Amplifying Genes In Saccharomyces cerevisiae In Order to Test for Increased Drug Resistance

Micaylon Moore Author

08/02/2021 Added

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When we crank up the dosage of a specific gene, does this increase resistance to our potential drugs? This research is geared towards creating new antifungals that have not developed resistance.

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[00:00:01.860]Hi, everyone.
[00:00:02.760]My name is Micaylon Moore
[00:00:04.070]and I'm an undergraduate biological scientist student
[00:00:06.780]at the University of Nebraska-Lincoln.
[00:00:09.140]This summer, I got the opportunity to work
[00:00:10.877]in the Riekhof Lab in the Beadle Center on campus.
[00:00:13.990]My project was amplifying genes in Saccharomyces cerevisiae
[00:00:17.270]in order to test for increased drug resistance.
[00:00:22.720]So, there are many human pathogenic fungi
[00:00:24.860]that exists in the general population
[00:00:26.670]and are especially harmful and dangerous
[00:00:28.340]to individuals with compromised immune systems.
[00:00:31.180]Some of the more frequently diagnosed fungal infections
[00:00:33.700]in immunocompetent individuals include ringworm,
[00:00:36.290]aspergillosis, and candidiasis.
[00:00:39.080]Anti-fungal medications are often of limited effectiveness
[00:00:41.990]due to fungi becoming more resistant
[00:00:43.730]to basic and classic anti-fungal drugs.
[00:00:46.760]Human and fungi are closely related
[00:00:48.460]which makes fighting off fungi difficult
[00:00:50.210]because you have to locate a target
[00:00:51.630]that kills off the fungi
[00:00:52.950]but is not harmful to the human
[00:00:54.430]and there are not many of these targets.
[00:00:56.840]Researchers need to find more antifungals
[00:00:58.820]and more targets for antifungals.
[00:01:03.950]So here in the lab,
[00:01:04.783]we have identified some compounds
[00:01:06.190]that have characteristics of antifungals under the names of
[00:01:08.517]Para-Nitrophenol Isocyanide, NPIC for short,
[00:01:11.960]and 2-Hydroxyethylhydrazine, HEH for short.
[00:01:15.560]NPIC is a compound we see here in the middle,
[00:01:17.280]which has been synthesized by Dr. David Berkowitz
[00:01:19.720]in the chemistry department
[00:01:20.820]at the University of Nebraska-Lincoln
[00:01:22.950]and it is derived from Xanthocilin,
[00:01:24.580]which is a naturally occurring compound on the left.
[00:01:26.760]HEH is the drug that we can see here the bottom right.
[00:01:30.450]This drug affects production of membrane lipids.
[00:01:35.060]So Saccharomyces cerevisiae is also known
[00:01:36.671]as baker's yeast or brewer's yeast
[00:01:38.850]and it is a great organism
[00:01:40.160]for lab experiments in genetic research.
[00:01:42.700]The single-celled eukaryotic yeast can replicate
[00:01:44.501]at a very high speed.
[00:01:45.802]It is relatively safe to use,
[00:01:47.740]and we have the ability to manipulate the genes
[00:01:49.890]with extensive genetic tools.
[00:01:54.270]So the question that I'm working on answering is
[00:01:56.350]when we crank up the dosage of a specific gene,
[00:01:58.350]also known as gene copy number,
[00:02:00.210]does this increase resistance to our potential drugs?
[00:02:03.170]We did this with a YEp13 plasmid
[00:02:05.320]that contains multiple Saccharomyces genes.
[00:02:07.800]The genes we are looking for and at
[00:02:10.610]are located in the tetracycline portion of the vector.
[00:02:13.670]Using the forward and reverse BamHI restriction enzymes,
[00:02:16.436]we can read approximately 800 to 1000 base pairs
[00:02:19.149]from this site.
[00:02:20.800]With this information, we can determine
[00:02:22.380]the sequence in between the sites
[00:02:24.100]ultimately determining the gene.
[00:02:28.810]So first we inserted the plasmids
[00:02:30.300]that contain random genes from Saccharomyces cerevisiae
[00:02:33.030]into competent E. coli cells where each cell picked up
[00:02:35.740]a unique set of Saccharomyces cerevisiae genes.
[00:02:38.920]I then picked colonies on selective media
[00:02:40.870]to obtain individual E. coli colonies
[00:02:43.130]with specific Saccharomyces genes.
[00:02:45.460]Next, I performed a mini-prep
[00:02:46.890]to isolate unique impure versions
[00:02:48.770]of individual plasmids.
[00:02:50.600]From here, I transformed those individual plasmids
[00:02:53.000]back into Saccharomyces.
[00:02:54.880]After this, I performed replica plating
[00:02:56.780]to hopefully obtain strands
[00:02:58.110]that are resistant to HEH and NPIC.
[00:03:03.400]We transformed E. coli with our Saccharomyces colonies
[00:03:06.010]to obtain single colonies
[00:03:07.250]and plated it onto LB + Ampicillin.
[00:03:09.850]After 24 hours of growth,
[00:03:11.875]the plates have an abundance of colonies.
[00:03:14.460]The plate reader concentrations demonstrate
[00:03:16.125]that there was a fair amount of DNA.
[00:03:21.270]The purpose of replica plating was
[00:03:22.910]to compare the master library plate
[00:03:24.660]and the drug plates to screen
[00:03:25.940]for resistant colonies.
[00:03:27.560]We used 7 micromolar NPIC plates,
[00:03:29.442]725 micromolar HEH plates, and a control.
[00:03:33.430]We tested other concentrations
[00:03:34.980]and decided to move forward
[00:03:36.150]with these concentrations.
[00:03:37.870]I performed the stamping method seven times
[00:03:39.650]for each of the five libraries.
[00:03:44.720]This here is a photo
[00:03:45.910]of the first of seven series I performed
[00:03:48.000]and I was expecting to see two or three colonies per plate,
[00:03:50.770]maybe 10, but definitely no more.
[00:03:52.810]I completed this experiment and it did not work
[00:03:54.950]so we needed to try another method of determining
[00:03:57.200]if we had transformed Saccharomyces cells
[00:03:59.320]with increased resistance to our drugs.
[00:04:04.420]Our next idea was to try the experiment in a liquid form,
[00:04:06.900]which would expose our cells to the drug
[00:04:08.810]and hopefully give them resistance,
[00:04:10.190]and I plated them out to see who survives.
[00:04:12.770]They were plated on 10 micromolar NPIC plates
[00:04:15.050]because they don't usually grow at that concentration.
[00:04:19.940]What I was hoping to see here is
[00:04:21.230]that the tubes that contained NPIC,
[00:04:23.540]developed NPIC resistant to yeast cells,
[00:04:25.400]and they grow in a higher abundance on the NPIC plates,
[00:04:27.910]but this was not the case.
[00:04:29.290]We tried two methods of testing the drug
[00:04:31.130]on our Saccharomyces that we thought was transformed,
[00:04:34.060]but it did not work and we decided to retransform E. coli
[00:04:36.910]with our Saccharomyces gene containing plasmid.
[00:04:41.810]I created three transformation mixes
[00:04:43.650]containing different amounts of DNA
[00:04:45.260]from a YEp13 library pool.
[00:04:47.880]I wanted to confirm
[00:04:48.790]that the plasma was inside of Saccharomyces
[00:04:51.010]by performing a transformation
[00:04:52.330]and taking the resulting plasmid
[00:04:53.870]and transforming it back into E. coli.
[00:04:56.100]Negative control with no plasmid should not grow at all
[00:04:59.180]because there is no plasmid to develop resistance,
[00:05:01.750]but as you can see, there is a lot of growth.
[00:05:04.380]After this last experiment did not work,
[00:05:06.187]I tried a new transformation
[00:05:07.880]with a new strain of E. coli, named XL1-Blue.
[00:05:11.610]One of the tubes was a no DNA control,
[00:05:13.680]one tube was a plasmid control,
[00:05:15.650]and five of the tubes contain library DNA.
[00:05:20.410]After numerous failed attempts,
[00:05:21.780]I decided to try using new Ampicillin stock.
[00:05:25.210]After creating new Ampicillin plates with the new drug,
[00:05:27.650]the Ampicillin worked and growth was inhibited.
[00:05:30.460]We can see in libraries 4 and 5 demonstrations
[00:05:32.867]that there are single colonies on the new Ampicillin
[00:05:35.460]while the old Ampicillin led to a lot of colonies.
[00:05:40.470]After exhausting all of our options,
[00:05:42.330]I wanted to confirm that my plasmid was still good.
[00:05:45.260]I use gel electrophoresis, which is a technique used
[00:05:48.010]to separate DNA fragments according to their size.
[00:05:51.040]In the gel without restriction enzymes,
[00:05:52.830]you can see the DNA,
[00:05:54.160]and in the gel with the restriction enzymes,
[00:05:55.762]the DNA had been split up into fragments
[00:05:58.330]by the restriction enzymes KPN1 and PBU2.
[00:06:01.680]We were able to confirm that our E. coli has
[00:06:03.470]now been transformed with our plasmids.
[00:06:07.750]So over the course of the summer,
[00:06:09.050]a lot of things did not go to plan
[00:06:10.480]because experiments did not produce the results
[00:06:12.870]that were expected.
[00:06:14.090]I had to go back and perform techniques multiple times
[00:06:16.760]and troubleshoot.
[00:06:17.930]We are now getting back on track
[00:06:19.260]and I recently received a sequence that worked,
[00:06:21.640]but it was just a vector, which is a good start.
[00:06:23.970]I'm going to continue this process
[00:06:25.620]and after determining possible genes
[00:06:27.560]that cause resistance from the plasmid,
[00:06:29.439]test out each individual gene to figure out
[00:06:31.870]which one is responsible.
[00:06:33.400]We can do this
[00:06:34.233]by separating each gene out
[00:06:35.657]into its own individual plasmid.
[00:06:37.890]I will redo stamping now that we have plasmids
[00:06:40.520]and DNA that we know works.
[00:06:42.220]Once I get resistant colonies and find the resistant genes,
[00:06:44.940]I'll work towards finding out why they are resistant.
[00:06:50.130]At this time, I would like to acknowledge
[00:06:51.910]and thank the Dr. Riekhof team for being so welcoming
[00:06:54.370]and allowing me this experience.
[00:06:56.970]I would also like to thank
[00:06:58.090]the Undergraduate Creative Activity and Research Experience,
[00:07:00.602]UCARE for short, at the University of Nebraska-Lincoln
[00:07:03.260]for allowing and providing funding for this opportunity.

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Amplifying Genes In Saccharomyces cerevisiae In Order to Test for Increased Drug Resistance

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