Competent Cell Production
As part of our Wet Lab experimentation, we prepared our own chemically competent DH5α cells. We then tested the competency of the cells via a calculation of bacterial transformation efficiency, for which we transformed the cells with the pHis17-mod0B plasmid and observed the number of colonies which grew on the plate.
Transformation efficiency (transformants/µg) is calculated as follows:
Number of colonies on the plate/ng of DNA plated X 1000 ng/µg
We calculated the transformation efficiency to be 8.72 x 10^6 cfu/ug of DNA
Growth Curves
It was ensured that equal amount of innoculum was added to each culture while setting up the growth curves by normalizing the the starting OD to 0.06.
All the cultures were setup in triplicate to reduce any chance of error and also make sure that there is no contamination growing in any particular culture. Each media had its own blank control also setup in triplicates which was normalized to the test reading at the end of the experimental readings.
While plotting the curve the average of the three triplicate reading was taken.
α-ketobutyrate curves:
For the generation of the standard curve of α-ketobutyrate we performed spectrophotometric absorbance readings of it hydrazone derivative. We had setup duplicates of every setup to account for any error that might take place and had also setup a control in which all the reagents were added as the test setup except α-ketobutyrate.
The detailed protocol is mentioned in our experiments page.
The following was the data obtained:
| Replicate number | Number of moles of α-ketobutyrate (umol) | Absorbance at 540 nm |
| 1 | 0.1 | 0.193 |
| 1 | 0.2 | 0.623 |
| 1 | 0.3 | 0.825 |
| 1 | 0.4 | 1.224 |
| 1 | 0.5 | 1.556 |
| 1 | 0.6 | 1.589 |
| 1 | 0.7 | 1.927 |
| 1 | 0.8 | 0.92 |
| 1 | 0.9 | 2.264 |
| 1 | 1.0 | 2.98 |
| 2 | 0.1 | 0.271 |
| 2 | 0.2 | 0.625 |
| 2 | 0.3 | 0.893 |
| 2 | 0.4 | 1.099 |
| 2 | 0.5 | 1.36 |
| 2 | 0.6 | 1.536 |
| 2 | 0.7 | 1.977 |
| 2 | 0.8 | 2.045 |
| 2 | 0.9 | 2.148 |
| 2 | 1.0 | 2.478 |
This is the final curve that was generated after plotting the above obtained data:
Interlab
We signed up for the Interlab Study 2022. However, we could not submit the results even after making a lot of plates for transformations for multiple cycles as the transformation of parts did not happen properly.
