Difference between revisions of "Part:BBa K3402000"
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| − | <p>Our part BBa_K33829010 | + | <p>Our part <a href="https://parts.igem.org/Part:BBa_K33829010">BBa_K33829010</a> and <a href="https://parts.igem.org/Part:BBa_K33829011">BBa_K33829011</a> are improved from the part-reporter GFP BBa_K3402000(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> |
| − | + | ==Characterization== | |
<h3>Construction of plasmid P-SS-yeGFP3-V5-4609-T</h3> | <h3>Construction of plasmid P-SS-yeGFP3-V5-4609-T</h3> | ||
Revision as of 05:26, 21 October 2021
yeGFP
The yeGFP can express green fluorescence protein, which can be used as a report gene to characterize the strength of promoters.
Usage and Biology
We link different promoters to yeGFP to express green fluorescent protein, so we will know the strength of promoters by testing different fluorescence intensity of different promoters
Contribution:IvyMaker-China 2021 iGEM Team
Our part BBa_K33829010 and BBa_K33829011 are improved from the part-reporter GFP BBa_K3402000(iGEM20_Jiangnan_China). We optimized the codon and added a stronger promoter BBa_K3829001 and terminator BBa_K3829000.
==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).
Construction of plasmid P-SS-yeGFP3-V5-5105-T
In our project, yeGFP was used to screen anchored proteins.
Fig.4 Structure of P-SS-yeGFP3-V5-5105-T.
Through restriction enzyme digestion verification and sequencing, the plasmid was successfully constructed (Figure 5).
Fig.5 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 6).
Fig.6 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