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             <h3>Results:</h3>
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             <h3>Results</h3>
  
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            <h4>Cloning</h4>
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            <p>
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                pTeTW3con2-ptet-mCherry--sfGFP-pT7 was cloned in two steps via a restriction and
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                ligation protocol. First, the mCherry gene was cloned into the backbone pTeTW3con2. Sequencing analysis
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                was carried out to test whether the cloning was positive before the next step started. Next, the sfGFP
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                gene was cloned into the backbone (pTeTw3con2-ptet-mCherry). The cloning fo the final product was
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                checked via sequencing.
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            </p>
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            <h4>Measuring the expression levels after single induction</h4>
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            <p>
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            </p>
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            <h4>Testing various dual induction strategies</h4>
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            <p>
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            </p>
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            <h4>Tuning the expression ratio</h4>
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            <p>
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            </p>
 
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 13:02, 18 October 2019

pT7 Superfolder Green Fluorescence Protein x pTet mCherry (Convergent)

Profile

Name pTeTW3con2-ptet-mCherry--sfGFP-pT7
Base pairs 1780
Molecular weight 26.7 kDa (mCherry) + 26.8 kDa (sfGFP)
Origin Synthetic
Parts tetA promoter, T7 promoter, T7 terminator, Lac Operator, RBS, mCherry, sfGFP
Properties Dual expression of mCherry and sfGFP as reporters to monitor expression levels of the tetA and T7 site.

Usage and Biology

This composite part is a dual expression plasmid with pTeTW3con2 as backbone. It was cloned to characterize the expression levels of the two convergent expression sites. The tetA promoter is inducible with anhydrotetracycline (AHT) and the site (BBa_K3187039) encodes the protein mCherry. The T7 promoter can be induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the site (BBa_K3187040) encodes the protein sfGFP.

The fluorescence of mCherry and sfGFP can be measured and acts as expression reporters.

Results

Cloning

pTeTW3con2-ptet-mCherry--sfGFP-pT7 was cloned in two steps via a restriction and ligation protocol. First, the mCherry gene was cloned into the backbone pTeTW3con2. Sequencing analysis was carried out to test whether the cloning was positive before the next step started. Next, the sfGFP gene was cloned into the backbone (pTeTw3con2-ptet-mCherry). The cloning fo the final product was checked via sequencing.

Measuring the expression levels after single induction

Testing various dual induction strategies

Tuning the expression ratio


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 76
    Illegal XbaI site found at 1729
    Illegal PstI site found at 805
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 76
    Illegal NheI site found at 810
    Illegal PstI site found at 805
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 76
    Illegal XhoI site found at 970
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 76
    Illegal XbaI site found at 1729
    Illegal PstI site found at 805
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 76
    Illegal XbaI site found at 1729
    Illegal PstI site found at 805
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1674