Part:BBa_K1957006

HyaC subunit of NiFe Hydrogenase

One of the three subunits that make up NiFe Hydrogenase in Shewanella oneidensis. Specifically this is the HyaC subunit, which is the outer periplasmic subunit. 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 14 to 16 have HyaC gene insert, which is 754bp. Ladder is Hyperladder Kb, sizes shown in bp

Colonies from lanes 14 and 16 were sent off for sequencing. After sequencing data came back and confirmed the correct gene insert, the DNA from lane 16 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 HyaC using Benchling. DNA sequence of screenshot starts at 355bp

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 176
  
• 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 690