Molecular Cloning Notebook
June:
- Extraction of the M13 bacteriophage plasmid
- Making 5X TBE Buffer stock
- PCR three of the helper phage genes of interest
- Ytk CamR
- M13 g2
- M13 g6
- Purify the gel extractions of the DNA fragments
- Check the DNA concentrations:
- Ytk CamR- 46.12 ng/ul
- M13 g2- 36.9 ng/ul
- M13 g6- 12.8 ng/ul
- Test compentency of chemically competent cells using a Ytk plasmid
- Plating on LB produced a lawn of cells
- Cells are competent
- Golden gate reactions
- Ytk + gblocks
- Ytk CamR + M13 g2 + M13 g6
- Chemical transformations into NEB5a
- Ytk plasmid + gblocks
- Ytk plasmid + Ytk CamR + M13 g2 + M13 g6
- Quadrant streaked transformed colonies onto carbenicillin
- Ytk plasmid + Ytk CamR + M13 g2 + M13 g6
July:
Some genomic regions still need to be isolated and assembled into the final helper phage. The experiments used are to get those parts and preserve the golden gated parts transformed into E. coli.
- Make new liquid LB stock
- Freezer stock NEB5a Ytk plasmid + Ytk CamR + M13 g2 + M13 g6
- PCR the remaining genomic parts
- PA15
- Redo the first three PCR products to get more genomic DNA
- Running into PCR failures
- Found out the thermocycler was not working properly
- Extract the plasmids for the twist parts
- Plasmid ori
- M13 ori
August:
- Freezer stock of transformed parts got destroyed
September:
Try and redo all the parts we lost to get the gp3 and gp8 genes of the helper phage.
- Amplified the gp3 and gp8 genomic regions of the main phage
Phage Testing Notebook
May
M13 Overlay Assay F+ host: May 28
- Did a standard agar overlay to plate phages and understand M13 plaque morphology
- Top Images are the view from the bottom of the plate
- Bottom Images are the top of the plates
- This procedure was repeated for a while and lysate was harvested from each (this accounts for the time gaps between pictures)
June
Spot Assay Using Phage Lysate Harvested June 18 To Estimate Phage Titer: June 21
- Did a standard agar overlay to plate phages and understand M13 plaque morphology
- Top Images are the view from the bottom of the plate
- Bottom Images are the top of the plates
- This procedure was repeated for a while and lysate was harvested from each (this accounts for the time gaps between pictures)
July
M13/K-12 Attachment Assay with Diluted Phage Lysate: July 16/17
- Standard agar overlay but the phage lysate harvested from the plates above was diluted to 10^-6 pfu using TMG as diluent
- The black dots mark individual plaques and the total counts (approximate) are written on the plates
- I am not entirely sure why the phage counts lessened, this may be due to different attachment times, or the phage lysate may have gotten too hot and killed some of the phages.
- 10 minute attachment time before plating
- The overlays below the top three use K-12 as the replacement for the M13 is harvested from using F+ as the host and the diluted phage lysate.
- Attachment time may vary between M13 and K-12, both variations of E.coli.
This was done to monitor the phage activity using a bacteria that is different from the bacteria used that produces the M13 phage to try and predict the phage activity if S. aureus was used instead of E. coli.
August
M13/K-12 Attachment Assay with Diluted Phage Lysate: August 8
- Phage count is still inconsistent, but it seems as though the phages prefer to use F+ as the pelleted bacteria compared to K-12, F+ is still used as the host for both, instead of M13 being pelleted and resuspended and chloroformed, K-12 is used.
- These findings may suggest a differing attachment time that is longer than 10 minutes between E. coli strains.
Spot Assay with Undiluted Phage Lysate: August 8
- negative control where F+ is added to the top agar and plated, allowed to cool, and then is spotted with 2 uL of undiluted phage lysate, this is done to test that the lysate has not lost pfu’s, we did this to see if that was why we had inconsistent plaque counts.
- A large plaque is seen in the middle of the plate, this affirms that the lysate is highly concentrated and that if pfu’s were lost, it was a minimal amount.
M13 Attachment Assay with Diluted Phage Lysate: August 14
- The attachment time was manipulated to see if there were more plaques with a longer attachment time or if the number remained, in this trial we found the highest attachment pfu count to be around 50 minutes
- Attachments longer than 5 minutes become very high. As written above, this heightens the phage’s virulence and range of hosts.
- We decided to look at attachment times under 5 minutes to prevent this, see information below.
Attachment Assay (1- 5 minute attachment): August 19
- An attachment assay with M13 using F+ as a host where the attachment times are in one-minute intervals starting at one minute and ending at 5 minutes.
- After the first minute, there were no plaque increases. The 1-minute attachment had relatively the same amount of plaques as the 5-minute attachment so the increasing attachment intervals appeared to have the same amount of pfus as the initial 1-minute attachment.
- This suggests that M13 binds to the phage lysate within the span of 1 minute when plated after 5 minutes. We found that anything higher than 5 minutes had too high of a phage titer to be entirely safe due to the virulence risk of the bacteriophage.
- This is important for understanding how long the phage/dye mixture would have to be exposed to infected skin in order for the phages to be utilized efficiently to target and kill only specific bacteria.
Attachment Assay with S. aureus: August 29
- A standard attachment assay where, instead of M13 or K-12 used, S. aureus is used and F+ remains used as the host.
- Faint, not completely clear plaques are observed showing that there is phage attachment with S. aureus within 5 minutes
- Not certain that these results provide an accurate portrayal of S. aureus phage attachment as there was remaining bacteria in the supernantant that caused the plates to have a very foul odor similar to spoiled milk
- This may account for the turbid plaques
September
Spot Assay Dilution using F+ as host and Phage diluted with TMG: September 3
- This was done to monitor phage activity, we found that anything lower than 1.2 x 10^6 did not have any plaques
- 2 uL of phage lysate was used and diluted, this was done to count the pfu for another phage harvest, we found that our concentration was relatively the same as the first harvest.
- This may be because of the controlled OD 600 dilutions of the bacteria before being pelleted for supernatant
Attachment Assay with S. aureus: September 17
- A standard attachment assay where, instead of M13 or K-12 used, S. aureus is used and F+ remains used as the host.
- The attachment, once again, was done allowing 1-5 minutes for attachment.
- No plaques were observed on these plates, some areas where the top agar formed a bubble and then popped the bubble may be confused as plaques.
- The reason for this is unknown and this procedure was repeated three separate times the first one having residual bacteria in the supernatant causing the plates to spoil.
- The second two times there were no/faint observable plaques, this may be due to excess chloroform being mixed with the supernatant, killing host bacteria though a lawn appears to have formed.
- The 5 minute attachment
- Results obtained using S. aureus proved inconclusive though we have evidence that M13 bacteriophages have the ability to bind to other bacteria within the E. coli variants.
