Week of 10/21/24

 

Introduction
Within the morph project, it was observed that P81 cells tend to age more in TGY than in R2B. This could be a result of the increased nutrient content causing enhanced metabolic activity and increased replication speed. Furthermore, the gram staining process for the morph project has been streamlined and the observations are more thoroughly and effectively documented. 
The twitch runs were further documented and the pictures have been uploaded to a google doc. 

Methods
For the morph project, the cells were packed to get better gram stains.
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
P81 appears to string out well under shaking conditions in TGY and under still conditions in R2B. 
Discussion
In TGY, we have a nutrient rich media. Perhaps, the increased replication rate increases the odds that a biofilm will form. Meanwhile, R2B is nutrient poor, so perhaps the gentler still conditions are more conducive towards cells aligning and connecting from end to end. 

Comments

Post a Comment

Popular posts from this blog

Week of 11/25/24

Image
  Introduction The use of proteinase K has been inconclusive thus far and so we have decided to increase the concentration to 20ug/ml. Proteinase K is optimized at 50-65 degrees Celsius so its activity is slowed at the incubation temperature of P81. Hopefully increasing the concentration will account for the decrease in temperature.  Additionally, lab is closing soon for winter break and winding down protocols are in effect. Media production has been halted to as needed only and freeze backs of samples are being created.  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 express...
Read more

Week of 11/18/24

Image
  Introduction The plans for twitch and morph were put into effect this week. We studied the effects of proteases upon the connected P81 cells while simultaneously studying the effects of chemoattracts upon twitching P81 cells.  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 co...
Read more

Week of 09/16/24

Image
  Introduction The transformation procedure was attempted again this week with a particular interest upon the OD value. We are attempting to perfect the procedure in every conceivable way so that any failure for P81 to transform can not be attributed to human error. Additionally, varying amounts of plasmid were used across experimental groups to test whether plasmid concentration has any effect. Methods A 278 ng per microliter plasmid extraction was used. The first experimental group was given 4 microliters while the second experimental group was given 8 microliters. The final experimental group was given 12 microliters of a 220.4 ng per microliter plasmid extraction.  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...
Read more