Difference between revisions of "Part:BBa K3447102"
 
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<partinfo>BBa_K3447102 short</partinfo>
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<partinfo>BBa_K3447102 short</partinfo><br>
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XynA is a xylanase gene derived from <i>Bacillus subtilis</i> 168 and encodes 1, 4-endonuclide xylanase. XynD gene, derived from Bacillus subtilis 168, can specifically hydrolyze the Arabinose residues on the O-2 xylan or O-3 xylan, and assist the hydrolysis of the xylan.<br>
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P<sub>C</sub> is a constitutive promoter that expresses the downstream <i>araC</i> gene. P<sub>BAD</sub> promoter is an inducible promoter, P<sub>BAD</sub> contains sequences of P<sub>C</sub>. It was verified by iGEM 2017 Amazonas_Brazil team that the induction effect of arabinose could be realized by using only <i>araC</i> and P<sub>BAD</sub>.<br><br>
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===Usage and Biology===
 
===Usage and Biology===
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In our project, we use them to hydrolyze xylan in culture medium to produce arabinose. For more information, please visit our Design page: <b>https://2020.igem.org/Team:Jilin_China/Design</b><br>
  
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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>
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==Characterization==
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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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To verify the construction of xynA-xynD, the digestion and agarose gel electrophoresis were performed by a standard protocol (Fig. 1A).<br>
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Moreover, Acidic Xylanase Assay (DNS demonstration method) was used to determine the enzyme activity of xylanase. As shown in Fig. 1B, the xylanase was produced as expected, and the enzyme activity is 0.105 U/mL (<b>Definition of enzyme activity</b>: 1 unit, the amount of enzyme required to hydrolysis xylan to produce 1 μmol of reducing sugar per milliliter of fermentation broth, under the condition of 50℃ and pH 4.8).<br>
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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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[[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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[[Image: T--Jilin_China--Results--2.jpg|thumb|center|700px|<b>Fig. 1 Xylanase could be produced by xynA and xynD. </b>(A) Digestion and electrophoresis of xynA-xynD. (B) The absorption curve of the xylose standard at 495 nm in DNS reaction. 5 g/mL xylan was added to the supernatant, which was centrifuged and collected in xylanase producing bacteria were incubated overnight in advance, and the enzyme activity was measured by DNS method at 50℃ for 30 mins. The concentration of xylose after hydrolyzation was determined by comparing to the standard curve.]]<br>
  
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Arabinose induces endotoxin expression: (链接a01+c07)<br>
 
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Arabinose induces green fluorescent protein expression: (链接a01+c09)<br><br>
 
  
 
==<b>Design</b>==
 
==<b>Design</b>==
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===Source===
 
===Source===
  
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We found this sequence data in the previous iGEM teams (Glasgow 2017) and in GenBank.<br><br>
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We found this sequence data in the previous iGEM part (<partinfo>BBa_K1175005</partinfo>) and in GenBank.<br>
  
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===References===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3447102 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3447102 SequenceAndFeatures</partinfo>

Latest revision as of 07:52, 27 October 2020


Arabinose production
XynA is a xylanase gene derived from Bacillus subtilis 168 and encodes 1, 4-endonuclide xylanase. XynD gene, derived from Bacillus subtilis 168, can specifically hydrolyze the Arabinose residues on the O-2 xylan or O-3 xylan, and assist the hydrolysis of the xylan.

Usage and Biology

In our project, we use them to hydrolyze xylan in culture medium to produce arabinose. For more information, please visit our Design page: https://2020.igem.org/Team:Jilin_China/Design

Characterization

To verify the construction of xynA-xynD, the digestion and agarose gel electrophoresis were performed by a standard protocol (Fig. 1A).
Moreover, Acidic Xylanase Assay (DNS demonstration method) was used to determine the enzyme activity of xylanase. As shown in Fig. 1B, the xylanase was produced as expected, and the enzyme activity is 0.105 U/mL (Definition of enzyme activity: 1 unit, the amount of enzyme required to hydrolysis xylan to produce 1 μmol of reducing sugar per milliliter of fermentation broth, under the condition of 50℃ and pH 4.8).

Fig. 1 Xylanase could be produced by xynA and xynD. (A) Digestion and electrophoresis of xynA-xynD. (B) The absorption curve of the xylose standard at 495 nm in DNS reaction. 5 g/mL xylan was added to the supernatant, which was centrifuged and collected in xylanase producing bacteria were incubated overnight in advance, and the enzyme activity was measured by DNS method at 50℃ for 30 mins. The concentration of xylose after hydrolyzation was determined by comparing to the standard curve.


Design

Design Notes

We added some synonymous mutations to avoid part rules.


Source

We found this sequence data in the previous iGEM part (BBa_K1175005) and in GenBank.


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