Part:BBa_K4012020

tSSA1 It is a terminator that ends the sequence that is resposible for the expression of tyrosine ammonia lyase

Secondary Structure

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Measurement

• [http://openwetware.org/wiki/Cconboy:Terminator_Characterization/Results How these parts were measured]

Obtaining the tSSA1 fragment and BsaI digested verification

Figure 1: [1] Results of yeast toolkit plasmids enzyme-digested verification

We construct tSSA1 with vector type8-pSB1K3-GFP and proceed digested verification using BsaI. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments tSSA1(238bp) matched with previous expectations by compared with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

tSSA1 in Level1 plasmid assembly

Figure 2: [2] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence DuLAR

The construction schematic of DuLAR sequence demonstrated as Fig.2. The initiation of the DuLAR sequence is done by promoter pPOP6, with termination done by tSSA1. The sequence ConL4 and ConRE are connector sequences within the Level 1 plasmid assembly. Furthermore, typr9-KVF and type9-VR are imposed to enact selection through colonies PCR, results shown in Fig.2 The band length 2500bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

tSSA1 in Level2 plasmid assembly

Figure 3: [3] A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway

tSSA1 is also involved in assembly of synthesis pathway of catechin, shown by Fig.3.

iGEM 2022 LINKS_China - contribution

Characterization

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).

Figure 1： promoters proved to express constant YPD throughout the experiment's time span(Reider Apel, A. et al.2017).

Collection of chromosomal locus
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.

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).

Usage for xylose-utilizing pathway
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).

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.

Usage for the production of shinorine and porphyra-334
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%.

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).

Usage and biology in the production of palythine
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).

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.

Usage and biology in the production of gadusol
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).

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.

Reference

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Jin C, Kim S, Moon S, Jin H, Hahn JS. Efficient production of shinorine, a natural sunscreen material, from glucose and xylose by deleting HXK2 encoding hexokinase in Saccharomyces cerevisiae. FEMS Yeast Res. 2021;21(7):foab053.

Chen M, Rubin GM, Jiang G, Raad Z, Ding Y. Biosynthesis and Heterologous Production of Mycosporine-Like Amino Acid Palythines. J Org Chem. 2021 Aug 20;86(16):11160-11168.

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Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, Tong GJ, Garber M, Nnadi O, Zhuang W, Hillson NJ, Keasling JD, Mukhopadhyay A. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 2017 Jan 9;45(1):496-508.

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Cress BF, Toparlak ÖD, Guleria S, et al. CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli. ACS Synth Biol. 2015;4(9):987-1000.