Team:iBowu-China/Contribution
Contributions
Contribution for Bronze Medal
This year, we engineered a gene circuit to express β-glucuronidase enzyme. In our experiments, we used two parts previously existed in the iGEM parts registry, and we made our contributions by "Add(ing) new documentation to an existing Part on that Part's Registry page" with new data collected in our work. The two parts are:
LacI (BBa_C0012), link here : http://parts.igem.org/Part:BBa_C0012
and
sfGFP (BBa_K515105), link here: http://parts.igem.org/Part:BBa_K515105
BBa_C0012
Introduction
This year team iBowu-China used two parts LacI and LacO, with iPTG as induction methods, to control the expression of sfGFP protein and β-glucuronidase protein in E. coli BL21(DE3)
Protocol
1. Transform the plasmids into E. coli BL21(DE3)
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 4ml LB medium with kanamycin. Add 1mM IPTG to all experimental groups as needed. Incubate at 37 degree Celsius in a shaker overnight.
3. Fluorescence. Acquire 100 ul bacteria culture and centrifuge at 10000g for 1 min. Aspirate the supernatant and resuspend with 100 ul ddH2O to get rid of the fluorescence background from LB medium. Add 100 µl sample solution into a sterile 96-well plate. Measure fluorescence with a microplate reader and then also measure OD600 for normalization.
Results
In the scenario where there is LacI in the plasmid and also where there exists LacO in the upstream gene of β-glucuronidase, this enzyme can be expressed in E. coli BL21 with Iptg induction. The enzymic activities and therefore the catalytic capabilities of the cells and the bacteria culture can therefore be controlled with different concentrations of induction substance. LacI can effectively work together with LacO to control gene expression in E. coli BL21(DE3). Our results confirmed 0.5 to 2mM Iptg can be effective and sufficient to release the suppression of gene expression exerted by LacI protein.
Figure. The expression of β-glucuronidase induced by Iptg with the existence of LacI can be measured by the enzyme activity. Higher the total activity can be seen directly by deeper purple color. The activity can also be measured at OD540 with OD600 normalization of bacteria concentration.
BBa_K515105
We used this plasmid as a positive comparison for control group in the measurement of green fluorescence. The plasmid can effectively produce green fluorescence.
Protocol:
The plasmid was obtained from BNDS-China 2021 team. It was then expressed both in E. coli DH5a and E. coli BL21(DE3) with Cm resistance. The bacteria culture was incubated without addition of any iPTG in both cases overnight at 37 degree Celsius. GFP was measured with a microplate reader and OD600 was taken for normalization of the concentration. A control group was also measured for background light intensity where the E. coli BL21(DE3) without this plasmid was cultured. A picture was taken to show the effect of green fluorescence protein expression.
Results:
The results show this part can produce green fluorescence protein highly effectively. In BL21(DE3), the expression is stronger than in DH5a. The intensity of the GFP light in BL21(DE3) is about 2x the intensity in DH5a. In both cases, the light intensity is much stronger than the background by about 100 fold. This part can be used for effective expression of green fluorescence protein. It is an ideal choice for control group, and it can also be engineered to express other sequences.
Figure. Green fluorescence protein expressed by E. coli DH5a and BL21(DE3) under visible light and their measured intensity normalized by their concentration (OD600). The third column was the control group where the E. coli BL21(DE3) without this plasmid was used.
Acknowledgement
