Team:Jiangnan China/Engineering
The core of our project is to construct a recombinant strain of S. Bombicola to produce the sophorolipid with our expectation. Step by step, we faced a lot of problems and failures. With careful consideration and effective improvement, finally we achieved engineering success.
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Promoters Screening
Background:
We know that involvement of strong promoters in carrier construction could enhance the expression level of the corresponding genes, while weak promoters correspond to inefficient expression.[1] So, how to acquire the promoters with different transcriptional level? How to know the expression intensity of different promoters?
Research:
Our research focused on finding an appropriate reporter gene to express in S. Bombicola. Then we can define a Promoters Screening Method to measure the translation intensity of different promoters. We found that the green fluorescent protein (GFP) was usually used for a template protein to determine promoter translation intensity and was convenient to observe and test,[2] so it may be a good choice.
Imagine
The green fluorescent protein (GFP) gene can be expressed in S. bombocola and be used as reporter gene to measure the translation level of different promoters.
Design
Design the GFP expression cassettes. One promoter was used to express GFP and one terminator was used to terminate its expression.
Bulid
After the comparison and analysis of S. bombicola’ s genome. Contrast analysis was carried out with DNAMAN to obtain about 1500 bp upstream of each gene initiation codon as its functional region of promoter. By designing specific primers, the genome of S. Bombicola ATCC 22214 was used as the template. Gene sequences of 14 promoters were obtained from Polymerase Chain Reaction (PCR).
Amplify the GFP gene and terminator Tsyn7 using PCR.
The fusion fragment “promoter-GFP-terminator” was assembled by fusion PCR and inserted into the plasmids pPHP to obtain the recombinant plasmids. These plasmids were respectively linearized.
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Fig1. GFP expression cassette
Test
The recombinant yeast was cultured on YPD plate added hygromycin B. After 48 hours of incubation, the existed colonies would be conducted fluorescence observation and transcription level analysis. However, none of the transformants showed fluorescence and expressed mRNA.
Learn
This result might be related to low conversion efficiency. But one more important reason is that S. bombicola is eukaryote, it is complex for heterologous gene to express in it.
Improve
We needed to adopt codon optimization[4] to improve the expression level of GFP. As a result, the GFP gene was successfully heterogeneously expressed in S. bombicola.
Research:
Some researches were done to find the most frequently used codon in S. bombicola.[5]
Imagine:
The green fluorescent protein (GFP) gene can be expressed in S. bombocola and be used as reporter gene to measure the translation level of different promoters.
Design
The optimum codons were used to optimize the GFP gene. Then we used one acquired promoter to express GFP in S. bombicola. If the GFP was successfully expressed in S. bombicola, then other promoters were also going to be used to express GFP, respectively.
Bulid
The 14 promoters obtained from the S. bombicola were amplified using PCR.
Obtain the GFP gene and terminator Tsyn7 by PCR.
The fusion fragment “promoter-GFP-terminator” was assembled by fusion PCR and inserted into the plasmid pPHP to obtain the recombinant plasmids. These plasmids were respectively linearized.
Test
After incubation for 48 hours, 14 kinds of transformants strains were acquired. Then through the analysis of fluorescence and transcription level, we successfully defined the intensity of different promoters.
Fig2. FITC-Mean of promoters
Fig3. RT-qPCR of promoters
Learn
The 14 promoters obtained from S. bombicola have shown different intensity. The promoter Ptef1 was the strongest promoter.
