Difference between revisions of "Part:BBa K3402050"
| Line 13: | Line 13: | ||
[[Image:Expression of yeGFP abc.png|700px|thumb|center|a) Fluorescence intensity of different promoters. b) Transcription levels of different promoters. c) GFP was successfully expressed in cells]] | [[Image:Expression of yeGFP abc.png|700px|thumb|center|a) Fluorescence intensity of different promoters. b) Transcription levels of different promoters. c) GFP was successfully expressed in cells]] | ||
| + | |||
| + | |||
| + | |||
| + | <html> | ||
| + | |||
| + | <h2>Improvement:IvyMaker-China 2021 iGEM Team</h2> | ||
| + | |||
| + | <p>Our part BBa_K33829011 is a recombinant yeGFP improved from the part-reporter GFP <a href="https://parts.igem.org/Part:BBa_K3402050">BBa_K3402050</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> | ||
| + | |||
| + | <h3>Construction of plasmid P-SS-yeGFP3-V5-5105-T</h3> | ||
| + | <p>In our project, yeGFP was used to screen anchored proteins.</p> | ||
| + | |||
| + | <img src="https://2021.igem.org/wiki/images/7/78/T--IvyMaker-China--Lab-06.jpg" style = "width:80%;"> | ||
| + | <br/><b>Fig.1</b> Structure of P-SS-yeGFP3-V5-5105-T. | ||
| + | <br/> | ||
| + | |||
| + | <p> | ||
| + | Through restriction enzyme digestion verification and sequencing, the plasmid was successfully constructed (Figure 2). </p> | ||
| + | |||
| + | <img src="https://2021.igem.org/wiki/images/f/f5/T--IvyMaker-China--lab31.jpg" style = "length:60%;"> | ||
| + | <br/><b>Fig.2</b> Verification of recombinant plasmids by restriction enzyme digestion. | ||
| + | M: DL 15000 DNA Marker； | ||
| + | 1：P-SS-yeGFP3-V5-5105-T double enzyme digestion (<i>Xba</i> Ⅰ & <i>EcoR</i> Ⅰ) | ||
| + | <br/> | ||
| + | |||
| + | <p>After the plasmid was successfully constructed, it was introduced into the target strain <i>Candida tropicalis</i> . And then the expression of yeGFP and the position of fluorescent was confirmed with CLSM (Figure 3).</p> | ||
| + | |||
| + | <img src="https://2021.igem.org/wiki/images/8/8b/T--IvyMaker-China--Lab-29.jpg" style = "width:70%;"> | ||
| + | <br/><b>Fig.3</b> Representative images of yeGFP (P-SS-yeGFP3-V5-5105-T) expression. | ||
| + | The yeast morphology observed under the bright field (Left). The yeast morphology observed under green fluorescence excitation wavelength (Middle). Merged image (Right). | ||
| + | <br/> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | <h3>References</h3> | ||
| + | |||
| + | <p>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.</p> | ||
| + | |||
| + | <p>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.</p> | ||
| + | |||
| + | <p>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.</p> | ||
| + | |||
| + | <p>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.</p> | ||
| + | |||
| + | <p>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. | ||
| + | </p> | ||
<!-- --> | <!-- --> | ||
Revision as of 09:19, 21 October 2021
report circuit
This device is composed of promoter Ptef1 (BBa_K3402007), reporter gene yeGFP (BBa_K3402000), terminator Tsyn7 (BBa_K3402001).
Usage and Biology
The green fluorescent protein (yeGFP) gene can be used as reporter gene and express in Starmerella bombocola. We conducted this device to test if the yeGFP can express in Starmerella bombocola, so we connected this device. Besides, the hygromycin resistance gene (hph) was built into the plasmid as the marker gene to determine if the transformation is successful. Two homologous arms are used to intsert this gene into PXA1 site.
As a result, the yeGFP was succefully expressed in Starmerella bombocola. Finally, we can link different promoters to yeGFP to know the strength of promoters by testing different fluorescence intensity and transcription levels analysis of different strains.
Improvement:IvyMaker-China 2021 iGEM Team
Our part BBa_K33829011 is a recombinant yeGFP improved from the part-reporter GFP BBa_K3402050 (iGEM20_Jiangnan_China). We optimized the codon and added a stronger promoter BBa_K3829001 and terminator BBa_K3829000.
Construction of plasmid P-SS-yeGFP3-V5-5105-T
In our project, yeGFP was used to screen anchored proteins.
Fig.1 Structure of P-SS-yeGFP3-V5-5105-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-V5-5105-T double enzyme digestion (Xba Ⅰ & EcoR Ⅰ)
After the plasmid was successfully constructed, it was introduced into the target strain Candida tropicalis . And then the expression of yeGFP and the position of fluorescent was confirmed with CLSM (Figure 3).
Fig.3 Representative images of yeGFP (P-SS-yeGFP3-V5-5105-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