Team:Grenoble-Alpes/Demonstrate
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DEMONSTRATE
The following page contains the results and succes that we actually obtained with our system.
Tests were performed on a biological transformation because we knew that the tricky part would be the cooling step of the thermal shock. In fact, the module could only go down to around 11 degrees so we looked out for literature about optimization of the transformation.
What literature has to tell us
As a reminder, a biological transformation or as it is also called a “temperature shock transformation” is typically a succession of a cold – hot –cold treatment from 0°C to 42°C and back to 0°C to induce the transfer of exogenous genetic material in competent bacteria.
According to a study [1] about the effect of Heat Shock Temperature, Duration, and Cold Incubation, “Escherichia coli BW25113 transformed with the pUC19 cloning vector at cold-step temperatures of 0°C, 4°C, 10°C, and 20°C demonstrated no significant difference in transformation efficiency. When the same strain was transformed using approximately a 20°C difference between the cold-step and hot-step treatments: 0 → 20 → 0°C and 20 → 42 → 20°C, no significant difference in transformation efficiency was observed between the two treatments.”
Considering the results of this study, we hypothesized that because the module has more than a 20°C difference between the cold-step and the hot-step (in fact, between 11°C and 42°C, there is a difference of 31°C), the transformation should succeed. However, the question was to know how well it would succeed. To test this, a few parameters could be changed: the DNA concentration or the type of competent bacteria for instance.
Petri dishes experiments
We first tried to transform pSB1C3-BBa_J04450 plasmids in competent DH5α bacteria and Top10 (two different E. coli strains) and growed colonies on petri dishes.
Goals:
- Verify that the heating and cooling modules work
- Test the efficiency of a transformation as a function of the DNA quantity and plot a curve similar to the following 24th figure.
- Test the efficiency of the module compared to a classical transformation using two different competent bacteria (Top10 and DH5α).
One of the main goal of the project was also to realize at least one biological step in our engineered system. Finally, we managed to realize a purification step with BL21 bacteria and using a lysis buffer instead of phages.
We wrote an entire Arduino computer code able to control the different elements of the system : the pipette, the linear guide, the rotative plate, the heating system, the actuator with the magnet holder. With the coordinated control of this different element, we successfully managed to realize the transformation. Below is the protocol that the Arduino code executes.
Protocol realized:
Add 200µL of Buffer 1 (Lysis Buffer)
Add 100µL of bacteria + wait 10 minutes
20µL of magnetic beads + wait 10 minutes
Bring magnets next to the tube to attract the magnetic beads and extract the waste liquid
Add 300µL of Buffer 1 (Lysis Buffer), flush, extract the liquid while magnets attract the beads and throw to the waste.
(Add 300µL of Buffer 2 (Wash 2), extract while magnets attract the beads and throw to the waste the liquid) * 2 times
Add 300µL of Buffer 3 (Elution), extract while magnets attract the beads and throw to the waste the liquid.
Eluate once more with 30µL of Buffer 3 + heat to 70°C during 5 minutes (DNA detaches from the beads)
Extract while magnets attract the beads and store the liquid (DNA).
The Arduino computer code allowing to realize this process is available here in this zip file.
We obtained with a nanodrop absorbance measurement a concentration of 38,4ng/uL and a purity of 1.87. The purity is between 1.8 and 2.0 so it means that the purity is acceptable for DNA. [4]. As for the concentration, we can’t tell if it is consistent or not because we did not measure the OD of the bacteria sample before the experience. Anyway it is not a value close to null so we still can tell that we extracted DNA.
That proves that our machine works ! And with more time, we could consider realizing the other steps of the biological process in the machine to have a proof of concept for the whole system. We could also spend more time to characterize the efficiency of the machine comparing to the process realized outside the machine.
Below is a video capturing the purification:
Our fluorescence sensor is able to detect fluorescence in samples where the optical density is at least of 0.014. You can find more explanations about our fluorescence unit here.