Week of 09/30/24

 

Introduction

Following a lack of growth on the experimental plates of the latest transformation run, we are currently shelving the transformation of Deinococcus aquaticus P81.

We are now focusing on the twitch assay and the morph assay. The twitch assay focuses upon the movement of Deinococcus aquaticus P81 via twitching mechanisms. Bacteria with pili extensions can drag themselves across surfaces, thus allowing them to move in search of nutrients even when they lack a flagella. This assay involves placing the bacteria directly onto plastic and tracking their movements over several weeks as they move towards the surrounding media in search of food. 

Additionally we are performing a morph assay. In this experiment, we are tracking a morphological change that we observe in P81. P81 elongates when grown in broth. The current hypothesis for this phenotypic change is that P81 forms a sort of biofilm in which the pili connections at the polar ends of the rods are connecting to other pili connections of other cells. This end to end connection creates long strands over time. To test this phenomenon, a protease can be used to attempt to break down the proteins within the pili connections. If an elongated string dissolves into individual rods after protease treatment, we can have evidence supporting the theory of pili connection. Alternatively, the cells could be failing to fully divide after replication. Evidence for this can be found by looking at the expression of the FtsZ gene within these cells. If this gene is inadequately expressed then it is possible that the cells are not dividing properly. 

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

Results

Gram stains revealed strings in shaken broths and rods in still broths. 

Media was made for the twitch motility assay. 

Discussion

The shaking motion of broths seems to induce biofilm formation. Increased metabolic activity due to increased aeration may result in greater expression of extracellular proteins. More experimentation is needed.