Difference between revisions of "Part:BBa K124002"

Revision as of 15:05, 12 October 2022

Yeast GPD (TDH3) Promoter

Regulatory region spanning 680 bp upstream of the start codon of the GPD1 gene in yeast.

Sequence and Features

Group: Tel-Hai 2017
Author: Yaakov Bulka
Summary: we added information about the promoter and on its mechanism.

This is a strong constitutive yeast expression promoter from glyceraldehyde 3-phosphage dehydrogenase. Also called TDH3 or GAPDH. Studies have found the promoter activity of TDH3 decreased significantly when glycerol or xylose was supplied as the carbon source and at high temperatures (42 °C) . Oxygen conditions had non-significant effect.

About the protein - the promoter was taken from the Glyceraldehyde-3-phosphate dehydrogenase peptide (GAPDH), that involves in glycolysis and gluconeogenesis. The protein is a tetramer that catalyzes the reaction of glyceraldehyde-3-phosphate to 1,3 bis-phosphoglycerate, detected in the cytoplasm and cell wall. GAPDH-derived antimicrobial peptides secreted by S. cerevisiae are active against a wide variety of wine-related yeasts and bacteria.

Partow, S., Siewers, V., Bjørn, S., Nielsen, J., & Maury, J. (2010). Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast, 27(11), 955-964.‏

Yang, C., Hu, S., Zhu, S., Wang, D., Gao, X., & Hong, J. (2015). Characterizing yeast promoters used in Kluyveromyces marxianus. World Journal of Microbiology and Biotechnology, 31(10), 1641-1646.‏

Tianjin 2021's characterization

Group: Tianjin 2021
Author: Ruiqi Liu
Summary: we characterized TDH3 Promoter’s strength in 4742 yeast.

GFP is an important reporter gene used in our project. We integrated GFP gene into yeast’s chromosomes, and used it to represent the degradation of chromosomes. In order to verify whether the TDH3 promoter can express the GFP signal intensity required in experiment, we characterized the TDH3 promoter’s efficiency. We constructed a GFP fragment that was controlled by the TDH3 promoter and bound it to the chromosome genome of 4742 yeast. We measured the GFP expression levels of yeast at 15h, 20h,25h and 30h using microplate analyzer. Meanwhile, we used 4742 yeast (no GFP fragment) as control group.

Fig 1. Gene part using GFP as reporter gene to characterize TDH3 promoter strength

We use GFP as reporter gene to test the intensity of TDH3 promoter, and draw a bar chart with fluorescence intensity /OD as the ordinate and time as the abscissa. The excitation/emission wavelength of GFP in the microplate detector was set as 488/535nm.

Fig 2. Characterization of TDH3 promoter’s strength

NEFU_China 2022's characterization

Group: NEFU_China 2022
Author: Qi Li
Summary: We characterized TDH3 Promoter’s strength in wat21 yeast.

mCherry is a red fluorescent protein derived from [mushroom coral] that is commonly used to label and trace certain molecules and cellular components. Compared with other fluorescence, the advantage of mCherry lies in its color and the most widely used green fluorescent protein (GFP) can be co-labeled, and mCherry also has excellent light stability compared with other monomeric fluorescent proteins, which is more excellent than other fluorescent protein labels. Among all the red fluorescent proteins, mCherry is quite popular and has been cited in over 200 articles.

Our team took mCherry as a reporter gene to characterize the strength of the promoter, detected its mCherry content by microplate reader, and obtained the following figure, in which P2 stands for the Yeast TDH3 promoter.

iGEM 2022 LINKS_China - contribution

In our project, we successfully employed a series of promoters and terminators from the yeast toolkit to express the necessary gene sequence and produce the target product. For our project's promoters, we selected pTDH3, pPGK1, and pTEF2 due to their stability in function. They all can be expressed efficiently and stably in YPD medium after homologous recombination in the yeast genome. Amongst the three promoters, pTDH3 is the most optimal due to its characteristics of high and stable expression , while the rest is followed by pPGK1, and pTEF2 (figure1).

[[Image:t-links-china-Contributions1|thumb|left|900px| Figure 1： promoters proved to express constant YPD throughout the experiment's time span(Reider Apel, A. et al.2017).

Our project used CRISPR cleavage and insertion experiments based on Apel's paper to successfully verify that the His3, 308, and 106 loci in the paper have high DNA insertion efficiency. We used His3 to produce xyl1, xyl2, and xyl3. For gadusol, we used 308 insertion position to produce EEVS and DDGS that further allows us to build our gene circuit for gadusol production. Last but not least, for shinorine and porphyra-334 production, we inserted gene at position 106 that further optimized the pathway for producing them.

[[Image:t-links-china-Contributions2|thumb|left|900px| Figure 2：Effect of chromosomal locus upon integration efficiency and reporter protein expression. We integrated a GFP reporter cassette into 23 chromosomal sites of S. cerevisiae to analyze the integration efficiency and reporter protein expression associated with each locus. (A) The GFP reporter cassette (PTEF1-GFP-TADH1) was integrated into each site (indicated by pink arrows) using a Cas9-sgRNA plasmid (pCut)(Reider Apel, A. et al.2017).

When we introduce exogenous xylose-utilizing pathway from Scheffersomyces stipitis to SC.L1（the S. Cerevisiae strain we constructed), we used pTDH3 to express xyl1 gene, pPGK1 for xyl2 gene, and pTEF2 for expressing the xyl3 gene; furthermore, we used tTDH1, tPGK1, and tSSA1 respectively for our gene circut. We used yeast toolkit (Lee, 2015) to assemble the Level 1 plasmid concluding the promoter, coding sequence, and terminator. Then we used PCR amplification to obtain the DNA fragment Xyl1, Xly2, Xly3 from Level1 plasmid, and two homology arm(LA and RA)from yeast genome(Fig.3B). The five fragments are transformed into the SC.L1 strain along with a pCRCT-His3 plasmid that will cut open the yeast genome at the His 3 position. The recombinant strains were identified by pcr and sequencing(Fig.3C and D), and the pCRCT plasmid was discarded using URA nutrient deficient medium. We name the strain with Xyl 1, 2, 3 inserted at His3 position SC.L2 (Figure 3).

[[Image:t-links-china-Contributions3|thumb|left|900px| Figure 3：Insertion of Xyl 1, Xyl 2, Xyl 3 to introduce a xylose-utilizing metabolic pathway. By transforming CRISPR-His3 plasmid pCRCT-His3, His3 Left Arm (LA), xyl1, xyl2, xyl3, and His3 Right Arm (RA), the three genes are inserted at His3 loci (A). We expanded the homogenous arms, xyl1, xyl2, and xyl3 genes through PCR and transformed them into L1 strains for it to be assembled in the genome. We performed colony PCR on the yeast colonies to determine the existence of LA-xyl1, xyl2, and xyl3-RA (C) and verified this result through the sequencing testing (D), obtaining the L2 strain.

In order to produce shinorine and porphyra-334, we used the promoter pTDH3 for ATP-grasp ligase (AGL) and pPGK1 for D-Ala-D-Ala ligase (ALAL). After selection, we used yeast toolkit (Lee, 2015) to assemble the Level1 plasmids containing only one gene(AGL or ALAL), and used Golden Gate assembly on the basis of Level1 to construct Level2 plasmid containing both AGL and ALAL. Finally, we transformed nine Level2 plasmids containing AGL and AlaL into SCL.3 strains that yield SC.L5 series. From our previous experiments, we already obtained plasmid Np5598-NlmysD to produce porphryra-334 and shinorine producing plasmid Np5598-Np5597 that can be transferred into the L3 and L6 strains. Through mass production of our design, we concluded that in comparison with the L3 assembly, porphyra-334 production in L6 yeast increased by 91.8%, and shinorine production increased by 70.9%.

[[Image:t-links-china-Contributions4|thumb|left|900px| Figure 4：Shinorine and porphyra-334 production after optimization. We transformed porphyra-334 producing plasmid Np5598-NlmysD and shinorine producing plasmid Np5598-Np5597 into the L3 and L6 strains (A) and compared the absorption spectrum after 72 hours of fermentation. The absorption peak at 334nm of L7 strains displayed significant improvement compared to L5 strains (B) . From OD334, we concluded that in comparison with the L5 assembly, porphyra-334 production in L6 yeast increased by 91.8%, and shinorine production increased by 70.9% (C).

After obtaining shinorine and porphyra-334, we produce the MAA palythine by removing a carboxyl group from shinorine. In order to produece palythine, we added pTEF2-NlmysH-tSSA1 to the Np5598-Np5597 combination to obtain the genetic circuit. We constructed the plasmid and transformed it into SC.L3 or SC.L6, obtaining L3:Np5598-Np5597-NlmysH and L6: Np5598-Np5597-NlmysH, a new circuit of NlmysH based upon the previous construction. We also compared the production in L3:Np5598-Np5597-NlmysH and L6:Np5598-Np5597-NlmysH using OD 320 value, which shows that L6 strains are much more productive than L3 strains. The OD 320 value of L6 is 2.62 times of L3 strains, whereas L3 strains barely had any increase in UV absorption at 320 nm. This shows that we have achieved palythine production, and also shows that our previous optimization of L3 is successful (Figure 5).

[[Image:t-links-china-Contributions5|thumb|left|900px| Figure 5: Palythine production in L6 and L3 strains. We transferred palythine plasmid Np5598-Np5597-NlmysH into the L3 and L6 strains (A). After 72 hours of fermentation, OD scanning results show that an absorption peak at was clear, but only in L6 strains (C). OD 320 value shows that L6 strains were much more effective in absorbing UV at 320 nm compared to L3 strains, and the value of OD320 of L6 is 2.62 times the control, justifying the production and optimization of palythine.

To produce gadusol, we chose the genes EEVS and MTOX from Danio rerio, which converts 4DG into gadusol. We used promoters pTDH3 to express EEVS and pPGK1 to express MTOX and inserted these genes into L2 yeast at position 308 to obtain L4 strain. Our project used CRISPR cleavage and insertion experiments based on Apel's paper, and we successfully verified that the His3, 308, and 106 loci in the paper have high DNA insertion efficiency, thus proving effectiveness for our production of gadusol (figure 6).

[[Image:t-links-china-Contributions6|thumb|left|900px| Figure 6:Insertion of DDGS and OMT at 308 position. By transforming CRISPR-308 plasmid pCRCT-308, LA, DDGS, OMT and RA, the two genes should be inserted at 308 position (A). We expanded the homogenous arms, DDGS and OMT genes through PCR and transformed them into L2 strains for it to be assembled in the genome. We performed colony PCR on the yeast colonies to determine the existence of LA-DDGS and OMT (C) and verified this result through the sequencing testing (D), obtaining the L3 strain.

Reider Apel, A., d'Espaux, L., Wehrs, M., Sachs, D., Li, R. A., Tong, G. J., Garber, M., Nnadi, O., Zhuang, W., Hillson, N. J., Keasling, J. D., & Mukhopadhyay, A. (2017). A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic acids research, 45(1), 496–508.