Difference between revisions of "Part:BBa K3038003"

(→‎FadM : Ecoli Long chain acyl CoA thioesterase)
 
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<div style="text-align:justify;">
  
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===FadM_Cter-FLAG : Ecoli Long chain acyl CoA thioesterase ===
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=FadM - Long-chain acyl CoA thioesterase=
  
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===Reference===
 
  
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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
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==Description==
  
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FadM is for <i>E. coli</i> long-chain acyl CoA thioesterase that is Thioesterase III.<br/>
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Thioesterase III (FadM) is a long-chain acyl-CoA thioesterase that is involved in the β-oxidation of fatty acids.
  
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https://biocyc.org/gene?orgid=ECOLI&id=G6244<br/>
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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.
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===GenBank===
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FadM : GenBank: P77712<br/>
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https://www.uniprot.org/uniprot/P77712
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===Protein Sequence ===   
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Without the Flag-tag :<br/>
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MQTQIKVRGY HLDVYQHVNN ARYLEFLEEA RWDGLENSDS FQWMTAHNIA
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FVVVNININY RRPAVLSDLL TITSQLQQLN GKSGILSQVI TLEPEGQVVA
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DALITFVCID LKTQKALALE GELREKLEQM VK
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Molecular size : 15.088 kDa (from nucleotide sequence)
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==Usage and Biology==
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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.
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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, and finally a terminater.  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.
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==Manipulations==
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===PCR amplification===
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Following the design of the synthetic gene, it is amplified by PCR thanks to the design of primers upstream and downstream of the sequence.
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 +
<center>https://static.igem.org/mediawiki/parts/7/72/T--Poitiers--TFadMPCRTab3.png <br>
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<strong>Electrophoresis photography following loads on agarose gel 0.8% of PCR products.</strong><br> The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is the NEB 1 kb Plus DNA Ladder. Lane 1 corresponds to the marker, lane 2 to the amplified FadM product.</center><br>
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===Enzymatic digestion and ligation in pSB1C3===
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After amplification of the synthetic gene, sample is purified, the amplicons are digested with restriction enzymes EcoRI and PstI. Similarly for the cloning vector pSB1C3. The insert (FadM) is then ligated into the plasmid.<br>
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<center>https://static.igem.org/mediawiki/parts/5/51/T--Poitiers--TLigationC3.png<br>
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<strong>Design of FadM/pSB1C3 with Geneious software.</strong><br> This map shows the pBAD promoter and its terminator flanking the coding sequence of the FadM protein. A Flag tag is also present in C-ter. Finally, in the plasmid is present and chloramphenicol resistance cassette.</center>
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===Cloning into thermocompetent cells JM109===
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The thermocompetent <i>E. coli</i> JM109 bacteria are then transformed and clones are obtained.
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<center> [[File:T--Poitiers--TFadMPCRcoloTab3.png|500px|center|]] <br>
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<strong>Clones on a selective LB medium (+ chloramphenicol 25 µg/mL) following the transformation of <i> E. coli</i> thermocompetent cells with the FadM/pSB1C3 ligations.</strong><br/></center>
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===PCR colony screening===
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After bacterial transformation, colony PCR is performed with the forward primer of FadM gene and reverse primer hybridizing into the plasmid. The PCR products are loaded on 0.8% agarose gel.
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<center>https://static.igem.org/mediawiki/parts/e/ec/T--Poitiers--TFadMclonesTab3.png <br>
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<strong>Electrophoresis photography following loads on agarose gel 0.8% of colony PCR products.</strong><br> The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is the NEB 1 kb Plus Ladder (left in the figure). Lane 1 to 10 corresponds to colony PCR performed other manipulations not study here, lane 1& to 20 corresponds to colony PCR performed on FadM/pSB1C3 ligation.</center><br>
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Clones 11, 12, 14, 16, 18, 19 and 20 have the right profile, an insert-vector fragment of 1200 pb.
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===Expression of the recombinant protein===
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NI: Not induced
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I: Induced
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M: Marker
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The last step consists in evaluating the enzymatic activity of the protein <i>in vitro</i>.
 +
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==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
  
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
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==<span class='h3bb'>Sequence and Features</span>==
 
<partinfo>BBa_K3038003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3038003 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K3038003 parameters</partinfo>
 
<partinfo>BBa_K3038003 parameters</partinfo>
 
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</div>

Latest revision as of 08:00, 21 October 2019

FadM - Long-chain acyl CoA thioesterase

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, and finally a terminater. 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.

T--Poitiers--TFadMPCRTab3.png
Electrophoresis photography following loads on agarose gel 0.8% of PCR products.
The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is the NEB 1 kb Plus DNA Ladder. Lane 1 corresponds to the marker, lane 2 to the amplified FadM product.


Enzymatic digestion and ligation in pSB1C3

After amplification of the synthetic gene, sample is purified, the amplicons are digested with restriction enzymes EcoRI and PstI. Similarly for the cloning vector pSB1C3. The insert (FadM) is then ligated into the plasmid.

T--Poitiers--TLigationC3.png
Design of FadM/pSB1C3 with Geneious software.
This map shows the pBAD promoter and its terminator flanking the coding sequence of the FadM protein. A Flag tag is also present in C-ter. Finally, in the plasmid is present and chloramphenicol resistance cassette.


Cloning into thermocompetent cells JM109

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

T--Poitiers--TFadMPCRcoloTab3.png

Clones on a selective LB medium (+ chloramphenicol 25 µg/mL) following the transformation of E. coli thermocompetent cells with the FadM/pSB1C3 ligations.

PCR colony screening

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

T--Poitiers--TFadMclonesTab3.png
Electrophoresis photography following loads on agarose gel 0.8% of colony PCR products.
The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is the NEB 1 kb Plus Ladder (left in the figure). Lane 1 to 10 corresponds to colony PCR performed other manipulations not study here, lane 1& to 20 corresponds to colony PCR performed on FadM/pSB1C3 ligation.

Clones 11, 12, 14, 16, 18, 19 and 20 have the right profile, an insert-vector fragment of 1200 pb.


Expression of the recombinant protein

NI: Not induced I: Induced M: Marker The last step consists 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


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]