Week of 09/02/24

 

Introduction

This week we were performing gram stains for the morphology project. This project looks at two different conditions: type of media and whether that media is inoculated while shaking or remaining still. Deinococcus aquaticus P81 cells have been observed to “elongate” when inoculated into a broth instead of upon a plate. The current theories explaining this morphological changes is that P81 might be attaching end to end by a biofilm or that P81 under mobile conditions fails to completely separate. 

This week we are inoculating multiple broths of TGY and R2B and observing whether or not the cells will continue to elongate as expected. 

Simultaneously, we are running another transformation procedure with P81 to attempt to insert the PRAD1 plasmid into P81. A successful transformation would give transformed P81 cells resistance to both ampicillin and chloramphenicol. 

At the same time we will be inoculating another run of SMA plates for the twitch motility assay. This assay would allow us to visualize the twitch motion of bacterial cells lacking a flagellum.

Methods

Morph Assay Protocol

1) Inoculate mother plate with sample

2) Inoculate R2A and TGY Plates and Broths

3) Incubate Plates and Broths

a. Some broths will be shaken and some kept still 

4) Gram Stain Plates and Broths after at least 24 hours

5) Record and compare results

6) If needed, introduce a protease to break apart pili connections

7) Re gram stain after 24 hours

8) If needed, run an RNA sequence to check gene expression

Twitch Motility Assay Protocol

1) Select media (e.g. 0.75% SMA and 0.5% agar)

2) Prepare a sterile hood by wiping everything down with ethanol

3) Melt media down

4) Wipe down all inoculating materials and insert them into the sterile hood

5) Dispense 10 ml of media into empty petri dishes containing five pronged molds to allow for insertion sites

a. Use a sterile 10 ml pipette

6) Let plates cool

7) Remove molds

8) Use UV light for 15 minutes to ensure sterilization of plates 

9) Inoculation center of plate (directly onto the plastic)

10) Dispense 100-150 microliters of chemoattractant media into one of the insertion sites

a. Use 0.5% agar media

11) Incubate Plates

12) Regularly Picture

Transformation Protocol

1) 1 ml cells OD value of 1 (late log)

2) Pellet

3) Resuspend in 100 microliters of TGY

4) Add 40 microliters of 0.3 CaCl2 mix

5) Ice 15 minutes

6) 30 microliter sample and about 10 microliters of plasmid (where the plasmid is 200-400 ng per microliter for a grand total of around 1 microgram of plasmid DNA)

7) Mix

8) Incubate at 30 degrees Celsius for 90 minutes while inverting every 15 minutes.

9) Add  2 ml of TGY and Incubate for 24 hours

10) Plate cells on TGYA and TGYA CM plates

11) Use Cells mixed with PCR water as a blank

Results

The gram stains for the morphology project revealed strings in the broths and rods on the plates.

The twitch motility assay ran into trouble because the 0.5% Agar plates failed to set.

The transformation procedure ended in no growth upon any chloramphenicol plates. 

Discussion

The gram stain procedure took way too long for the morphology project. The gram stains may have to be broken up over the course of several days. There was also some staph contamination upon several plates and as such it is imperative that aseptic techniques are utilized at every possible opportunity.

While the protocol calls for 0.5% Agar, it may be wiser to bump the composition up to 1%. The 0.5% agar is simply too inconsistent.

The transformation protocol showed growth upon the control plates but no growth upon the experimental plates. We were unable to get a successful transformation from this trial. Perhaps it may be wise to try a higher dose of the plasmid during our next trial run.