Part:BBa_K1957005
HyaA subunit of NiFe Hydrogenase
One of the three subunits that make up NiFe Hydrogenase in Shewanella oneidensis. Specifically this is the HyaA subunit, which is the membrane bound catalytic subunit. This gene encodes one out of the three subunits that can be used to make the entire NiFe hydrogenase construct.
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Figure 2: Diagnostic Digest of HyaA NiFe Hydrogenase subunit. Lane 2 contains the HyA insert ligated into pSB1C3 i.e. BBa_K1957005 (610). Lane 3 has BBa_K1957005 digested with EcoR1 and Pst1 (DD). Lane 4 has BBa_K1957005 digested with just EcoR1 (E). Lane 5 has BBa_K1957005 digested with just Pst1 (P). Sizes of HyaA is 1215bp and pSB1C3 is 2029bp. Ladder is Hyperladder Kb, sizes shown in bp - 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1
Illegal BsaI.rc site found at 1151
DNA of the colony on lane 10 (Fig. 1) was sent off for sequencing. After sequencing confirmation it was submitted into the iGEM registry. Due to time constraints we were unable to assemble the full HyaABC gene cluster into the pBAD expresison vector.
Restriction site incompatibility, [1000], is with regards to MoClo or Golden Gate cloning. This restriction site could be used in to assemble the gene cluster into a single expression vector. The Bsal restriction enzyme sites flank the coding sequence, and digestion with Bsal should therefore 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.
Sequence and Features
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