Difference between revisions of "Part:BBa K1957003"

Latest revision as of 18:45, 13 October 2016

HydB (N-terminally tagged) subunit of FeFe Hydrogenase

One of the three subunits that make up FeFe Hydrogenase in Shewanella oneidensis. Specifically, this is the HydB subunit which is the central subunit. This basic part has a strep-tag attached to its N-terminus for consequent protein assays. This unit is one out of the three subunits that can be used to make a the entire FeFe hydrogenase construct.

Restriction site incompatibility, [1000], is with regards to MoClo or Golden Gate cloning. This restriction site was used in 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 1: Screenshot of sequencing data of colony aligned with HydB N-terminus using Benchling. DNA sequence of screenshot starts at 125bp.

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 369

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 <partinfo>BBa_K1957003 short</partinfo>
-One of the three subunits that make up FeFe Hydrogenase in Shewanella oneidensis. Specifically, this is the HydB subunit which is the central subunit. This basic part has a strep-tag attached to its N-terminus for consequent protein assays. This unit is one out of the three subunits that can be used to make a the entire FeFe hydrogenase construct.
+One of the three subunits that make up FeFe Hydrogenase in ''Shewanella oneidensis''. Specifically, this is the HydB subunit which is the central subunit. This basic part has a strep-tag attached to its N-terminus for consequent protein assays. This unit is one out of the three subunits that can be used to make a the entire FeFe hydrogenase construct.
+Restriction site incompatibility, [1000], is with regards to MoClo or Golden Gate cloning. This restriction site was used in 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
+[[File:T--NRP-UEA-Norwich--Sequencing_Data_609_HydBNterm_.jpg|thumb|none|'''Figure 1: Screenshot of sequencing data of colony aligned with HydB N-terminus using Benchling'''. DNA sequence of screenshot starts at 125bp.]]
-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.
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