Difference between revisions of "Part:BBa K3040014"
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<partinfo>BBa_K3040014 short</partinfo>
 
<partinfo>BBa_K3040014 short</partinfo>
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==Background==
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FadD promoter  is one of the regulator in the enzymes of fatty acid biosynthesis in E. coli. It is composed of two fadR recognition sites  (The sequence is slightly different from the fadR recognition site in fadBA promoter), one ArcA binding site and one CRP binding site.<br><br>
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LacI repressor and its operator together form a genetic switch, the lac operon . The switch functions when inducer molecules alter the conformation of the repressor in a specific manner. In the presence of a particular metabolite such as lactose, the repressor undergoes a conformational change that reduces its affinity for the operator.<br>
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                <img style="margin:20px auto 5px auto;" src="https://static.igem.org/mediawiki/parts/d/dd/T--NTHU_Taiwan--fadD_promoters_of_E.coli.png" width="100%">
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                <p style="color:Gray; padding:0px 30px 10px;">Figure1. The fadD promoters of E. coli.</p>
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pFadD promoter is one of the regulator in the enzymes of fatty acid biosynthesis in E. coli. It is composed of two fadR recognition sites, and one CRP binding site. The fatty acid metabolism system of E. coli is consist of many parts, fadD, fadL, fadR, fadA, etc. Interestingly, each enzyme in this family has its own different sequence of fadR recognition site in its promoter, which makes these promoter having different strength, yet can still be interchangeable for us to apply in our system.  
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==Mechanism and Design==
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Based on this fact, we tried pFadD promoter to take place of the weak pFadBA promoter. With two fadR recognition sites native in pFadD, we assume a better result in the sensitivity and a lower leakage in our pFadD promoter. Yet, we further modified pFadD promoter by replacing its CRP binding site with a FadR recognition site of the sequence of pFadBA promoter.
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Having its own different sequence of fadR binding site makes pFadD promoter having different strength with pFadBA. Based on this fact, we tried pFadD promoter to take place of the weak pFadBA promoter. Moreover, with two fadR recognition sites and various repressor binding sites, native in pFadD, we assume a better result with the lower leakage in our pFadD promoter.<br><br>
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==Reference==
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Moreover, we further modified pFadD promoter by replacing its CRP binding site with a LacI repressor binding site, which makes the rfp expression was tightly repressed in the absence of IPTG. Therefore, the hybrid promoter can be fully activated only when both the fatty acids( in our case, we add oleic acid) and IPTG are present. By doing so, we expect to achieve the lower leakage than the natural acyl-CoA responsive promoter pfadBA submitted by iGEM12_NTU-Taida (BBa_K817002).<br>
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FENG, Youjun; CRONAN, John E. Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes. PloS one, 2012, 7.9: e46275.
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==Expression in E. coli==
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The fadD-lac promoter was used to transform E. coli DH5α.<br>
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==Result==
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We can see clearly that pFadD_Lac, pFadD promoter with an additional lac binding site, has relatively low leakage and has 2-3 fold increase in expression as the fatty acid concentration rises. Though compared to the original promoter pFadBA, both pFadD and pFadD_FadR also has reduction in leakage, we assumed them not functioning since they show no changes in expression as the concentration of fatty acid rise.<br>
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        <div class="col-lg" style="margin:auto;text-align:center;">
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                <img style="margin:20px auto 5px auto;" src="https://static.igem.org/mediawiki/parts/8/8d/T--NTHU_Taiwan--Protein_Expression.png" width="50%">
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                <p style="color:Gray;  padding:0 10% 30px;">Figure 5. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac (n=3).</p>
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                <img style="margin:20px auto 5px auto;" src="https://static.igem.org/mediawiki/parts/b/b0/T--NTHU_Taiwan--Protein_Expression_of_fadD.png" width="50%">
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                <p style="color:Gray; padding:0 10% 30px;">Figure 6. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac after 16 hours of induction under different fatty acid concentration (n=3).</p>
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3040014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3040014 SequenceAndFeatures</partinfo>
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3040014 parameters</partinfo>
 
<partinfo>BBa_K3040014 parameters</partinfo>
 
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Revision as of 11:08, 19 October 2019


pFadD promoter with pLac regulating downstream RFP

Background

FadD promoter is one of the regulator in the enzymes of fatty acid biosynthesis in E. coli. It is composed of two fadR recognition sites (The sequence is slightly different from the fadR recognition site in fadBA promoter), one ArcA binding site and one CRP binding site.

LacI repressor and its operator together form a genetic switch, the lac operon . The switch functions when inducer molecules alter the conformation of the repressor in a specific manner. In the presence of a particular metabolite such as lactose, the repressor undergoes a conformational change that reduces its affinity for the operator.

Figure1. The fadD promoters of E. coli.

Mechanism and Design

Having its own different sequence of fadR binding site makes pFadD promoter having different strength with pFadBA. Based on this fact, we tried pFadD promoter to take place of the weak pFadBA promoter. Moreover, with two fadR recognition sites and various repressor binding sites, native in pFadD, we assume a better result with the lower leakage in our pFadD promoter.

Moreover, we further modified pFadD promoter by replacing its CRP binding site with a LacI repressor binding site, which makes the rfp expression was tightly repressed in the absence of IPTG. Therefore, the hybrid promoter can be fully activated only when both the fatty acids( in our case, we add oleic acid) and IPTG are present. By doing so, we expect to achieve the lower leakage than the natural acyl-CoA responsive promoter pfadBA submitted by iGEM12_NTU-Taida (BBa_K817002).

Expression in E. coli

The fadD-lac promoter was used to transform E. coli DH5α.

Result

We can see clearly that pFadD_Lac, pFadD promoter with an additional lac binding site, has relatively low leakage and has 2-3 fold increase in expression as the fatty acid concentration rises. Though compared to the original promoter pFadBA, both pFadD and pFadD_FadR also has reduction in leakage, we assumed them not functioning since they show no changes in expression as the concentration of fatty acid rise.

Figure 5. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac (n=3).

Figure 6. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac after 16 hours of induction under different fatty acid concentration (n=3).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 30
    Illegal suffix found in sequence at 230
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 30
    Illegal SpeI site found at 231
    Illegal PstI site found at 245
    Illegal NotI site found at 36
    Illegal NotI site found at 238
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 30
    Illegal suffix found in sequence at 231
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 30
    Illegal XbaI site found at 45
    Illegal SpeI site found at 231
    Illegal PstI site found at 245
    Illegal AgeI site found at 863
    Illegal AgeI site found at 975
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters