Contribution

Overview

Gene transcription in the host cells is regulated by different promoters and then regulation the corresponding protein expression. So, it is important to use a proper promoter in different host cells of different species. In order to carry forward the spirit of iGEM, and inherit and spread the value of iGEM, we specially searched the iGEM Biological Parts library for related lipase and picked a biological part BBa_K319003, TEF1 promoter, submitted by iGEM10_uOttawa in 2010, and they measured the intensity of different kinds of promoters. Our team chose the TEF1 promoter to construct a plasmid that could be used to regulate our target protein in Saccharomyces cerevisiae, and adding data of the usage of TEF1 promoter. This information can be a good reference for future iGEM teams working on lipases.

Add new experimental data to an existing Part BBa_K319003, TEF1 promoter

TEF1 promoter, which is a stronger promoter and shows higher expression levels in Saccharomyces cerevisiae or other kinds of yeast cells.in this project, we amplified this promoter from the plasmid pHCas9-Nours and fused it with the ATF1 gene fragment and CYC1 terminator to regulate the expression of ATF1 to construct our engineered strain.

a) Construction of TEF1-ATF1 expression plasmids

Firstly, we amplified three fragments, which were the TEF-1 promoter, CYC1 terminator, and ATF1. The results indicated that the target DNA strands are successfully amplified (Figure 1).

Figure 1. Gel electrophoresis to identify the target DNA fragments. A. TEF1 promoter (400 bp), B. ATF1 DNA fragment (1575 bp).

In order to build our plasmids, The ATF1 was amplified from the genome of S. cerevisiae, and the TEF1 promoter and the CYC1 terminator were amplified from the pHCas9-Nours plasmid. Next, we fused the three fragments by PCR and extracted the recombinant DNA fragment (Figure 2). Then, we digested the target fragments and the pYES2 vector with SpeI and SalI, and we used T4 DNA ligase to ligate the fragments and the vector. Then we transformed the recombinant plasmids into E. coli DH5α competent cells and coated on the LB (Amp+) solid plates.

Figure 2. Gel electrophoresis results of the target gene fragments.

We verified the colonies through colony-PCR (Figure 3), and then we inoculated single colonies (1, 2, 5, and 7) and we send the constructed recombinant plasmid to a sequencing company for sequencing.

Figure 3. verification of the recombinant plasmids by colony-PCR.

Add new information to the Part BBa_K4278724, BBa_K4278723, and BBa_K4278717

a) BBa_K4278724, BAT2:

BAT2, which catalyzed the deamination of BCAAs (branched-chain amino acids), which was thought to be the first step in the degradation of BCAAs. It was reported that BAT2 affected the production of higher alcohols in an opposite manner, and when knockout of the BAT2 gene in a single-gene-deletion manner decreases the production of higher alcohols which would accumulate and increase its toxicity in the long-term fermentation process. This makes it particularly important to control the content of higher alcohols during fermentation

b) BBa_K4278723, pYES2-ATF1

This part is a composite part named pYES2-ATF1, and it is built up of the pYES2 shuttle vector (BBa_K4278716) and TEF1 pro-ATF1-CYC1 ter (BBa_K4278719). The pYES2 vector is one of the most commonly used Saccharomyces cerevisiae vectors, which can shuttle in E. coli and S. cerevisiae. The vector is a high-copy-number plasmid. When expressed in the prokaryotic system, the Amp+ resistance can be used to screen the right colony, while transformed into the S. cerevisiae, the strain should be cultured at 28-30℃. This plasmid backbone can be used to express different proteins in the future.

c) BBa_K4278717, l-tef1-atf1-cyc1-tefp-nrsr-teft-r

This composite part is the recombinant DNA fragment constructed by TEF1 promoter (BBa_K4278701), ATF1 DNA fragment (BBa_K4278702), CYC1 terminator (BBa_K4278703), TEF promoter (BBa_K4278705), NrsR DNA fragment (BBa_K4278706), and TEF terminator (BBa_K4278707). This DNA fragment also contained a homologous arm designed according to BAT2, we fused the 503bp upstream sequence of BAT2 (BBa_K4278700) at the 5’-terminal of the composite DNA sequence, and we fused the 501bp downstream sequence of BAT2 (BBa_K4278708) at the 3’-terminal. The homologous arms were used to replace the BAT2 gene fragment in the S. cerevisiae genome in a recombinant way.

Reference

Zhang J., Zhang C., Wang J., Dai L., Xiao D. (2014) Expression of the Gene Lg-ATF1 Encoding Alcohol Acetyltransferases from Brewery Lager Yeast in Chinese Rice Wine Yeast. In: Zhang TC., Ouyang P., Kaplan S., Skarnes B. (eds) Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012). Lecture Notes in Electrical Engineering, vol 249. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-37916-1_5.
Ma L, Huang S, Du L, Tang P, Xiao D. Reduced Production of Higher Alcohols by Saccharomyces cerevisiae in Red Wine Fermentation by Simultaneously Overexpressing BAT1 and Deleting BAT2. J Agric Food Chem. 2017 Aug 16;65(32):6936-6942. doi: 10.1021/acs.jafc.7b01974.
Lilly M, Bauer FF, Styger G, Lambrechts MG, Pretorius IS. The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates. FEMS Yeast Res. 2006 Aug;6(5):726-43. doi: 10.1111/j.1567-1364.2006.00057.x.
Dai L., Zhang C., Zhang J., Qi Y., Xiao D. (2014) Effects of IAH1 Gene Deletion on the Profiles of Chinese Yellow Rice Wine. In: Zhang TC., Ouyang P., Kaplan S., Skarnes B. (eds) Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012). Lecture Notes in Electrical Engineering, vol 249. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-37916-1_42.
Choi YJ, Lee J, Jang YS, Lee SY. Metabolic engineering of microorganisms for the production of higher alcohols. mBio. 2014 Sep 2;5(5):e01524-14. doi: 10.1128/mBio.01524-14.
孙中贯,刘琳,王亚平,王雪山,肖冬光.酿酒酵母高级醇代谢研究进展[J].生物工程学报,2021,37(2):429~447.
于洪梅. 气相色谱法分析啤酒中 5 种高级醇的方法研究[J]. 食品研究与开发, 2018, 39(18):5