The columns labelled A1 and A2 were minipreps conducted with the new kit and the columns labelled B1 and B2 were minipreps conducted with the old kit. Thus, for the gel image shown above, we conducted two mini-prep with an old and a new kit. The second well which had the initial diluted plasmid DNA had no trace of DNA on its column, while the next four wells which had the concentrated plasmid had traces of DNA on their columns.įigure 6: Gel electrophoresis results comparing two different plasmids obtained with different miniprep kits to the NEB 1kb MW ladderĪnother suspicion we had about the results we had was whether the mini-prep kit we used was problematic. From the gel image shown below we were correct. After the mini prep, we ran a gel, to confirm if we were correct. We performed a digestion, ligation, and transformation for only the puc19 plasmid to obtain a very concentrated plasmid after we conducted a mini prep. Thus, we sought to find out if our suspicion was correct. We suspected the plasmid might have been over diluted to undergo digestion and ligation. We then suspected something might have been wrong with the concentration of the plasmid used. No color change was observed for this investigation, however. We plated the culture with the gold particles on ampicillin plates to observe growth and color change over a period. We took the colony and added it to ampicillin media, added some gold particle-chippings and grew it overnight. However, we observed very few colonies for the Au sensing module. 2c and 3c depict the vectors and inserted fragments with their various sizes (base pairs), which enabled the team to visualize how the insertion was done and the size of the various components.Īfter ligation, we transformed the parts from the three tubes from ligation with the competent cells and allowed for overnight growth.ġ.3 OBSERVATION, RESULTS AND TROUBLESHOOTĪfter the overnight growth, we did not observe any growth or colonies on our agar plates for Arsenic and Fe sensing modules. This resulted in complete plasmids with various sensing module inserts, as shown in figures 1b, 2b, and 3b. We also cut the fragments with EcoR1 and PSt1 to maximize ligation efficiency. By inserting, we cut the plasmid with the EcoR1 and Pst1 to create sticky ends to facilitate ligation. We used restriction and insertion cloning to insert these fragments into the plasmid (pUC 19).
This was done successfully, as represented in figures (1a,2a, and 3a). In this approach, we first performed a linear ligation between the sensing module and kill switch fragments (Au and UV, As and UV, Fe part 1 & 2 and UV) to evaluate if it's feasible to be ligated. PRELIMINARY SIMULATIONSīefore performing this experiment in the Lab, the team utilized Snapgene to simulate the digestion and ligation of the various parts. And finally, the result for theįirst try of the hydrogel encapsulation and some proposed future experiments to obtain the expected results. Then, we explain the corrections of the digestion and ligation protocols resulting from this troubleshooting.Īlso, we present the results obtained from the final try, continued with miniprep, PCR and gel electrophoresis to characterize the part. We also explain the troubleshooting of these initial experimentsĭetermining the issues with the ligation and digestion. Ligations, transformations, gel electrophoresis, and making competent cells. Science, mechanical and electrical engineering majors) started practicing in the lab with digestions, Our less experienced team with biological lab work (70% of the team are computer The genetic parts for our project in silico with the SnapGene application and sent them out for
With lots of research and iterations, the team designed Showing the digestion and ligation of our parts. The second part presents the preliminary experiments Simulated agarose gel electrophoresis in SnapGene. To separate the DNA fragments according to size and charge after a restriction digest, the team First, we describe the construction and characterization of the various sensing modules cloned in the E-coli strain using The results outline the experimental work carried out over 1-2 months of lab work.
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