA gene cluster into a single expression vector please see the NRP-UEA 2016 iGEM teams wiki.

Figure 2: Screenshot of sequencing data of colony aligned with HyaB N-terminally tagged using Benchling. DNA sequence of screenshot starts at 1018bp

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 282
  Illegal BamHI site found at 1479
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1067
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI.rc site found at 1766
  Illegal SapI.rc site found at 37