Team:Uppsala/Proof
Proof of concept
As a proof of concept we did 2 rounds of experiments on our heat shock chip, one where we compared its transformation efficiency to that of regular heat shock transformation done off-chip and one where we compared transformation efficiency on the chip at different temperatures.
Heat Shock Chip
During the summer we made over 30 chips and numerous successful heat shock transformations using only 8.4 ng of DNA and 5.9 μL of competent cells.
Bacterial growth was seen on nearly all of the chip transformed plates. In other words, the transformation worked on our chip and it greatly reduced the amount of reagents needed since colony growth was observed on plates were only 6mL of cell/DNA suspension was heat shocked. The number of colonies for each trial was added by counting all of the five plates for each trial as one replicate. That left three replicates for each chip. Calculations of colony forming units per microliter DNA were made and the mean for each chip is shown in Figure 1. We found that the transformation efficiency was much higher in the conventional heat shock than on our chip.
The plates with bacteria transformed on the chip showed many colonies, considering the small amount of cells and DNA that were plated. A large variation in number of colonies between different trials was observed. That is, two transformations carried out on the chip in the same manner could yield a varying number of colonies.
The cleanliness of our chip was good as little to no colonies grew on the plates with only SOB run through the chip nor on the plates with only cells (negative control).
Raw data
Here the raw data of every transformation is presented alongside the comparison transformations we did.
| Trial. | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
|---|---|---|---|---|---|---|---|---|---|
| Chip model | 1 | 2 | 3 | ||||||
| No. of colonies | 964 | 1096 | 505 | 911 | 1074 | 55 | 85 | 771 | 1029 |
| Transformation efficiency (cfu/ng DNA) | 34.9 | 39.6 | 18.2 | 33 | 38.8 | 1.99 | 3.07 | 27.9 | 37.2 |
| DNA concentration(ng/µl) | 47 | 47 | 47 | 0.56 | ||
|---|---|---|---|---|---|---|
| No. of Colonies | >1000 | 170 | 72 | 64 | ||
| Transformation efficiency (cfu/ng DNA) | 42.5 | 304 | 129 | 114 | ||
According to the results presented in Table 1 and Table 2 the heat shock chip did not reach higher efficiency than conventional methods, at least with the comparison metrics used. There was also very high variation in the transformation efficiency among the experiments done on on the chip(which were done using similar/identical reagents). The efficiency varied from 1,99 in experiment number 8 up to 39,6 in experiment number 4, which is a 20-fold variation in efficiency. On a per plate basis the highest efficiency we had was 123 000 and the lowest was 0 (8 out of 50 plates had 0 colonies), with the average transformation efficiency being 36.
During the experiments we could identify several potential causes which could cause this large variation of results. The setup of the chip involved running heated water from a beaker through the chip and this means that there can be a chance that there is higher or lower thermal leakage than expected, leading to the transformation efficiency being affected. Also we were not sure whether heat shocking at 42 degrees for 35-40 seconds is the optimal way to do it. Since the heat is conducted much better and faster through the chip from the heating channels and because the cells are in a much smaller volume of media the cells are heated more rapidly maybe a lower/higher temperature should be used a shorter/longer time. Furthermore we noticed that there was a very large variance due to the human factor(i.e. who was actually conducting the experiment) and that experience in the procedure played a large role in how successful the transformation was. This was likely due to the primitive and direct way cells were fed into and out of the chip (using a syringe to push in/out cells and eye-measuring to see whether cells have entered the chip makes for inaccurate timing and a lot of back and forth).
Results for the temperature comparisons
For each transformation at a set temperature the amount of formed colonies can be viewed in figure 3. Figure 3 shows the data points for the triplicates at each temperature having a large spread of the amount of formed colonies. Only the transformations at temp. 55 ◦C showed formed colonies for each of the three replicates.
The highest average value for formed colonies was at 55 ◦C for the heating fluid, giving 30 colonies whilst heat shocking at 65 ◦C and 75 ◦C yielded 21 and 14.7 colonies. The average (avg) number of colonies at each temperature drops slightly when going from 55 ◦C, 65 ◦C to 75 ◦C since the latter two contain replicates with zero number of colonies.
The avg value for the number of colonies at each temperature was used to determine the average transformation efficiency, represented by the cfu/µg value and is presented in Table 3.
| Temperature (°C) | Transformation efficiency (cfu/ng DNA) |
|---|---|
| 55 | 4.11 |
| 65 | 2.88 |
| 75 | 2.01 |
