Difference between revisions of "Part:BBa K3038003"

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(→‎Design)
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Thanks to Geneious software we have designed a gene with a promoter and a C-term tagged with a Flag tag. This construct doesn't heve a terminator because of it is a composite part that will be assembled with another gene with a terminator (3A Assembly). The promoter is inducible to arabinose. This allows a controlled expression of the synthetic gene to avoid any effect of toxicity. In addition, arabinose is an inexpensive inducer and very present in the laboratories of our university. The tag allows to purify and detect the protein in the host strain by using specific columns.
 
Thanks to Geneious software we have designed a gene with a promoter and a C-term tagged with a Flag tag. This construct doesn't heve a terminator because of it is a composite part that will be assembled with another gene with a terminator (3A Assembly). The promoter is inducible to arabinose. This allows a controlled expression of the synthetic gene to avoid any effect of toxicity. In addition, arabinose is an inexpensive inducer and very present in the laboratories of our university. The tag allows to purify and detect the protein in the host strain by using specific columns.
 +
 +
==Manipulations==
 +
===PCR amplification===
 +
Following the design of the synthetic gene, it is amplified by PCR thanks to the design of primers upstream and downstream of the sequence.
 +
 +
===Cloning design in pSB1A3===
 +
 +
===Cloning into pSB1A3===
 +
 +
After amplification of the synthetic gene, sample is purified, the amplicons are digested with restriction enzymes EcoRI and PstI. Similarly for the cloning vector pSB1A3 according to the protocol described above. The insert is then ligated into the plasmid.
 +
 +
 +
===Cloning into thermocompetent cells JM109===
 +
 +
The thermocompetent E. coli JM109 bacteria are then transformed and clones are obtained.
 +
 +
===PCR colony screening===
 +
 +
After bacterial transformation, colony PCR is performed with the forward and reverse primer hybridizing into the plasmid. The PCR products are loaded on 0.8% agarose gel.
 +
 +
===Expression of the recombinant protein===
 +
NI : Not induced
 +
I: Induced
 +
M: Marker
 +
The last step consist in evaluating the enzymatic activity of the protein in vitro.
  
 
==Reference==
 
==Reference==

Revision as of 08:47, 19 October 2019

Description

FadM is for E. coli long-chain acyl CoA thioesterase that is Thioesterase III.
Thioesterase III (FadM) is a long-chain acyl-CoA thioesterase that is involved in the β-oxidation of fatty acids.

https://biocyc.org/gene?orgid=ECOLI&id=G6244

For the competition, FadM is tagged in Cterm by a FLAG tag. This tag allows the purification of the protein in order to test the activity.

GenBank

FadM : GenBank: P77712
https://www.uniprot.org/uniprot/P77712

Protein Sequence

Without the Flag-tag :
MQTQIKVRGY HLDVYQHVNN ARYLEFLEEA RWDGLENSDS FQWMTAHNIA FVVVNININY RRPAVLSDLL TITSQLQQLN GKSGILSQVI TLEPEGQVVA DALITFVCID LKTQKALALE GELREKLEQM VK

Molecular size : 15.088 kDa (from nucleotide sequence)

Usage and Biology

The enzyme is able to hydrolyze a number of related substrates. The best substrate is 3,5-tetradecadienoyl-CoA, which is a minor side product of oleate β-oxidation that is resistant to further degradation. The hydrolysis product, 3,5-tetradecadienoate, is released into the growth medium [Ren04a, Nie08]. Thioesterase III is expressed upon growth on oleic acid as the sole source of carbon [Ren04a, Nie08]. FadM is a member of the fad regulon; expression is induced by a number of fatty acids, with C18:1 as the best inducer [Feng09b]. Reports disagree on whether [Nie08a] or not [Feng09b] conjugated linoleic acid (CLA) induces an even higher level of expression of fadM.

Design

Thanks to Geneious software we have designed a gene with a promoter and a C-term tagged with a Flag tag. This construct doesn't heve a terminator because of it is a composite part that will be assembled with another gene with a terminator (3A Assembly). The promoter is inducible to arabinose. This allows a controlled expression of the synthetic gene to avoid any effect of toxicity. In addition, arabinose is an inexpensive inducer and very present in the laboratories of our university. The tag allows to purify and detect the protein in the host strain by using specific columns.

Manipulations

PCR amplification

Following the design of the synthetic gene, it is amplified by PCR thanks to the design of primers upstream and downstream of the sequence.

Cloning design in pSB1A3

Cloning into pSB1A3

After amplification of the synthetic gene, sample is purified, the amplicons are digested with restriction enzymes EcoRI and PstI. Similarly for the cloning vector pSB1A3 according to the protocol described above. The insert is then ligated into the plasmid.


Cloning into thermocompetent cells JM109

The thermocompetent E. coli JM109 bacteria are then transformed and clones are obtained.

PCR colony screening

After bacterial transformation, colony PCR is performed with the forward and reverse primer hybridizing into the plasmid. The PCR products are loaded on 0.8% agarose gel.

Expression of the recombinant protein

NI : Not induced I: Induced M: Marker The last step consist in evaluating the enzymatic activity of the protein in vitro.

Reference

Engineering of Bacterial Methyl Ketone Synthesis for Biofuels. Ee-Been Goh,a,c Edward E. K. Baidoo,a,c Jay D. Keasling,a,c,d and Harry R. Beller. Appl Environ Microbiol. 2012 Jan; 78(1): 70–80. doi: 10.1128/AEM.06785-11. PMCID: PMC3255637. PMID: 22038610

Activity

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
    COMPATIBLE WITH RFC[1000]