Team:NU Kazakhstan/Characterization

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Characterization

Characterization

iGEM NU Kazakhstan team had chosen the part BBa_K861173 which has the glucose repressible promoter BBa_K118011. Increasing the glucose concentration will result in the decreased activity of the cAMP cyclase, which will downregulate the activity of the downstream gene. In this case it is the RFP encoding sequence which excitation/emission wavelength is 584/607, according to BBa_E1010. Also, RFP has a long folding time which is equal to 24 hours. Choice of media is important, as yeast extract in the LB media contains some amounts of glucose, which can affect the expression of the RFP. Therefore, M9 minimal media was chosen for this procedure, where the glycerol is used as a carbon source.
BL21 expressing strain was chosen for the expression of this part. First, the transformed with this part bacteria were incubated overnight in LB broth liquid culture. After that, the ODs at 600nm wavelength were measured. Next, the culture was split into five 15ml falcon tubes. Then, the cultures were spun down for 10 minutes at 4000rpm and the supernatants were removed. After that, the M9 media or LB broth were added to the falcon tubes, so that the absorbance in each tube will be 0.02. To measure the effect of glucose on the fluorescence, 4 different concentrations (0 g/l, 0.2 g/l, 1 g/l, and 5 g/l) of glucose in M9 media had been prepared, as well as 1 LB media without any additional glucose.
After the preparation of the following concentrations, 0h measurement was performed. For that 250 ul of the media from each falcon tube, as well as the blanks were transferred into a 64 well plate. Then, the measurement of absorbance and fluorescence were performed with Varioskan Flash, which was calibrated based on the interlab protocol. The measurements were taken at 0h, 6h, 12h, 24h and 36h periods.

The calibration was measured according to the iGEM 2019 Plate Reader Abs 600 (OD) calibration and iGEM 2019 Plate reader Fluorescence calibration. Rhodamine B was used as the standard to obtain the calibration plot. The obtained results you can see on figures 1-4.
Figure 1. Particle standard curve
Figure 2. Particle standard curve, log scale
Figure 3. Rhodamine B standard curve
Figure 4. Rhodamine B standard curve (log scale)

BL 21 E. coli strain cells were incubated in m9 media with different concentrations of glucose (0-5mg/L) and in LB media with no glucose added. The absorbance and fluorescence were measured after 0, 6, 12, 24 hours of incubation. The value of MEFL/particle was calculated using the provided by iGEM 2019 Excel file. Figures 5 and 6 show the overall trend dependent on time of incubation and concentration of glucose.
Figure 5. The activity of RFP upon the increase in concentration of glucose
Figure 6. The activity of RFP within different incubation time

As it can be seen from the graphs, part works as expected. The high concentration of glucose decreases the expression of RFP. Also, the expression of protein reaches its highest point after 24 hours of incubation, as the folding of this protein takes 24 hours. It can be also seen that due to the presence of glucose in LB, the expression is reduced even without the usage of additional glucose.
The difference is also seen after incubation of cells in LB with 1g/L and 5 g/L concentrations of glucose. As it can be seen, there is the substantial expression of RFP with lower concentration.
Figure 7. Cells incubated with different concentrations of glucose