Week 21: March 27, 2023 - March 31, 2023

 Researching Deinococcus Species 

After inoculating new species and failing to see growth after multiple days, we were beginning to become frustrated since it was slowing down our project. There seemed to be a lot of waiting around to see when a species would be ready to work with while the current species we were currently testing and growing in the incubator. 

This made me question if we were doing something wrong or if there was more information that we could find out about the Deinococcus genus. I decided to go on a search to learn more about Deinococcus in order for us to be more productive and prepare more for the following weeks. 

Coming into this project, I was introduced to two species of the Deinococcus genus, Deinococcus radiodurans and Deinococcus aquaticus. Both of these species seemed to have a similar growth time of about 48 hours in a 30°C environment. Our newly inoculated species were placed in the same conditions, for the same amount of time, and using the media. For both D. radiodurans and D. aquaticus, we grew them in tryptone-glucose-yeast extract medium, or TGY, and they seemed to have no problem growing. Our newer species, however, were showing minimal and slow growth. 

In order to learn more about the Deinococcus genus, I went through the list of species that we wish to work with in the future in order to find their MIC and began to read up on them. I put together a table of the basic information for each species in hopes that it might help us when beginning to work with them. 



Deinococcus Genus Basic Information    

Species Name

Growth Temperature

Media

Shape

Gram Pos or Neg

Motile

pH

Incubation Period

D. indicus

30°C

TSB

rod-shaped

negative

No

6-7

-

D. caeni

28°C

R2A/R2B/TGY

rod-shaped

negative

No

6-9.5 (optimum: 7)

-

D. roseus

28°C

R2A/R2B/TGY

rod-shaped

positive

No

5-9 (optimum: 7)

2-3 days

D. aquatilis

28°C

R2A/R2B/TGY

rod-shaped

positive

No

8-9

-

D. GoB

28°C

TSB

coccus-shaped

positive

No

7.0-8.0 (optimum: 7.5)

-

D. grandis

26-30°C

PYEA

-

positive

-

-

-

D. aquaticus

28°C

R2A/R2B/TGY

rod-shaped

negative

No

6-9 (optimum: 7)

-

D. Budensis

-

-

-

-

-

-

-

D. Metalli

28°C

R2A/R2B/TGY

-

-

No

-

-

D. Daejeonensis

28°C

R2A/R2B/TGY

coccus-shaped

positive

No

6-8.5 (optimum: 7.25)

-

D. Pimensis

28°C

R2A/R2B/TGY

-

positive

-

-

-

D. Sonorensis

28°C

TGY or Glycerol-Yeast Extract Peptone

-

-

-

-

-

D. Xinjiangensis

28°C

TSB or Glycerol Yeast Extract Peptone

coccus-shaped

positive

No

5-8 (optimum: 7)


D. Hapiensis

-

-

-

-

-

-

-

D. Hohokamensis

28°C

R2A/R2B/TGY

-

-

-

-

-

D. Misasensis

37°C

Rich Medium

-

positive

-

-

-


Not all of the information was available for each of the species but we did get some insight on different things that we could try. For example, D. indicus is a species that was showing significantly less growth than the other species we had inoculated. I found that a media that is commonly used for growing D. indicus is Tryptic Soy Broth, or TSB. It is also a non-motile bacteria. This means that it does not have a lot of movement on its own.

Perhaps we were not encouraging its growth by trying to grow it in TGY. Even though Dr. Tuohy said that they have found that bacterias who typically grow in TSB have had the same if not better results when placed in TGY, we thought it might be a good idea to try growing D. indicus in TSB. This will allow us to narrow down the list of possible reasons why the species are experiencing such slow growth. 

Comments

Post a Comment

Popular posts from this blog

Spring 2024: Week 14 - April 29, 2024 - May 3, 2024

Image
  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 sam...
Read more

Spring 2024 Week 2: January 29, 2024 - February 2, 2024

Image
  Introduction:  This week we continued our work with E. coli pRHAM. Last friday, we completed a plasmid extraction in order to get the pRHAM plasmid out. This week, we focused on confirming that we successfully extracted the plasmid before moving onto using that sample in PCR. If both of these go smoothly, we will be able to begin assembly, using Gibson Assembly, at the beginning of next week.  Methods: 1/29/2024  Using gel electrophoresis was our way to confirm if we had extracted the pRHAM plasmid. We mixed 2 μL of UV blue dye with 10 μL of our plasmid sample in an Eppendorf tube. We spun our tubes down in the pop spinner to make sure it was all one homogenous liquid. Since the plasmid’s size is 947 kb, we added a 1 kb ladder into one of the wells so that we could compare the size of our band to the ladder. We let the gel run and checked it under the UV light.   1/30/2024 Using our plasmid sample, we ran a short-amp PCR. We mixed 8 μL of mastermix ...
Read more

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

Image
  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℃ f...
Read more