Spring 2024: Week 14 - April 29, 2024 - May 3, 2024
Introduction
Last week we attempted Gibson Assembly for the first time. As we interpreted the results closely, we seem to have been successful. We had a faint band at around 2,500 bp and our fragment was 2,527 bp. Since the band was faint, we are going to attempt to amplify it this week.
Methods
4/29/24
We kept our broth culture of D. metalli in the fridge and the biofilm has continued to form. Since it is one of the few species in the Deinococcus genus that forms a biofilm, we were interested in seeing it under the microscope. We decided to do a gram stain on it.
4/30/24
In order to amplify our Gibson product, we decided to run it through PCR again. This time, we did Q5 PCR to get a higher yield than what we previously got. We ran two different samples. The first sample was 48 µL of mastermix + 2 µL of our 3-way Gibson that had been put through PCR last week. The second tube was the original Gibson product that had not been put through PCR.
At the same time, we ran long-amp PCR on our pRAD1 plasmid because we did not have enough of our vector sample to work with. If we are successful with this PCR reaction, we will need more of our vector sample to run another Gibson Assembly reaction with our 3-way assembly + our vector (pRAD1).
5/1/24
Since we will need to check our PCR reactions to make sure that we got the results that we are looking for, we will be running a gel. We will need 9 wells so we decided to run a larger gel that had 15 wells. It holds 120 ml so we adjusted the measurements for the TAE buffer and the agarose accordingly. 120 ml of TAE buffer and 1.2 g of agarose were microwaved together to create the gel. The well that contained the ladder had a total of 10 µL while the other wells had 2 µL of loading dye and 10 µL of sample. We loaded the wells as follows:
1 kb ladder
PCR Gibson (wells 2 &3)
Original Gibson assembly (wells 4 &5)
Long-amp PCR (wells 6-9)
Prep media for both projects
5/2/24
We decided to nanodrop all of our samples so that we know each of their concentrations. We checked our original fragment extractions as well as our Gibson Assembly product and our Long-amp products.
Since we did Gibson Assembly without cleaning up our fragments, we decided to try cleaning up one of them to see if it would help with the purity or not. We were worried about the cleanup having an adverse reaction so we only tried it on one of our Gibson samples. We also picked two of the Long-amp products and cleaned them up.
Results
4/29/24
Fig. 1 : Potential picture of D. metalli's biofilm. 
Fig. 2: D. metalli under the mircroscope forming an almost complete circle.
Fig. 3: D. metalli coming together in lines.
Fig. 4: Potential picture of D. metalli's biofilm.5/1/24
5/2/24
Original Fragments
Gibson Product and Long-amp Product
Long-amp cleaned through PCR cleanup kit
Discussion
4/29/24
Looking at D. metalli under the microscope was interesting. I am not sure if any of these pictures properly show its biofilm. Whether I was able to see the biofilm or not, it is interesting to see the different things D. metalli does in nutrient-rich broth. The cells line up with each other and form what we can only describe as a filamentous structure. This is the first time I have seen it form as circle (Fig. 2).
5/1/24
The bands that we saw on the fragments that had already been through PCR after the Gibson protocol seemed to disappear after going through Q5 PCR. This is the opposite of what we expected to happen. Interestingly, the samples that were put through only Gibson and Q5 PCR had bright bands. Our Long-amp fragments also had extra bands that we did not expect. We put them through the PCR cleanup kit in hopes of improving its purity.
5/2/24
Our Long-amp fragments cleaned up nicely. Both their A260/A280 and A260/A230 improved. After checking all of our samples on the nanodrop, we are able to get an idea for which samples we will be working with going forward. It also allows us to see if the concentrations had anything to do with the Q5 PCR working for the samples that came straight from Gibson versus those who went from Gibson to PCR to Q5 PCR.
Conclusion
We saw multiple successes this week. By doing Q5 PCR, we are able to see which method works. We now know that we can perform Gibson and then put the samples into Q5 PCR. This will allow us to amplify the samples while keeping the assembly in tact. Next week, we plan on performing Gibson Assembly with the 3-way assembled fragment and the pRAD1 vector.
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