Difference between revisions of "Team:Grenoble-Alpes/Demonstrate"

DEMONSTRATE

• PROJECT
  • BIOLOGY
  • ENGINEERING
  • 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.

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:

1. Add 200µL of Buffer 1 (Lysis Buffer)
2. Add 100µL of bacteria + wait 10 minutes
3. 20µL of magnetic beads + wait 10 minutes
4. Bring magnets next to the tube to attract the magnetic beads and extract the waste liquid
5. Add 300µL of Buffer 1 (Lysis Buffer), flush, extract the liquid while magnets attract the beads and throw to the waste.
6. (Add 300µL of Buffer 2 (Wash 2), extract while magnets attract the beads and throw to the waste the liquid) * 2 times
7. Add 300µL of Buffer 3 (Elution), extract while magnets attract the beads and throw to the waste the liquid.
8. Eluate once more with 30µL of Buffer 3 + heat to 70°C during 5 minutes (DNA detaches from the beads)
9. 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.