Difference between revisions of "Part:BBa K3829010"
 
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<p>Our part BBa_K33829010 is a recombinant yeGFP improved from the part reporter GFP <a href="https://parts.igem.org/Part:BBa_K3402000">BBa_K3402000</a> (iGEM20_Jiangnan_China). We optimized the codon and added a stronger promoter <a href="https://parts.igem.org/Part:BBa_K3829001">BBa_K3829001</a> and terminator <a href="https://parts.igem.org/Part:BBa_K3829000">BBa_K3829000</a>. </p>
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<p>Our part BBa_K33829010 is a contribution to the part-reporter GFP <a href="https://parts.igem.org/Part:BBa_K3402000">BBa_K3402000</a> (iGEM20_Jiangnan_China).</p>
 
   
 
   
 
<h2>Characterization</h2>
 
<h2>Characterization</h2>
  
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<h3>Construction of plasmid Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-4609-T</h3>
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<h3>Construction of plasmid P-SS-yeGFP3-V5-4609-T</h3>
 
<p>In our project, yeGFP was used to screen anchored proteins.</p>
 
<p>In our project, yeGFP was used to screen anchored proteins.</p>
  
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<img src="https://2021.igem.org/wiki/images/d/d9/T--IvyMaker-China--Lab-05.jpg" style = "width:80%;">
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<img src="https://2021.igem.org/wiki/images/d/d9/T--IvyMaker-China--Lab-05.jpg" style = "width:70%;">
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<br/><b>Fig.1</b> Structure of Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-4609-T
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<br/><b>Fig.1</b> Structure of P-SS-yeGFP3-V5-4609-T.
 
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Through restriction enzyme digestion verification and sequencing, the plasmid was successfully constructed (Figure 2). </p>
  
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<img src="https://2021.igem.org/wiki/images/e/e4/T--IvyMaker-China--Lab-32.jpg" style = "length:30%,width:70%;">
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<br/><b>Fig.2</b> Verification of recombinant plasmids by restriction enzyme digestion.
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M: DL 15000 DNA Marker；
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1：P-SS-yeGFP3-V5-4609-T double enzyme digestion (<i>Xba</i> Ⅰ & <i>EcoR</i> Ⅰ)
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<p>The plasmid was linearized and transferred to <i>Candida tropicalis</i> . The transformants were screened out by uracil deficiency. And then cultivated the transformants and observed the fluorescence with confocal laser scanning microscopy (CLSM). As a result, green fluorescence was observed on the cell surface, indicating that yeGFP was expressed and the anchor protein 4609 performed well. (Figure 3).</p>
  
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<img src="https://2021.igem.org/wiki/images/2/2c/T--IvyMaker-China--Lab-33.jpg" style = "length:60%;">
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<br/><b>Fig.3</b> Representative images of yeGFP (P-SS-yeGFP3-V5-4609-T) expression.
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The yeast morphology observed under the bright field (Left). The yeast morphology observed under green fluorescence excitation wavelength (Middle). Merged image (Right).
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<h3>References</h3>
 
<h3>References</h3>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3829010 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3829010 Sequence And Features</partinfo>
  
  

Latest revision as of 10:11, 21 October 2021


P-ss-yeGFP-V5tag-Anchor protein 4609-T

Our part BBa_K33829010 is a contribution to the part-reporter GFP BBa_K3402000 (iGEM20_Jiangnan_China).

Characterization

Construction of plasmid P-SS-yeGFP3-V5-4609-T

In our project, yeGFP was used to screen anchored proteins.


Fig.1 Structure of P-SS-yeGFP3-V5-4609-T.

Through restriction enzyme digestion verification and sequencing, the plasmid was successfully constructed (Figure 2).


Fig.2 Verification of recombinant plasmids by restriction enzyme digestion. M: DL 15000 DNA Marker； 1：P-SS-yeGFP3-V5-4609-T double enzyme digestion (Xba Ⅰ & EcoR Ⅰ)

The plasmid was linearized and transferred to Candida tropicalis . The transformants were screened out by uracil deficiency. And then cultivated the transformants and observed the fluorescence with confocal laser scanning microscopy (CLSM). As a result, green fluorescence was observed on the cell surface, indicating that yeGFP was expressed and the anchor protein 4609 performed well. (Figure 3).


Fig.3 Representative images of yeGFP (P-SS-yeGFP3-V5-4609-T) expression. The yeast morphology observed under the bright field (Left). The yeast morphology observed under green fluorescence excitation wavelength (Middle). Merged image (Right).

References

1.Eisenhaber, Birgit, et al. "A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe." Journal of molecular biology 337.2 (2004): 243-253.

2.Möller, Steffen, Michael DR Croning, and Rolf Apweiler. "Evaluation of methods for the prediction of membrane spanning regions." Bioinformatics 17.7 (2001): 646-653.

3.Smith MR, Khera E, Wen F. “Engineering Novel and Improved Biocatalysts by Cell Surface Display.” Ind Eng Chem Res, volume 53, issue 16, 29 April 2015, pp. 4021-4032.

4.Tanaka T, Yamada R, Ogino C, Kondo A. “Recent Developments in Yeast Cell Surface Display toward Extended Applications in Biotechnology.” Appl Microbiol Biotechnol, volume 75, issue 3, August 2012, pp. 577-591.

5.Andreu C, Del Olmo ML. “Yeast Arming Systems: pros and cons of different protein anchors and other elements required for display.” Appl Microbiol Biotechnol, volume 102, issue 6, Mar 2018, pp. 2543-2561.

Sequence and Features BBa_K3829010 Sequence And Features