"
Team:WashU/Notebook/Yeast
From 2010.igem.org
Week of 6/21
6/24
plated Yeast Gal-YFP MATa, trp1, leu2::GAL1-YFP URA3/Kanr and Yeast YF4 MATa Prototroph on a Kan plate and a YPD plate
Started a liquid culture of Yeast YF4 MATa Prototroph in YPD
Week of 6/28
6/29
A practice transformation was preformed at the Cohen Lab with Ilaria. The OD600 was 0.651 which was low, but used in order to speed up the experiment. The PMKV005 loxKanlox Plasmid (McCusker 2005 Yeast, 21: 163-171) was used into transform wildtype yeast. The plates used were Kan positive plates created on 6/24. However it was later found that these plates might not correctly select for drug resistance. Two plates were made, one with the transformant and one with a control. The attached procedure was used and annotated during the experiment
7/1
• Examined the Sample and Control plates from the practice transformation on June 29th. Two colonies had grown on the Sample plate and none showed on the control plate. The low colony number could be due to a low OD600 (0.651) used in the transformation. • Plated the two colonies on a G418 plate in order to conduct the first round of purification
7/2
PRACTICE YEAST TRANSFORMATION Colonies grew on the first purification plate for both colony 1 and 2. Two colonies were plated from both transformed colony 1 and transformed colony 2 onto a second and final purification plate -- PLATES: YPD: all yeast grew His-: only 1&2 (WT) yeast grew, (4,6,11,12 did not grow) Kan:3,4,7 grew- 1&2 were selected against but some colonies were visible Ura-: only 1&2 (WT) yeast grew, (11 did not grow)
Amp: bacteria 8,9,10 grew (other amp plate WT control did not grow)
conclusion: all the resistance plates work in that they select against those w/o the resistance; however, there was some low level of growth on some of these plates where there was not supposed to be
Week of 7/12
7/12
streaked out wild type yeast strain FY4 onto a YPD plate and inoculated about 5 mL of YPD- incubate and shake Brian: The FY5 freezer stock was accidentally allowed to thaw and therefor a second freezer stock was started by growing an overnight culture inoculated from the original freezer stock. The freezer stock was made according to protocol from the cohen lab.
7/13
Plated FY4 yeast onto a YPD plate to grow up for a practice transformation in case the plate from the freezer stock does not grown since there were no visible growths on it. The FY4 was taken from a Ura- plate plated on 6/29 because it had the best colonies out of all the plates in the refrigerator. A freezer stock of FY4 was made according to protocol from an overnight culture of the FY4 strain since the original stock was destroyed when plating on 7/12.
7/14
Yeast colonies were observed on the FY4 plated on 7/12 from the FY4 freezer stock. This plate was brought to the Cohen lab along with liquid YPD-4 and they were used to inoculate a 5ml overnight culture.
7/15
Yeast Transformation Alice and Brian went to the Cohen Lab to preform a yeast transformation. Ilaria showed us how to use the nano-drop using the following procedure:
1. Wipe the nanodrop with a kim-wipe 2. Click the shortcut on the computer that is identicle to the logo on the nano drop 3. Choose Nucleic Acid in the program 4. Put on 1 μL of dH2O to clean it 5. Cleantop and bottem with kim wipe 6. Put on 1 μL of blank 7. Clean with Kim wipe 8. Put on 1 μL of plasmid 9. Make sure sample type is set to DNA 50 10. Look at the graph, a spike at 130 is due to salts, a spike at 260 is due to DNA, a spike at 280 is due to protein. Record the 260/230 ratio and 260/280 ratios, you want then both to be above 1, somewhere between 1-2 is good
A DNA concentration of ~1ng/ μL was measured which is extremely low, and therefor the plasmid should not be used to preform a yeast transformation. This could be a poissible explination for the extremely low transformation efficiency of Transformation-1. Because of this it is necessary to amplify the plasmid in E. coli. Amplification of PMKV005 The PMKV005 was to be amplified in E. coli to increase its concentration. Vorachek-Warren et. al. 2004 showed that PMKV005 was derived from the pAG26 plasmid which had ampicillin resistance. The competent cells were obtained from NEB thawed on ice and transformed with the following protocol.
1. Pipette 1 μL of plasmid into the cells. Flick the tubes very gently; 2. Let them site on ice for 10 minutes; 3. heat shock at 42 degC for 45 seconds; 4. Return tubes on ice; 5. add 250 μL SOC media (LB works too but SC was used); 6. grow cells at 37 degC 225 rpm for 1 hour; Placed in incubator directly to the left of the shaker in the cohen lab 7. Plate 50 μL on a antibiotic resistant plate (which you have warmed up with the lid slightly open for about 20 minutes); 8. incubate overnight at 37 degC.
7/16
Plated a colony from the PMKV005 e. coli transformation on 7/15 into LB for an overnight culture.
7/17
Conducted a Miniprep from the o/n culture of PMKV005 transformed E. coli made on 7/16. Conducted using the Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge protcol on page 22 of the attached manual. All places when centrifugation is between 30-60 seconds 60 seconds were used.
The 3 LB plates from the Mol. Bio. group were also examined and no colonies were observed.
7/18
Made an overnight culture of the FY4 strain using YPD-2
Week of 7/19
7/19
-Prepared an o/n culture of FY4 strain from plate made on 7/12 for transformation on 7/20 -Nanodropped the PMKV005 plasmid purified on 7/17 and found a concentration of 22.8 μL -Conducted a Yeast Transformation following the protocol received from Ilaria FY4 cells were used from culture started on 7/18. 1. Cell inoculated at 8:55 and put in shaker at 37 degC until 12:30 when it was transported over to the cohen lab. OD600 taken at 12:50 measure 0.318. At 2:45 a final OD600 of 0.631 was reached. -The T-mix was prepared x6 and the salmon sperm was boiled at x9 because some boils off 9,10. The cells were transformed with the following mixtures. Vial # Concentration of DNA (ng/μL) Volume of DNA (μL) Mass of DNA (ng) Volume of T-mix
1 22.8 0.877 20 359 2 22.8 0.439 10 359.5 3 .228 8.77 2 351 4 .228 0.877 0.2 359 5 0 0 0 360
Our first round of Kan Plates were used to plate the colonies.
7/20
Attempted to conduct a transformation back at our lab. However we were unable to get an OD600 reading. The nano-drop gave inconsistent and negligible results. Using a spectrophotometer an OD of just 0.004 was recorded, on two separate trials. The following procedure was used for the spectrophotometer. An o/n culture of the FY4 strain plated on 7/12 was made.
1.Turn on the instrument by rotating the 0%T dial clockwise. You should hear it click when it turns on. 2.Allow the spec to warm up for at least 15 minutes. Given the long warm up time we will likely be leaving this device on during periods of heavy use. Please do not turn the device off if other users are likely to use it. 3.After the warm-up period, set the wavelength to 600 nm (top knob). 4.Set the filter lever to the appropriate position for the selected wavelength (bottom). 5.Press the MODE button until the display mode is set to FACTOR. 6.Using the INCREASE/DECREASE buttons ensure that the factor is set to 1.0. 7.Press the MODE button until the display mode is set to TRANSMITTANCE. 8.With no cuvette in the holder and the lid closed, turn the 0%T knob until the measured transmittance is 0.0. This is zeroing the device. 9.Fill a cuvette with 3.0 mL of the medium used to grow the cells (e.g. YPD) and wipe the sides with a KimWipe to eliminate fingerprints and dust particles. 10.Place the cuvette in the compartment. 11.Align the guide mark of the cuvette with the guide mark on the compartment. 12.Close the lid of the compartment. 13.Adjust the TRANSMITTANCE to 100.0 using the 100%T knob (right). This is essentially the equivalent of when you TARE a balance. 14.Remove the sample. You may choose to leave this cuvette of YPD for other users as they will likely be performing the same procedure. Note: the spectrophotometer must be blanked for each user even if a common cuvette is being used. 15.In a new cuvette add 3.0 mL of your cell culture sample. 16.Wipe the cell with a KimWipe and insert into the compartment as in Step 10. 17.Record the value shown in the data display (make sure MODE is ABSORBANCE). Absorbance is a synonym for OD (optical density). 18.When all measurements are obtained, turn the O%T (left knob) counterclockwise to switch the spec off. 19.Clean your cuvettes thoroughly and dry them completely.
Taken from http://fg.cns.utexas.edu/fg/course_notebook_chapter_nine.html
7/21
Measured the OD600 of the overnight FY4 culture using a spectrophotometer and a nanodrop Spectrophotometer gave a reading of 1.220 Nano drop gave readings of .282, 0.347, 0.360
The nanodrop has a 1mm pathlength while the spectrophotometer has a 1 cm pathlength so there is an expected factor of 10 difference We later became aware that the spectrophotometer cannot measure saturated cultures accurately so we conducted a more thorough experiment. The culture was diluted 5 fold and the absorbance of both the saturated and diluted cultures were measured.
Results Absorbance OD600 Spectrophotomer Saturated Culture 1.055 1.055 Spectrophtometer 1/5 dilution .221 1.105 Nanodrop Saturated Culture Trial 1 0.131 1.31 Nanodrop Saturated Culture Trial 2 0.181 1.81 Nanodrop Saturated Culture Trial 3 0.133 1.33 Nanodrop Saturated Culture Trial 4 0.159 1.59 Nanodrop Saturated Culture Trial 5 0.201 2.01 Nanodrop 1/5 dilution trial 1 0.155 1.55 Nanodrop 1/5 dilution trial 2 2.2 22 Nanodrop 1/5 dilution trial 3 0.712 7.12 Nanodrop 1/5 dilution trial 4 -0.677 -6.77
From these results we have chosen not to use the nanodrop to measure OD600 of cell cultures because it gives very sporadic readings instead the spectrophotometer will be used.
-An overnight culture of the FY4 strain was started using YPD-1 -The FY5 strain was plated from freezer stock and from the plate with strains 1-12 on it 3.Gal-YFP 4.Gal-CFP 5.Control YFP 6.Control CFP 7. Gal: CFP-YFP
7/22
YPD: The YPD made in the cohen lab on 7/19 was divided up into separate bottles Transformation: A culture was started using 50 ml of the remainder of the YPD from the first batch and 750 μL of overnight culture. An OD600 reading was taken 6 hours later and it recorded only .8 Therefor the transformation was aborted due to slow growth. Growth Curve Analysis: A 50ml culture was started with an OD600 of 0.10 in a 250 ml flask, capped with a medal top and place on the shaker at 100rpm in 30 degC. OD600 readings were taken every hour. -Overnight culture OD600: 5X dilution OD600=0.392 therefor the OD600 of the overnight culture is 1.69 -1.96* culture volume/50ml=0.1 therefor the culture volume used was 2.55ml
Time Time Passed OD600 Notes 9:40:00 0 0.113 10:40:00 1:00:00 0.14 11:40:00 2:00:00 0.18 12:42:00 3:02:00 0.252 13:46:00 4:06:00 0.354 14:40:00 5:00:00 0.485 15:39:00 5:59:00 0.642 16:40:00 7:00:00 0.845 17:37:00 7:57:00 1.356 Used 3x dilution 18:39:00 8:59:00 1.938 Used 3x dilution 19:10:00 9:30:00 2.136 Used 3x dilution 23:02:00 13:22:00 4.536 used 6x diltution
7/23
Contamination Detection The overnight culture created on 7/23 was examined under a light microscope for contamination. 100 μL of o/n culture was placed in a microcentrifuge tube and then vortexed to seperate the cells. 10 μL of the culture from the microcentrifuge tube was then placed on a glass slide and covered with a coverslip. The cells were observed at 40x with a Nikon Eclipse 200 microscope. Small circular cells were observed moving across the screen at a slow pace. The cells formed groupings of 2-3. Nothing other
of note was observed.
Conclusions It was concluded that no observable contamination of the cell culture was seen.
Yeast Transformation with pMKV005: attempt #2 Overnight culture created on 7/22 was brought to an OD of 0.174 to grow for around 5 hours to a final OD of 0.7. At this time, the yeast was transformed with 20 ng, 2ng, and 0.2 ng of pMKV005 and plated on YPD/Kan plates. A control was also made. No colonies were seen on any of the plates after 2 days of incubation.
