Difference between revisions of "Part:BBa K1957005"

Revision as of 14:43, 9 October 2016

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 unit is one out of the three subunits that can be used to make a the entire NiFe hydrogenase construct.

Figure 1: PCR Colony of HyaA NiFe Hydrogenase subunit. Lanes 10 to 12 have HyaA (610) gene insert, which is 1215bp. Ladder is Hyperladder Kb, sizes shown in bp
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

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 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.

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 1151

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 <li style="display: inline-block;"> [[File:T--NRP-UEA-norwich--Gel_digest_diagnostic_610_.jpg|thumb|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]] </li>
-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 ligate the construct into the pBAD vector.
+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 i.e. Golden Gate cloning. As we used this in downstream cloning experiments the restriction site could not be avoided.
+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.
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