Spring 2024: Week 15 - May 6, 2024 - May 10, 2024

 Introduction 

During our last week in the lab, we attempted to perform Gibson Assembly with the 3-way fragment we previously assembled and the vector (pRAD1 plasmid). We are also preparing for the summer semester when we will also continue our work with D. metalli. 

Methods 

5/6/24 

We are running out of pRAD1 plasmid extractions. We will need to extract more since we will continue to use it for this project and also for our D. metalli project. We inoculated E. coli pRAD1 into a flask of 25 ml of LB broth. We added ampicillin into the broth before inoculating. Since we plan on extracting within 24 hrs, we placed the flask in the shaking incubator overnight. 

We also performed Gibson Assembly. This time we are attempting to assemble the 3-way fragment and the pRAD1 vector. If it assembles correctly, we will have a fragment that is 3,500 kb. We put the 3-way fragment and the vector in a PCR tube with the Gibson mastermix. The tube was incubated at 50℃ for an hour. 

5/7/24

We started off doing plasmid extractions on E.coli pRAD1 using the Zyppy Miniprep Plasmid Kit. In total, we had four total reactions. 

The primers were ready on this day so we ran PCR on the Gibson product from 5/6/24. We ran two reactions. In one tube, we added 2 µL of sample + 23 µL of the forward primer (Chloramphenicol primer). In the other, we added 2 µL of sample + 23 µL of the reverse primer (Ampicillin primer).

After the PCR reactions were done, we ran the samples on a gel. For the forward primer, we will be looking for a fragment that is 3,503 bp. For the reverse primer, we will be looking for a fragment that is 4,681 bp. 10 µL of ladder was loaded into a well. The other two wells had 2 µL of sample and 10 µL of primer.  We loaded the wells as follows:

• 1kb ladder

• Forward primer (Chloramphenicol)

• Reverse primer (Ampicillin)

Results 

5/6/24

Since we had to wait for the primers to be ready, we placed the tube in the -20℃ until it was ready to use. We will not know if it was successful until we run it through Q5 PCR and a gel.

5/7/24

pRAD1 Extractions

Sample	ng/µL	A260/A280	A260/A230
MC 1	44.9	1.90	1.97
MC 2	42.0	1.89	2.02
KM 1	40.9	1.88	1.93
KM 2	52.0	1.84	1.68

Fig. 1: 1kb ladder, Chloramphenicol primer + Gibson product, Ampicillin primer + Gibson product 

Discussion 

The results of the gel were confusing to us at first. We did not believe that we had a successful assembly due to the lack of bands in the spots where we would expect to see them. Upon further discussion, we came to the conclusion that there is a chance that the assembly might be successful because of the smearing that is present in both of the wells with the sample. An unsuccessful assembly would not give us any smearing and potentially, it wouldn’t show a band at all. 

Conclusion 

When we come back for the summer semester, we will use this sample to attempt to transform DH5a E.coli and D. radiodurans. If it is successful, we will have white D. radiodurans colonies instead of their typical reddish-orange color.