A Year in the Lab: Further Water Collection and Plasmid Isolation

Monday 24th February

My week began with preparation for further water collection, by autoclaving 2 500ml Duran flasks. I also retrieved and re set up the water filtration system on my bench ready for water processing. I checked to make sure I had enough MLSA plates containing antibiotic, membrane filters and LB broth containing the same concentration of antibiotic as the MLSA plates.

Tuesday 25th February

I began the day by collecting my autoclaved Duran flasks, extendable rod and gloves, then headed out to the River Witham to collect samples from the same location as previously. Once finished I returned to the lab and began filtering my collected water through filter membranes and applying these to MLSA plates, using the same method as before. I then incubated these overnight at 44ºC.

IMG_3958

I also decided to prepare LB agar plates containing antibiotic ready for transformation. I used the same method as for MLSA plates with antibiotic, however I adjusted the amount of LB agar powder used according to the product information.

Wednesday 26th February

Today started by removing the incubated MLSA plates and inoculating single colonies from the plates into separate LB broths containing antibiotic. These were then incubated overnight with agitation at 37ºC.

I then collected and prepared the required equipment and reagents to conduct plasmid isolation by alkaline lysis, this included:

  • Solutions 1 (GTE), 2 (SDS/NaOH) and 3 (KOAc)
  • Isopropanol (2-propanol)
  • 70% Ethanol
  • TE Buffer
  • 1kb Ladder
  • Pure water (H2O)
  • Loading Buffer
  • Gel electrophoresis tank
  • 1x TAE Buffer
  • Eppendorf tubes

Thursday 27th February

I began today by removing the incubated LB broths and inoculating each onto a nutrient agar slope, in order to add them to my isolate collection and allow continued access to colonies. I then started plasmid isolation through alkaline lysis:

How to conduct alkaline lysis

  • Place KOAc on ice to allow pre chilling.
  • Centrifuge 3ml (1.5ml at a time) of each overnight LB broth culture in separate 1.5ml eppendorfs for 2 minutes at 30,000 rpm.

IMG_5494

  • Store the rest of the culture at 4ºC.
  • Pour off the supernatant from each tube and dry the remaining pellet.
  • Add 100μl of GTE solution and re-suspend by pippetting.

IMG_5499

  • Add 200μl of SDS/NaOH solution and mix by inverting the tube a few times.
  • Add 150μl of pre chilled KOAc, vortex for 10 seconds to mix then place on ice for 5 minutes.
  • Centrifuge for 5 minutes at 30,000 rpm.
  • Pour the supernatant from each tube into new tubes and throw away the remaining pellet.
  • Add 900μl of isopropanol and store on ice for 10 minutes.
  • Centrifuge for 5 minutes at 30,000 rpm.
  • Pour off the supernatant from each tube and dry the remaining pellet.
  • Add 1ml of 70% ethanol to each tube, vortex briefly then centrifuge for 5 minutes at 30,000 rpm.
  • Add 50μl of TE buffer and vortex briefly.
  • Samples are now ready for gel electrophoresis.

Gel electrophoresis was conducted as before, using a 1% agarose gel and 1x TAE buffer running at 110V for 1.5 hours. Each sample was prepared by combining: 5μl of sample DNA with 3μl loading buffer and 7μl pure H2O, before pippetting 15μl into each well. Samples were run alongside a 1kb ladder as reference. As this was my first attempt at plasmid isolation by alkaline lysis I wasn’t sure how good my results would be, but I was pleasantly surprised!

plasmid isolation gelMy first attempt at plasmid isolation through alkaline lysis! 

I ran 6 samples against a 1kb ladder, as can be seen the gel image above. If plasmid isolation isn’t conducted completely correctly excess genomic DNA can be seen near the top of the gel, larger than 10kb because it is too big to move further through the gel. Excess RNA can be seen near the bottom of the gel, less than 0.5kb because it is small enough to move quickly through the gel. What I am interested in is the presence of bands between these two – in this case around 1.0-2.0kb. The two clear bands indicate that the bacterial samples processed contained plasmids which could be isolated by alkaline lysis and then visualised via gel electrophoresis. This is important to my research as I am specifically looking for Escherichia coli isolates which have the ability to transfer plasmids containing information regarding the production of ESBLs and therefore antibiotic resistance.

1kb ladder NEB1kb ladder reference bands (New England BioLabs (NEB))

These plasmid positive samples can then be combined with competent cells for transformation, allowing plasmids to be taken up and incorporated into the recipient chromosome. These samples can then be used for PCR, in order to amplify any of the 5 types of CTX-M gene (Bla), if present.

I also decided to conduct antibiotic susceptibility testing on each of these 6 samples. As before, each was spread plated onto a separate Mueller Hinton agar plate, allowed to absorb for 10 minutes before applying 8 different antibiotic discs to each. These plates were then incubated overnight at 37ºC.

IMG_5004

Friday 28th February

Today was a very quiet day for me in the lab and only involved the removal of my antibiotic susceptibility plates from the incubator and observing/recording the zone clearing. As before, I recorded this information to the nearest mm and added it to the prepared table in my lab book. Antibiotic susceptibility was as I expected, showing resistance to amoxycillin, ampicillin, ceftriaxone and cefotaxime, as well as intermediate susceptibility to several others.

Next week…

I am really pleased with how my first attempt at plasmid isolation went! Next week I hope to conduct alkaline lysis on all the samples within my Escherichia coli  isolate collection, in order to visualise how many actually carry plasmids. I also hope to add a few more samples to my collection and then proceed to transforming those which carry plasmids into competent cells.

Now my restriction enzyme has arrived I also hope to continue work on perfecting my PFGE bacterial plugs and running protocol, beginning with trying out my newly made batch of plugs. I also hope to start PCR soon, however this will depend on how long my primers take to arrive after I order them at the beginning of next week.

A Year in the Lab…

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