Part:BBa_K2224001:Experience
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Applications of BBa_K2224001
Part Description:
This is the experiment report of SMS_Shenzhen team in August, 2017.
We, 2017 SMS iGEM Team, have submitted this part AND characterized the function and effectiveness of this part. BBa_K2224001 is a composite part with the backbone pSB1C3. The CDS of this part is Lecanicillium lecanii strain 432 endochitinase (chit1), a chitinase originate from verticillium lecanii, a kind of entomogenous fungus. With a GeneBank identifier DQ412944 in NCBI. This enzyme helps catalyze chitin. SMS students optimized the codons and removed introns to fit the original sequence into E. coli expression system.
Experiment Abstract
The main purpose of this experiment is to measure the activity of a target enzyme, Lecanicillium lecanii strain 432 endochitinase (chit1), in TOP10 expression system.Testing the effectiveness of these enzymes’ expression in E. coli expression system is a fundamental portion of the whole project. According to previous researches, we expected that the expression activity of this chitinase in Top10 system should appear effective and show a co-relation to time. The data we obtained in this experiment include bacteria density in culture(in ABS) and enzyme activity(in U/10^4 cells). As our speculation, the expression results should confirm our expectation.
The original gene sequence of this chitinase DQ412944 was published on NCPI. SMS students have processed the CDS. Designed primer with restriction sites, modified the CDS for standardization. We then synthesized this plasmid with the backbone pSB1C3.
Experiment Princeple
We used a Chitinase activity measurement kit to measure the activity of this chitinase quantitatively. The enzyme activity was defined by the amount of N- acetyl glucosamine that produced by Chitinase' s hydrolysis to chitin divided by the amount of cells. Since the product, N- acetyl glucosamine, can lead to light absorbtion peak at 585nm and the light absorption can be detected by Spectrophotometer, we can figure out the relative activity of enzyme DQ412944. Enzyme activity in TOP10 E. coli expression system related to time shown below. (ABS is for absorbance, U is for active unit)
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