Team:CUHKSZ/pages/wet-lab
Wet Lab
Overview
1. Obtain Engineered E.coli.
2. Obtain Culture System of Acetobacter Pasteurianus.
3. Test the Metabolic Properties of Co-culture System
Construction of Engineered E.coli
Achievement: iGEM CUHKSZ 2021 team modified pUC19 plasmid as the vector to endue E.coli the capability of decompose ethanol.
We carried out E. coli culture and stored E. coli frozen stocks in glycerol, and measured the growth curve of E. coli by spectrophotometry. The E. coli bacteria was cultured in LB broth and the absorption value is measured under 560 nm wave length. The result is consistent with the theoretical value, with a long logarithmic growth period.
| Time(h) | 0 | 1 | 2 | 3 | 3.5 | 4 | 4.5 | 5 | 5.5 | 6.5 | 7.5 |
| OD560 | 0.061 | 0.136 | 0.154 | 0.580 | 1.089 | 1.485 | 1.788 | 2.008 | 2.221 | 2.322 | 2.666 |
But the initial experiment found no colony in the medium containing ampicillin during the transformation experiment, and this result remained after several repetitions, even when the concentration of bacteria was increased. We speculate that there are a number of reasons. Therefore, for these possible reasons, 1. Incorrect antibiotic or antibiotic concentration 2. Cells are not viable (i.e., competent cells are not viable and has low transformation efficiency) 3. Wrong heat-shock protocol 4. Construct is too large. So we also began to conduct some exploratory experiments to solve the problem. For example, if it is because of the incorrect concentration of antibiotics, we designed a series of concentration gradients from scratch to conduct experiments to explore the appropriate concentration of antibiotics. Due to the time limit, we turn to E. coli BL21, which has a higher protein expression level. The transformation was successful, and the transformed BL21 and acetic acid bacteria are cultured in large amount to receive PSA test.
Explore A Suitable Culture System of Acetobacter Pasteurianus
After obtaining a stable culture system of E. coli, we began to explore a suitable culture system of acetobacter pasteurianus to prepare for the subsequent co-culture system. Since acetobacter cannot grow normally in LB broth, it is necessary to explore a suitable system for acetobacter and verify that the system can be cultured properly for E. coli. According to relevant literature and our own experiment experience, the acetobacters grow very slowly even in the favorable environment, and the growth is very different in different media, such as in GY medium and V50 medium. Therefore, we turned into a qualitative measure of the growth of acetobacters, that is, by observing the color of the culture media, instead of quantitative measure using spectrophotometer. The culture media component we chose is: 10 g/L tryptone, 5 g/L yeast extract, 20 g/L glucose, and the bacteria are cultured in a 30 °C incubator with aeration. In order to further improve the growth rate, we also added ethanol in the culture media, and set four comparisons, each contained 0%, 2%, 4% and 6% of anhydrous ethanol, respectively. After 24 hours of culture, the bacteria grew the most rapidly in the media containing 2% ethanol; the growth rate sharply decreased when the ethanol concentration reached 6%.
Test of Co-culture System
The alcohol digestion test is to measure whether the alcohol can be effectively digested in the co-culture system of transformed E. coli and acetobacter. We choose to use a hand-held ethanol-meter to measure whether the alcohol concentration was significantly reduced after 16 hours of co-culture of bacteria group, since the table-top ethanol-meter has lower accuracy in the measurement of low concentration alcohol, and OD measurement is easily affected by other metabolites. The experimental systems are alcohol with volume fraction of 2%, 4% and 6%, respectively, which are divided into bacteria group and sterile group for control. However, alcohol digestion was not detected in the first experiment. We suppose that is probably resulted from the low concentration of bacteria or the low expression of target protein. So, we conduct a follow-up experiment which increased the concentration of bacteria and added IPTG to promote the expression of recombinant plasmid. However, the ethanol concentration increased after 16 hours of incubation.
