Part:BBa_K2220004:Experience

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Applications of BBa_K2220004

We ligated this part to shuttle plasmid pYD1 and did the colony PCR to verify the efficiency(Fig.1). Meanwhile, the sequencing results further confirmed that we successfully cloned the expression vectors.

Fig.1 Electrophoresis result of BBa_K2220004 expression vector.

We successfully transfected the correct carrier into S.cerevisiae EBY100 and induced expression.By culturing and inducing control group(EBY100-pYD1) and experimental group(EBY100-FliC(XynA)) in same initial concentration in SG-CAA medium (plus 5% xylan), the same volume supernatant were collected every two hours from 10h to 18h to examine the concentration of reducing sugar by DNS method. The results are shown as below.

Fig.2 Xylnase enzyme activity assay curve.

The value of OD₅₄₀ are positive correlation with the concentration of reducing sugar under DNS method. So here we use the value of OD₅₄₀ to estimate the concentration of reducing sugar .Before 14h, engineered yeast were consuming the galactose in medium, so the OD₅₄₀ values of two groups are similarly decreasing. After 14h, the OD₅₄₀ of Control group is still decrease, while the value of experimental group are change to increase, and decrease again after 16h, that means FliC(Xylanase) proteins have been displayed in certain quantity at the beginning of 14h, and degraded the xylan of culture medium to xylose, so the concentration of reducing sugar increased. After a period, yeast absorption rate of xylose and galactose was greater than that of xylan decomposed by enzyme, so the concentration of reducing sugar decreased again. Thus, from here we concluded that our FilC(XynA) are successfully displayed on the yeast surface.

Here are the protocols we used.

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