Difference between revisions of "Part:BBa K2407303"

Revision as of 17:51, 26 October 2017

β-carotene gene

A composite part of three key genes to produce β-carotene which can be expressed in Saccharomyces Cerevisiae.

Introduction

After reviewing the literature, we understand that carotene can be overexpressed in Saccharomyces cerevisiae. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB and crtI from X. dendrorhous was sufficient to enable carotenoid production. crtYB, crtI, and crtE can complete the expression of β-carotene, and the cell is orange.

Components of the Mating switcher

To characterize our Mating Switcher, we built a gene route to switch the expression from RFP to β-carotene. In this route, we combined RFP with TEF-1 promoter. To prevent leaky expression, we choose two kinds of terminators——ADH1 and Ura3‘s. So β-carotene’s expression is controlled by promoter-vox-RFP-Terminators-vox structure. Before the mating-type switch, our yeast presents reddish color due to RFP’s expression. After the Mating Switcher, with the deletion of RFP and terminators flanked by vox locus, β-carotene expresses and the strains take on an orange color. In Saccharomyces cerevisiae, these two colors are easy to distinguish. In this way, we can easily visualize the function of our switcher, as well as measure its efficiency and error rate.

This part was amplified by using the primers indicated by René Verwaal (2007), Some of those primers are:

• crtYB-F CGC GGATCC ATGACGGCTCTCGCATATTAC
• crtYB-R TGCG GTCGAC TTACTGCCCTTCCCATCCGC
• CRTI-F GCG GGATCC ATGGGAAAAGAACAAGATCAGG
• CRTI-R TGCG GTCGAC TCAGAAAGCAAGAACACCAACG
• crtE-F CGC GGATCC ATGGATTACGCGAACATCCTC
• crtE-R TGCG GTCGAC TCACAGAGGGATATCGGCTAG

We designed different promoters in the middle of each carotene genes, and used overlap PCR to combine them together.

Sequence

The part consists of TEF1 promotor, crtE, tTDH3 promotor, crtI, tFBA1 promotor and crtYB. The sequence of the part is shown below.

Sequence and Features