Difference between revisions of "Part:BBa K3447102"
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Arabinose operon is composed of AraC protein, P<sub>BAD</sub> and P<sub>C</sub>. The <i>araC</i> gene translates from the P<sub>C</sub> to the left.<br>
 
Arabinose operon is composed of AraC protein, P<sub>BAD</sub> and P<sub>C</sub>. The <i>araC</i> gene translates from the P<sub>C</sub> to the left.<br>
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We used the P<sub>C</sub> promoter to express AraC constitutively, and constituted the sensing part of the arabinose sensing system.<br>
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We used the P<sub>C</sub> promoter to express AraC constitutively, and constituted the sensing part of the arabinose sensing system.<br><br>
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In the arabinose operon, it is regulated by the AraC operon and activates the expression of downstream arabinose metabolism genes in the presence of arabinose inducers.<br>
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The L-Arabinose operon is naturally present in <i>E. coli</i>.The regulatory protein AraC ACTS as a dimer. The operon contains two cis-type control elements: The PC promoter for the synthesis of AraC and the P<sub>BAD</sub> for the synthesis of the enzyme needed for the catabolism of L-Arabinose. PBAD contains three half sites, each of which binds to a subunit of ARAC-O2, I1, and I2. The O1 site is composed of O1L and O1R half sites that bind two subunits. Half of O2 is in the araC coding region.
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AraC gene expression product belongs to araC protein family, which is a type of two monomers with dimer structure, and each monomer with DNA binding sites of the spiral screw - corner - functional areas. It’s amino terminal determines the inducibility and it’s carboxyl terminal determines the DNA binding ability. Through this helix-rotation-helix structure, araC contacts and binds to four large sulcus regions adjacent to the DNA. <br>
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We connected the target genes downstream of P<sub>BAD</sub> to constitute the effect part of arabinose sensing system, and constructed the whole arabinose sensing system on the same plasmid with P<sub>C</sub> and <i>araC</i>.<br>
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[[Image: Diagram of araC binding with α-L-Arabinose.|center|frame|100px|<b>Figure 1. Diagram of araC binding with α-L-Arabinose. </b> (a)α-L-arabinose, α-D-arabinose and β-D-arabinose structures. (b) A wild-type AraC combined with L-Arabinose (LA) binds to the crystalline structure of the pocket. The amino acids identified here are residues that play an important role in ligand binding.]]<br><br>
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The dimerization domain is formed by folding into a pocket, combining arabinose, and dimerized by an antiparallel coiled coil. AraC stimulates RNA polymerase binding to DNA and the rate of formation of open complexes. The presence or absence of arabinose may alter the DNA contact of the upstream subunit of homodimer AraC, thereby affecting the binding of RNA polymerase. In the absence of L-arabinose, the DNA-binding domain (DBD) of the AraC dimer binds the I<sub>1</sub> and O<sub>2</sub> halves (separated by 210 bases) and inhibits transcription by forming a DNA ring upstream of the dimer. After P<sub>BAD</sub> binds to L-arabinose, the dimer changes the conformation, making DBD bind to the adjacent I<sub>1</sub> and I<sub>2</sub> halves, thus leading to transcriptional activation through interaction with RNA polymerase in P<sub>BAD</sub>. The regulatory characteristics of AraC are due to the sensitive conversion between the two conformation Pr and Pi, and the specific interaction with the inducer.<br>
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[[Image: Schematic diagram of araBAD gene induced expression mechanism|center|frame|100px|<b>Figure 2. Schematic diagram of araBAD gene induced expression mechanism</b> ]]<br><br>
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[[Image: AraC action mechanism diagram|center|frame|100px|<b>Figure 3. AraC action mechanism diagram</b> ]]<br><br>
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In our project, we used this part together with P<sub>C</sub> and P<sub>BAD</sub> to form the arabinose sensing system (链接a01), and achieved different functions by linking different target genes downstream of P<sub>BAD</sub>.<br>  
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Arabinose induces endotoxin expression: (链接a01+c07)<br>
 
Arabinose induces endotoxin expression: (链接a01+c07)<br>
 
Arabinose induces green fluorescent protein expression: (链接a01+c09)<br><br>
 
Arabinose induces green fluorescent protein expression: (链接a01+c09)<br><br>

Revision as of 06:47, 25 October 2020


Arabinose production

PC is a constitutive promoter that expresses the downstream araC gene. PBAD promoter is an inducible promoter, PBAD contains sequences of PC. It was verified by iGEM 2017 Amazonas_Brazil team that the induction effect of arabinose could be realized by using only araC and PBAD.

Usage and Biology

Arabinose operon is composed of AraC protein, PBAD and PC. The araC gene translates from the PC to the left.
We used the PC promoter to express AraC constitutively, and constituted the sensing part of the arabinose sensing system.

AraC gene expression product belongs to araC protein family, which is a type of two monomers with dimer structure, and each monomer with DNA binding sites of the spiral screw - corner - functional areas. It’s amino terminal determines the inducibility and it’s carboxyl terminal determines the DNA binding ability. Through this helix-rotation-helix structure, araC contacts and binds to four large sulcus regions adjacent to the DNA.

File:Diagram of araC binding with α-L-Arabinose.
Figure 1. Diagram of araC binding with α-L-Arabinose. (a)α-L-arabinose, α-D-arabinose and β-D-arabinose structures. (b) A wild-type AraC combined with L-Arabinose (LA) binds to the crystalline structure of the pocket. The amino acids identified here are residues that play an important role in ligand binding.


The dimerization domain is formed by folding into a pocket, combining arabinose, and dimerized by an antiparallel coiled coil. AraC stimulates RNA polymerase binding to DNA and the rate of formation of open complexes. The presence or absence of arabinose may alter the DNA contact of the upstream subunit of homodimer AraC, thereby affecting the binding of RNA polymerase. In the absence of L-arabinose, the DNA-binding domain (DBD) of the AraC dimer binds the I1 and O2 halves (separated by 210 bases) and inhibits transcription by forming a DNA ring upstream of the dimer. After PBAD binds to L-arabinose, the dimer changes the conformation, making DBD bind to the adjacent I1 and I2 halves, thus leading to transcriptional activation through interaction with RNA polymerase in PBAD. The regulatory characteristics of AraC are due to the sensitive conversion between the two conformation Pr and Pi, and the specific interaction with the inducer.

File:Schematic diagram of araBAD gene induced expression mechanism
Figure 2. Schematic diagram of araBAD gene induced expression mechanism


File:AraC action mechanism diagram
Figure 3. AraC action mechanism diagram


In our project, we used this part together with PC and PBAD to form the arabinose sensing system (链接a01), and achieved different functions by linking different target genes downstream of PBAD.


Arabinose induces endotoxin expression: (链接a01+c07)
Arabinose induces green fluorescent protein expression: (链接a01+c09)

Design

Design Notes

We added some synonymous mutations to avoid part rules.


Source

We found this sequence data in the previous iGEM teams (Glasgow 2017) and in GenBank.

References

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 22
    Illegal NheI site found at 45
    Illegal NheI site found at 166
    Illegal NheI site found at 891
    Illegal NheI site found at 914
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 76
    Illegal BsaI.rc site found at 570
    Illegal BsaI.rc site found at 1657
    Illegal BsaI.rc site found at 1817
    Illegal SapI.rc site found at 624