Difference between revisions of "Part:BBa K5109001"

Latest revision as of 14:48, 27 September 2024

Dehalogenase type II intracellular expression tool

This part is a tool created 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_K5109009) is a coding sequence of a Dehalogenase enzyme to which we added bases at the ends, to insert the sequence of some desired restriction sites. 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.

Usage and Biology

We started our experiments using E. coli strain Dh5a, but we weren't able to obtain any colonies. The first positive responses arrived when we switched from Dh5a to TOP F' strain: since this strain contains the expression cassette of LacI, which is constitutively expressed, we figured out that the absence of LacI induces a constant production of the protein that might result too stressful for the cells, or instead even toxic. We decided to continue our cloning using this strain.

Dehalogenase type II was chosen in the optic of a bioremediation project, to try using it to degrade PFAS molecules. Dehalogenases are known to be able to break C - F bond, releasing fluoride ion, therefore we choose to go on with the experiments focusing on the extracellular expression of Dehalogenase type II, to relieve stress on the host cells by allowing the enzyme to function outside of the cell, by expressing it with a surface display system that links it to the outer membrane surface.

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 772
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 778
Illegal BsaI.rc site found at 804

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 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 (PARTE CREATA
-BBa_K5109009
-Dehalogenase type II with NdeI and BamHI at the ends
-Coding sequence of Dehalogenase type II from part BBa_K3347010, with added bases to insert NdeI cutting site upstream and BamHI cutting site downstream.
 Dehalogenase type II (BBa_K5109009) is a coding sequence of a Dehalogenase enzyme to which we added bases at the ends, to insert the sequence of some desired restriction sites. 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.
-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.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+Usage and Biology
 We started our experiments using E. coli strain Dh5a, but we weren't able to obtain any colonies. The first positive responses arrived when we switched from Dh5a to TOP F' strain: since this strain contains the expression cassette of LacI, which is constitutively expressed, we figured out that the absence of LacI induces a constant production of the protein that might result too stressful for the cells, or instead even toxic.
 We decided to continue our cloning using this strain.