Difference between revisions of "UW-Stout/UV Light SP22"

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(Created page with "To knock out genes in S. cerevisiae, we used a Cas9-assisted homologous recombination approach: * Build an S. cerevisiae shuttle vector expressing Cas9 and a guide RNA targeti...")
 
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−
To knock out genes in S. cerevisiae, we used a Cas9-assisted homologous recombination approach:
+
==Introduction==
−
* Build an S. cerevisiae shuttle vector expressing Cas9 and a guide RNA targeting the gene of interest.
+
We will be stressing multiple strains of yeast cells with UV light exposure. Using the following calibration protocol and knockout gene protocol.  
−
* Use PCR to make a linear URA3 cassette flanked by homologous sequences upstream and downstream of the Cas9 target site.
 
−
* Transform BY4735 with the plasmid and PCR DNA.  Select on Ura dropout plates.
 
−
* Use PCR to verify the URA3 insertion.
 
  
−
==Resources==
+
===Materials===
 +
*Phosphate Buffered Saline (PBS)
 +
*Growth Media (Agarose)
 +
*Yeast Strains, Knockouts, and Wild Type
  
−
* Background strain: [https://www.atcc.org/en/Products/All/200897.aspx BY4735].  BY4735 is a [https://www.ncbi.nlm.nih.gov/pubmed/9483801 MATalpha 6-way auxotroph based on S288C], bearing non-revertable deletions in [[ADE2]], [[HIS3]], [[LEU2]], [[MET15]], [[TRP1]] and [[URA3]].
+
===Equipment===
−
* Plasmids: Knock-out plasmids were built using the [https://pubs.acs.org/doi/10.1021/sb500366v Dueber lab's yeast toolkit.]  Additional assembly details are below.
+
*P-1000 Micropipette
 +
*P-10 Micropipette
 +
*Bachur & Associates Santa Clara, CA 95050 Model LS-100-3 UV Light Exposure System set at 400 Watts
 +
*Incubator set to 30°C
 +
*Fine Tip Sharpie
 +
*60mm Culture Dish
 +
*Vortexer
 +
*Gloves
 +
*Hair Net
 +
*Coverall Top
 +
*Boot Covers
  
−
==Construction of Cas9/sgRNA plasmids==
+
==Calibration Protocol==  
−
# We used the Yeast Toolkit to build a L2 plasmid containing the following functional parts:
+
  '''''Safety Note''''': When conducting any experiment it's important to wear the proper PPE for yourself as well as to protect your experiments in our lab we wore a lab coat and gloves. While we were in our cleanroom doing the UV Light Exposure we wore additional equipment including hair nets, coverall top, boot covers, and gloves.
−
#* Cas9 driven by the constitutive PGK1 promoter
 
−
#* an sgRNA cassette with a GFP dropout
 
−
#* a LEU2 yeast selection cassette
 
−
#* a CEN6/ARS4 yeast origin of replication
 
−
#* an kanamycin E. coli selection casette
 
−
#* a ColE1 E. coli origin of replication
 
−
# For each gene we targeted, we used [https://benchling.com/ Benchling] to select 20-bp targeting sequences adjacent to PAMs.  We attempted to find targeting sequences in the first 1/4 of the gene to ensure gene disruption.
 
−
# We designed and synthesized oligonucleotides as per the YTK instructions, annealed them, then used a GoldenGate reaction with Esp3I to clone them into the expression plasmid.  We verified the plasmid using a traditional restriction map.
 
  
 +
===Calibration Experiment 1===
 +
In our First calibration experiment, we conducted UV light exposure based on time, the intervals we used are as follows: 1 second, 3 seconds
 +
5 seconds, 10 seconds, 20 seconds, 50 seconds, 100 seconds, 200 seconds, 500 seconds, and 1000 seconds.
  
−
==Construction of a URA3 knockout cassette==
+
#Calculate the amount of pure cell culture and amount of PBS in order to get 1000 cells per mL
−
# We used [https://benchling.com/ Benchling] to identify 40 bp upstream and downstream of the Cas9 target site.
+
#Using a P-10 Micropipette, pipette the amount of cell culture needed into a 1.5mL micropipette tube.
−
# We designed primers against the URA3 selection cassette on plasmid YTK74 (from the Dueber lab's yeast toolkit) to append those 40 bp to cassette upstream and downstream.
+
#Using a P-1000 Micropipette, pipette the amount of PBS calculated into the same micropipette tube as step 2
−
# We synthesized those oligonucleotides, performed the PCR, verified the PCR product using gel electrophoresis and purified it using a silica spin column.
+
#Mix the PBS and cell culture either by hand or by using a Vortexer
 +
#Pipette 100uL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it.  
 +
#Add Glass beads and shake for 30 seconds, then remove glass beads
 +
#Turn on the UV Light, set it to 400 Watts
 +
#Label cell culture with interval time (1 Second, 2 Seconds, etc.)
 +
#Remove the top off of the cell culture plate
 +
# Expose the single-cell plate for the designated time under the UV Light
 +
#Repeat Steps 8-10 until out of intervals.  
 +
#Place cell culture plate in 30 degrees C incubator for 48 hours
 +
#See Results
  
−
==Transformation of S. cerevisiae==
+
===Calibration Experiment 2===
−
# We used [https://www.ncbi.nlm.nih.gov/pubmed/17401330 Gietz' PEG/SS DNA/LiAc protocol] to co-transform the Cas9/sgRNA plasmid and the URA3 cassette.  
+
Due to the results of calibration 1, the time gaps left us needing to do another experiment to test the time between 500 seconds and 1000 seconds. We did new time intervals of 550 seconds, 600 seconds, 650 seconds, 700 seconds, 750 seconds, 800 seconds, 850 seconds, and 950 seconds.
−
# We plated on leucine dropout plates to measure transformation efficiency, and uracil dropout plates to isolate clones in which the genes had been disrupted.
 
  
−
==Verification of gene disruption==
+
#Calculate the amount of pure cell culture and amount of PBS in order to get 1000 cells per mL
−
# We used a [https://www.ncbi.nlm.nih.gov/pubmed/21548894 lithium acetate / SDS method] to extract genomic DNA from putative knockout strains
+
#Using a P-10 Micropipette, pipette the amount of cell culture needed into a 1.5mL micropipette tube.
−
# We used [https://benchling.com/ Benchling] to design PCR primers upstream and downstream of the knockout site.
+
#Using a P-1000 Micropipette, pipette the amount of PBS calculated into the same micropipette tube as step 2
−
# We used PCR followed by gel electrophoresis to determine if the URA3 cassette had inserted at the desired site.
+
#Mix the PBS and cell culture either by hand or by using a vortexer
 +
#Pipette 100uL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it.
 +
#Add Glass beads and shake for 30 seconds, then remove glass beads
 +
#Turn on the UV Light, set it to 400 Watts
 +
#Label cell culture with interval time (550 Seconds,600 Seconds, etc.)
 +
#Remove the top off of the cell culture plate
 +
# Expose the single-cell plate for a designated time under the UV Light
 +
#Repeat Steps 8-10 until out of intervals.
 +
#Place cell culture plate in 30 degrees C incubator for 48 hours
 +
#See Results
 +
 
 +
==Calibration Results==
 +
 
 +
===Calibration Experiment 1 Results===
 +
 
 +
'''Results'''
 +
1 Second
 +
[[File:Ashton1Sec.jpg|200px|]]
 +
500 Seconds
 +
[[File:Ashton500Sec.jpg|200px|]]
 +
1000 Seconds
 +
[[File:Ashton1000Sec.jpg|200px|]]
 +
 
 +
 
 +
'''Interpretation'''
 +
We found that the experiment we conducted was inconclusive. The 500 Second plate had too many yeast cells for us to count and make an accurate count and the 1000-second plate didn't have any yeast colony's on it, we concluded that the point we want must be between the 500-second mark and the 1000 second mark.
 +
 
 +
===Calibration Experiment 2 Results===
 +
 
 +
'''Results'''
 +
 
 +
[[File:ASHTONMIKOLOSKIYeast Colonys Over Time.png|400px|]]
 +
*550 seconds = 483 colonies
 +
*600 seconds = 279 colonies
 +
*650 seconds = 198 colonies
 +
*700 seconds = 40 colonies
 +
*750 seconds = 54 colonies
 +
*800seconds = 13 colonies
 +
*850 seconds = 4 colonies
 +
*900 seconds = 2 colonies
 +
*950 seconds = 1 colony
 +
 
 +
 
 +
 
 +
'''Interpretation'''
 +
We found that the point we were looking for was at the 600-second mark, with this in mind this will be the amount of time we will use in the knockout protocol.
 +
 
 +
==Knock-out Protocol==
 +
 
 +
'''''Safety Note''''': When conducting any experiment, it's essential to wear the proper PPE for yourself and protect your experiments. In our lab, we wore lab coats and gloves. While we were in our cleanroom doing the UV Light Exposure, we wore additional equipment, including hairnets, coverall top, boot covers, and gloves.
 +
 
 +
#Calculate the amount of pure cell culture and amount of PBS in order to get 200 cells per mL
 +
#Using a P-10 Micropipette, pipette the amount of cell culture needed into a 1.5mL micropipette tube.  
 +
#Using a P-1000 Micropipette, pipette the amount of PBS calculated into the same micropipette tube as step 2
 +
#Mix the PBS and cell culture either by hand or by using a vortexer
 +
#Pipette the full 500uL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it.
 +
#Pipette the full 500uL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it. This one will not be exposed to UV light and will be designated as the control
 +
#Add Glass beads and shake for 30 seconds, then remove glass beads
 +
#Label cell culture with the Gene Number
 +
#Turn on the UV Light, set it to 400 Watts
 +
#Remove the top off of the cell culture plate
 +
# Expose the single-cell plate for 600 seconds under the UV light, keeping the control away from the light
 +
#Repeat Steps 8-10 until finished
 +
#Place cell culture plate in 30 degrees C incubator for 48 hours
 +
#See Results
 +
 
 +
==Knock-out Results==
 +
[[File:ASHTONMIKOLOSKIFinal Project Graph.png|350px|]]
 +
 
 +
*Knock-out Strain Wild Type
 +
 
 +
*Knock-out Strain 1:[[YDL109C]]
 +
 
 +
*Knock-out Strain 2:[[YGL146C]]
 +
 
 +
*Knock-out Strain 3:[[YOR111W]]
 +
 
 +
*Knock-out Strain 4:[[YHL029C]]
 +
 
 +
*Knock-out Strain 5:[[YDR307W]]
 +
 
 +
*Knock-out Strain 6:[[YNL058C]]
 +
 
 +
*Knock-out Strain 7:[[YCL049C]]
 +
 
 +
*Knock-out Strain 8:[[YGR079W]]

Latest revision as of 11:55, 3 May 2022

Introduction

We will be stressing multiple strains of yeast cells with UV light exposure. Using the following calibration protocol and knockout gene protocol.

Materials

  • Phosphate Buffered Saline (PBS)
  • Growth Media (Agarose)
  • Yeast Strains, Knockouts, and Wild Type

Equipment

  • P-1000 Micropipette
  • P-10 Micropipette
  • Bachur & Associates Santa Clara, CA 95050 Model LS-100-3 UV Light Exposure System set at 400 Watts
  • Incubator set to 30°C
  • Fine Tip Sharpie
  • 60mm Culture Dish
  • Vortexer
  • Gloves
  • Hair Net
  • Coverall Top
  • Boot Covers

Calibration Protocol

 Safety Note: When conducting any experiment it's important to wear the proper PPE for yourself as well as to protect your experiments in our lab we wore a lab coat and gloves. While we were in our cleanroom doing the UV Light Exposure we wore additional equipment including hair nets, coverall top, boot covers, and gloves.

Calibration Experiment 1

In our First calibration experiment, we conducted UV light exposure based on time, the intervals we used are as follows: 1 second, 3 seconds 5 seconds, 10 seconds, 20 seconds, 50 seconds, 100 seconds, 200 seconds, 500 seconds, and 1000 seconds.

  1. Calculate the amount of pure cell culture and amount of PBS in order to get 1000 cells per mL
  2. Using a P-10 Micropipette, pipette the amount of cell culture needed into a 1.5mL micropipette tube.
  3. Using a P-1000 Micropipette, pipette the amount of PBS calculated into the same micropipette tube as step 2
  4. Mix the PBS and cell culture either by hand or by using a Vortexer
  5. Pipette 100uL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it.
  6. Add Glass beads and shake for 30 seconds, then remove glass beads
  7. Turn on the UV Light, set it to 400 Watts
  8. Label cell culture with interval time (1 Second, 2 Seconds, etc.)
  9. Remove the top off of the cell culture plate
  10. Expose the single-cell plate for the designated time under the UV Light
  11. Repeat Steps 8-10 until out of intervals.
  12. Place cell culture plate in 30 degrees C incubator for 48 hours
  13. See Results

Calibration Experiment 2

Due to the results of calibration 1, the time gaps left us needing to do another experiment to test the time between 500 seconds and 1000 seconds. We did new time intervals of 550 seconds, 600 seconds, 650 seconds, 700 seconds, 750 seconds, 800 seconds, 850 seconds, and 950 seconds.

  1. Calculate the amount of pure cell culture and amount of PBS in order to get 1000 cells per mL
  2. Using a P-10 Micropipette, pipette the amount of cell culture needed into a 1.5mL micropipette tube.
  3. Using a P-1000 Micropipette, pipette the amount of PBS calculated into the same micropipette tube as step 2
  4. Mix the PBS and cell culture either by hand or by using a vortexer
  5. Pipette 100uL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it.
  6. Add Glass beads and shake for 30 seconds, then remove glass beads
  7. Turn on the UV Light, set it to 400 Watts
  8. Label cell culture with interval time (550 Seconds,600 Seconds, etc.)
  9. Remove the top off of the cell culture plate
  10. Expose the single-cell plate for a designated time under the UV Light
  11. Repeat Steps 8-10 until out of intervals.
  12. Place cell culture plate in 30 degrees C incubator for 48 hours
  13. See Results

Calibration Results

Calibration Experiment 1 Results

Results 1 Second Ashton1Sec.jpg 500 Seconds Ashton500Sec.jpg 1000 Seconds Ashton1000Sec.jpg


Interpretation We found that the experiment we conducted was inconclusive. The 500 Second plate had too many yeast cells for us to count and make an accurate count and the 1000-second plate didn't have any yeast colony's on it, we concluded that the point we want must be between the 500-second mark and the 1000 second mark.

Calibration Experiment 2 Results

Results

ASHTONMIKOLOSKIYeast Colonys Over Time.png

  • 550 seconds = 483 colonies
  • 600 seconds = 279 colonies
  • 650 seconds = 198 colonies
  • 700 seconds = 40 colonies
  • 750 seconds = 54 colonies
  • 800seconds = 13 colonies
  • 850 seconds = 4 colonies
  • 900 seconds = 2 colonies
  • 950 seconds = 1 colony


Interpretation We found that the point we were looking for was at the 600-second mark, with this in mind this will be the amount of time we will use in the knockout protocol.

Knock-out Protocol

Safety Note: When conducting any experiment, it's essential to wear the proper PPE for yourself and protect your experiments. In our lab, we wore lab coats and gloves. While we were in our cleanroom doing the UV Light Exposure, we wore additional equipment, including hairnets, coverall top, boot covers, and gloves.
  1. Calculate the amount of pure cell culture and amount of PBS in order to get 200 cells per mL
  2. Using a P-10 Micropipette, pipette the amount of cell culture needed into a 1.5mL micropipette tube.
  3. Using a P-1000 Micropipette, pipette the amount of PBS calculated into the same micropipette tube as step 2
  4. Mix the PBS and cell culture either by hand or by using a vortexer
  5. Pipette the full 500uL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it.
  6. Pipette the full 500uL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it. This one will not be exposed to UV light and will be designated as the control
  7. Add Glass beads and shake for 30 seconds, then remove glass beads
  8. Label cell culture with the Gene Number
  9. Turn on the UV Light, set it to 400 Watts
  10. Remove the top off of the cell culture plate
  11. Expose the single-cell plate for 600 seconds under the UV light, keeping the control away from the light
  12. Repeat Steps 8-10 until finished
  13. Place cell culture plate in 30 degrees C incubator for 48 hours
  14. See Results

Knock-out Results

ASHTONMIKOLOSKIFinal Project Graph.png

  • Knock-out Strain Wild Type
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