Spring 2024: Week 12 - April 15, 2024 - April 19, 2024

 Spring 2024: Week 12 - April 15, 2024 - April 19, 2024

Introduction

With the conference done, we have decided to continue to work on our Deinococcus radiodurans project. While we are not done working with Deinococcus metalli, we plan to work on both projects simultaneously. Since we are still working on planning out our work with D. metalli, we are able to jump back into working on our original project. 

Our goal is to be able to knock out the CrtI gene in D. radiodurans (D. rad) which is the gene responsible for giving the bacteria its reddish-orange color. If we are successful, we will have white D. rad colonies. Before spring break and before starting our work with D. metalli, we had some setbacks on our D. radiodurans project. We were ready to do Gibson Assembly but our sample concentrations were not matching up with our calculations. When we checked our original fragment concentrations on the nanodrop, they were lower than what they originally were. 

Now that we are focusing on this project again, we are going to work toward doing Gibson Assembly. We will need to run our original fragments through PCR in hopes that we will be able to use them and not have to go back and extract each fragment from their original bacteria. 

Methods

4/15/2014 

We started off the week by prepping the media that we will need to start the project back up. We no longer had any of the bacteria we needed since we had to clear out any media, broth cultures, or plate cultures that would go back during spring break. Since we will be growing D. rad and E. coli we made two different types of media. We made 100 ml of TGY broth and 200 ml of TGY agar. We also made 200 ml of LB broth and 200 ml of LB agar. 

4/16/2024 

Using the autoclaved media that we made the day before, we poured plates. For D. rad, we poured regular TGY plates. The LB agar plates will be used to grow two different types of E. coli that will each require a different antibiotic. Ampicillin was added to 100 ml of LB agar and kanamycin was added to the other 100 ml of LB agar. The ampicillin plates will be used to grow E. coli pRAD1 and the kanamycin plates will be used to grow E. coli pRHAM. 

Results 

This week was spent as a preparation and planning period to get back on track for our project. We made enough media and plates to get us started on the project. We did not do anything that gave us results this week. 

Discussion 

We are looking into doing Circular PCR as a form of assembly. I have spent time reading up on it and trying to become familiar with it. At the moment, we are not sure if we will use it alongside Gibson or try it out before Gibson and see what the results are. 

    As for our D. metalli project, we presented our poster to everybody in the lab and got feedback. We want to attempt to transform D. metalli and we will be planning the project while continuing to work on our current project. 

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