Team:HokkaidoU Japan/Promoter/Results

From 2013.igem.org

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<h1>Result</h1>
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<h2>-35 region randomization</h2>
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<p>We randomized -35 region by PCR primers with random hexamer region. The template DNA was consensus_promtoer-B0034-mRFP1-B0015 (about 1,000 bp). We assayed the constructed sequences and isolated 10 distinct promoters. We sequenced the randomized promoter sequences to confirm that only -35 regions was changed.</p>
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</p>
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<div class="fig fig400">
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  <img src="https://static.igem.org/mediawiki/2013/c/c1/HokkaidoU2013_promoter_Result-fig1.png">
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  <div>Figure  randomized promoter sequences.</div>
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</div>
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<h2>promoter assay; mRFP1, LacZ and Kanamycin resistance gene</h2>
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<h3>mRFP1</h3>
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<p>mRFP1 expressing JM109 colonies were resuspend to 2 ml LBC liquid culture.
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After cultivation (180 rpm shaking at 37C) for 12 hrs, we measured OD650 with micro titer plate reader. We avoided using 600 nm because mRFP1 absorbs 600 nm. mRFP1 expression was measured with FIM.
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All 10 of the promoters were characterized. Five promoters were used as a reference.
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</p>
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<p>Reference promoters are following<p>
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<ul>
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  <li>BBa_R0010: pLac</li>
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  <li>BBa_R0040: TetR</li>
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  <li>BBa_J23106: constitutive promoter family member (1185 arb. unit)</li>
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  <li>BBa_J23112: constitutive promoter family member ( 1 arb. unit)</li>
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  <li>Negative control: not protein expression construct</li>
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</ul>
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<div class="fig fig400">
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  <img src="https://static.igem.org/mediawiki/2013/7/73/HokkaidoU2013_promoter_Result-fig2.png">
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  <div>Figure  mRFP1 assay result</div>
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</div>
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<h3>promoter selection by modeling</h3>
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<p>We chose 5 of 10 promoters by the value of theoretical transcription efficiency. This efficiency is affected by binding energy in our assumption.</p>
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<div class="fig fig400">
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  <img src="https://static.igem.org/mediawiki/2013/b/b2/HokkaidoU2013_promoter_Modeling_fig5.png">
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  <div>Figure  Theoretical transcription efficiency distribution</div>
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</div>
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<h3>LacZ&alpha;</h3>
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<p>
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We selected five promoters from our original family to model. LacZ&alpha;
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Only these promoters were characterized using LacZ assay.
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LacZ (&beta;-galactosidase) activity was measured with &beta;-galactosidase assay kit. (OZ Biogenesis
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http://www.funakoshi.co.jp/data/datasheet/OZB/GC-10002.pdf )
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DH5&alpha; strain was used.
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</p>
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<div class="fig fig400">
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  <img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_promoter_Result-fig4.png">
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  <div>Figure  &beta;-galactosidase assay result</div>
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</div>
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<p>
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These data was compared with modeling data (logarithm of transcription efficiency, t. e.).
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BBa_K1084010 and BBa_K1084009 couldn't be characterized by mRFP1 assay.
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</p>
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<div class="fig fig800">
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  <img src="https://static.igem.org/mediawiki/2013/e/eb/HokkaidoU2013_promoter_Result-fig5.png">
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  <div>Figure  Comparison of assay results and modeling data</div>
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</div>
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<h3>Kanamycin resistance gene</h3>
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<p>Kanamycin resistance gene is expressed by these promoters as POK construct.</p>

Revision as of 02:07, 28 September 2013

Maestro E.coli

Promoter

Result

-35 region randomization

We randomized -35 region by PCR primers with random hexamer region. The template DNA was consensus_promtoer-B0034-mRFP1-B0015 (about 1,000 bp). We assayed the constructed sequences and isolated 10 distinct promoters. We sequenced the randomized promoter sequences to confirm that only -35 regions was changed.

Figure randomized promoter sequences.

promoter assay; mRFP1, LacZ and Kanamycin resistance gene

mRFP1

mRFP1 expressing JM109 colonies were resuspend to 2 ml LBC liquid culture. After cultivation (180 rpm shaking at 37C) for 12 hrs, we measured OD650 with micro titer plate reader. We avoided using 600 nm because mRFP1 absorbs 600 nm. mRFP1 expression was measured with FIM. All 10 of the promoters were characterized. Five promoters were used as a reference.

Reference promoters are following

  • BBa_R0010: pLac
  • BBa_R0040: TetR
  • BBa_J23106: constitutive promoter family member (1185 arb. unit)
  • BBa_J23112: constitutive promoter family member ( 1 arb. unit)
  • Negative control: not protein expression construct
Figure mRFP1 assay result

promoter selection by modeling

We chose 5 of 10 promoters by the value of theoretical transcription efficiency. This efficiency is affected by binding energy in our assumption.

Figure Theoretical transcription efficiency distribution

LacZα

We selected five promoters from our original family to model. LacZα Only these promoters were characterized using LacZ assay. LacZ (β-galactosidase) activity was measured with β-galactosidase assay kit. (OZ Biogenesis http://www.funakoshi.co.jp/data/datasheet/OZB/GC-10002.pdf ) DH5α strain was used.

Figure β-galactosidase assay result

These data was compared with modeling data (logarithm of transcription efficiency, t. e.). BBa_K1084010 and BBa_K1084009 couldn't be characterized by mRFP1 assay.

Figure Comparison of assay results and modeling data

Kanamycin resistance gene

Kanamycin resistance gene is expressed by these promoters as POK construct.

Methods
Conclusion

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