Part:BBa_K5109001
Dehalogenase type II intracellular expression tool
This part is made for the inducible expression of enzymes of interest, in this case the enzyme is Dehalogenase type II. The part is structured with the biobrick prefix and suffix at the ends, allowing cloning via RFC 10. The chosen promoter is pTac (BBa K864400), an inducible promoter consisting of the sequence of pTac and LacO: the sequence includes the Lac Operon, repressing the expression in presence of LacI protein, and allowing it in presence of IPTG inducer. This part is to be used in bacterial strains already containing a gene cassette for constitutive LacI expression, or in alternative, to be cloned inside strains via co-trasformation with a plasmid carrying the LacI expression cassette. This way expression of the enzyme is blocked until IPTG is added. If the trascription is not blocked, constitutive expression of the enzyme of interest has shown to interfere with the growth of cells, preventing the formation of colonies. RBS.1 (BBa_B0030) is a strong RBS, based on Ron Weiss thesis. It presents a high non-modularity with promoters and protein coding parts. Dehalogenase type II (BBa_K3347010) is a coding sequence of a dehalogenase enzyme. We obtained the sequence from the previous work of USAFA 2020 team: thanks to their work, the sequence which comes originally from Delftia acidovorans, has been codon optimized for the expression in Escherichia coli. The sequence does not present terminator: this is due to complications in phase of synthesis. Therefore we provided BsaI cutting sites in order to allow Golden Gate cloning to insert other terminators. Otherwise, the sequence can be used by cloning it via RFC 10 inside a plasmid that contains a terminator right after the suffix sequence: in our work, we used pJump 29-1A (BBa_K5109060). After cloning it inside a plasmid used as expression vector, this part can be used to interchange different enzymes of interest: NdeI cutting site is located upstream the coding sequence of the Dehalogenase, while BamHI cutting site is located downstream the coding sequence. When a different enzyme is used to replace the dehalogenase, the enzyme of interest has to be either synthetized with NdeI and BamHI sites at the ends, or amplified with tailed primers that could insert those sites. Then, a directed cloning can be done, via restriction with NdeI and BamHI and subsequent ligation. NdeI cutting site is also specifically designed to be used to insert a coding sequence upstream the enzyme sequence: in our case, this is necessary to insert the sequence of a membrane proteine, building a surface expression tool.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 772
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 778
Illegal BsaI.rc site found at 804
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