Wednesday June 9 2021
- Team Members: Amogh, Bonnie, Elizabeth
- Protocols Followed: (Non-His Tag) Alpha Factor + mNeon Green PCR with DpnI Digestion and Gel Electrophoresis
- Purpose: Incorporate alpha factors, which will aid endocytosis into yeast, into the existing mNeon plasmid using primers.
- Results:
Figure 1: PCR Gel
- Lane 1: 10 kb DNA ladder, Lane 2 and 3: PCR product
- Gel was not run long enough to confirm the product was 7.5kB as expected, as the ladder was too compacted.
Monday June 14, 2021
- Team Members: Bonnie, Elizabeth
- Protocols Followed: Pouring Plates, Making Overnight Cultures
- Purpose: To prepare for future experiments
- Results:
- Autoclaved/Made LB, water, and poured plain, no abx agar plates
- Plates are parafilmed and in USB fridge
- LB and water are on the counter in main USB room
Wednesday June 16, 2021
- Team Members:
- Bonnie and Elizabeth did the first half of making chemically competent cells with BL21 plain overnights
- Steven, Amogh, and Carolyn finished this up
- Protocols Followed: Making Competent Cells, Making Overnight Cultures, Making Glycerol Stocks
- Purpose: To prepare competent cells to be used for transformation
- Results:
- Made and autoclaved 80% glycerol
- Ran PCR with new MA76 plasmid using GoTaq
- PCR failed, no bands
- Known errors:
- Possible extension time error, adjusted for day 2
- TBE buffer was added after sampled were added to wells, could have caused them to wash out
Thursday June 17, 2021
- Team Members: Bonnie, Elizabeth, Amogh, Alec
- Protocols Followed: PureLink MiniPrep Kit
- Purpose: To purify out encapsulin plasmid for sequencing for confirmation of alpha factor addition
- Results:
- Completed plasmid mini-preps of MA76 and TG27 strains from Cassie using the PureLink quick plasmid miniprep kit
- Samples were taken to nanodrop in Anthony's lab
- Samples are stored in USB lab freezer (-20 C)
- Reran same PCR with GoTaq, made extension time longer according to online protocol (from 4 min to 7.5 min)
- Holding at 4 C until tomorrow
- Cells were transported back to USB freezer
Friday June 18, 2021
- Team Members: Elizabeth, Bonnie, Adam, Alec
- Protocols Followed: His Tagged PCR: Alpha Factor + mNeon Green
- Purpose: Digest PCR Product, Make a New Batch of PCR Product
- Results:
- Attempted to dpnI digest Thursday's 06/17/21 PCR product, however contents of a mislabeled tube were added instead of dpnI.
- Reran new PCR with GoTaq to replace the one that was incorrectly digested. Holding at 4C until Monday 06/21/21.
- Filled and autoclaved 4 boxes of 0.1-10ul and 3 boxes of 1-20ul pipette tips
Wednesday June 23, 2021
- Team Members: Elizabeth, Bonnie, Anya
- Protocols Followed: Pouring Plates Protocol, Chemical Transformation Protocol
- Purpose: Purify Plasmid for Transformation to reproduce plasmid
- Results:
- Made 4 LB/Agar/Ampicillin Plates for transformation (Followed Pouring Plates Protocol, adding 100ul amp for 10ml LB/Amp after autoclaving)
- Performed Gel Extraction on the first PCR product gel from June 9th (Platinum Taq PCR product)
- Completed transformation (following Chemical Transformation Protocol) with two eppendorf tubes, one labeled BL21 and another labeled BL21 control (no DNA added)
- Plates with ampicillin were streaked with transformed cells and are in 37 C incubator (not the shaking one)
Thursday June 24, 2021
- Team Members: Amogh, Elizabeth, Bonnie
- Protocols Followed: Chemical transformation protocol, mNeon+His+SpyTag plasmid Protocol
- Purpose: To test for why transformation was unsuccessful
- Results:
- Transformation plates from yesterday have no growth.
- Today, I did a transformation of the comp cells with pUC19 to "test" if the comp cells are alright to use. Will check the plate tomorrow with growth (sitting at 37 in the incubator).
- Used chemical transformation protocol
- Made a couple more ampicillin LB plates which are in the fridge.
- Made glycerol stocks from the three plates in the fridge. Those are now in the -80 and should be used for overnights/plates in the future.
- There's also another restreaked BL21 plate in the incubator for no particular reason other than to have an extra on hand especially if we need to make more comp cells.
- Amogh did a DpnI digest on a green sample from the freezer thinking that it was the PCR product for the mNeon+His+SpyTag plasmid, but it wasn't actually a PCR product (gel was run the next day with no bands).
Friday June 25, 2021
- Team Members: Bonnie, Elizabeth
- Protocols Followed: Gel Electrophoresis
- Purpose: To confirm whether or not the PCR was successful
- Results:
- Moved newly streaked BL21 plate and transformation plate from yesterday (Thursday June 24, 2021) into the fridge.
- Made AMP plates (~50). Labeled and parafilmed.
- Cleaned freezer of old primers.
- Made and ran gel of PCR product from yesterday (Thursday June 24, 2021). The gel showed no bands in the PCR product column, and on further investigation, we found the PCR tube that should have been digested on Thursday still in the freezer. This means that the PCR digest with dpnI performed on Friday was not performed on the correct PCR product.
- The correct PCR product was digested with dpnI. A new gel was made and run.
- When visualized, no bands showed.
- Correct PCR Product/GoTaq WITH Purple Loading Gel (Couldn't see bands under UV light):
- Correct PCR Product/ GoTaq WITHOUT Purple Loading Gel (Still couldn't see bands under UV light):
Figure 2: PCR Results
- We now have one gel's worth of gelred left. May need to get a stain if gelred does not come in soon.
- Streaked the two plates from Cassie (MA76 and TG27). These will be left out on the counter until Monday (June 28th, 2021).
Monday June 28, 2021
- Team Members: Bonnie, Elizabeth, Amogh
- Protocols Followed: mNeon+Histag+Spytag plasmid using High Fidelity Platinum Taq DNA polymerase, Gel Electrophoresis
- Purpose: Attempt to confirm whether or not PCR was successful in adding alpha factor to the mNeon plasmid
- Results:
- Made overnights for comp cells for BL21 cells
Figure 3: PCR Results
- Did PCR on the mNeon+Histag+Spytag plasmid using High Fidelity Platinum Taq DNA polymerase
- Performed a DpnI digest on completed PCR product
- Used PCR product to run gel electrophoresis
Tuesday June 29, 2021
- Team Members: Bonnie, Elizabeth, Amogh
- Protocols Followed: Making Competent Cells, Chemical Transformation
- Purpose: To attempt to transform plasmid into competent cells to amplify plasmid quantity
- Results:
- Made competent cells using the BL21 overnight cultures from yesterday (6/28).
- Transformed the competent cells using the PCR product from yesterday (6/28) and from the gel extraction from (6/09), along with. a positive and negative control. Plated on Amp+ plates.
Wednesday June 30, 2021
- Team Members: Bonnie, Elizabeth, Amogh, Carolyn
- Protocols Followed: Chemical Transformation
- Purpose: Reattempting successful transformation of modified T4 encapsulin plasmid
- Results:
Figure 4: New Plates
- Redo Transformation from yesterday 6/29 with the following plate:
- pUC19 (Carolyn's BL21)
- pUC19 (MSBT BL21)
- PCR Product From 6/28 (Carolyn's BL21)
- PCR Product From 6/28 (MSBT BL21)
- PCR Product From 6/28 (MC1091)
- Negative Control (MSBT BL21)
Friday July 2, 2021
- Team Members: Bonnie, Elizabeth, Amogh
- Protocols Followed: Invitrogen Miniprep Kit Protocol
- Purpose: Miniprep plasmids from transformation for sequencing to confirm presence of alpha factor
- Results:
- Mini Prepped overnight cultures from yesterday, using invitrogen miniprep kit (protocol in box)
- Some cultures did not grow very well, so we only mini prepped cultures B, C, D, E, H
- Used Anthony's nanodrop to measure concentrations of plasmid
- B: 52.244 ng/ml
- C: 62.679 ng/ml
- D: 35.689 ng/ml; gave us error message
- E: 39.187 ng/ml
- H: 62.921 ng/ml
- Concentrations of D and E were too low and probably would not give good results, so we did not send them in to sequencing
- Sent samples B, C, and H in for sequencing through Eurofins
Monday July 12, 2021
- Team Members: Bonnie, Elizabeth, Amogh
- Protocols Followed: Gel Electrophoresis
- Purpose: Gel extract the PCR product to remove extraneous DNA
- Results:
- Made and ran a gel with the PCR product from 06/28/21 then performed gel extraction in order to clean up the pcr and send it in for sequencing. Epindorf tube of this is in the freezer labelled "Purified PCR Prod. for Seq." and contains 12ul of solution
- Made re-streaked and made overnight from the transformed plate.
- Results as of Tuesday 07/13/21 at 10am. Streak towards the bottom of the plates labeled A-H is the streak done on 07/12/21. The streak towards the top of those same plates is from 07/01.
Figure 5: New Plates
Tuesday July 13, 2021
- Team Members: Bonnie, Elizabeth, Amogh, Alec
- Protocols Followed: Invitrogen Miniprep Kit Protocol, Making Overnights Protocol
- Purpose: Collect plasmids from overnights to send for sequencing
- Results:
- More Eppendorf tubes were autoclaved.
- OD of a randomly selected overnight was 2.148. All of the overnights appear to be about the same turbidity. The corresponding streak plates all showed growth as well.
- 4 of the overnight cultures were selected and mini-prepped. These are in the freezer labelled A-D with the date (7-13) labelled on the side of the tube. Do not confuse it with the previous minipreps form 07/01 or 07/02, which also have tubes labelled B, C, and D.
- The other 4 overnights were left in the incubator to continue growing along with the overnight of the old Cassie plate (not the re-streak of the Cassie plate which should be miniprepped tomorrow).
- The 4 overnights that were in the shaking incubator were centrifuged for 5 mins and then the LB was decanted. The pellets were then stored in the freezer.
- The old Cassie plate was used to make an overnight dish which is in the shaking incubator. This should be mini prepped tomorrow.
Wednesday July 14, 2021
- Team Members: Bonnie, Elizabeth, Amogh, Alec
- Protocols Followed: Making Overnights
- Purpose: Assess minipreps for ability to sequence and increase plasmid concentration in our minipreps
- Results:
- Made overnights with amp for expression, purification, and isolation protocol.
- Nano-Drop of Minipreps of plasmid from yesterday (7/13):
- A 40.209 ng/uL
- B 27.69
- C 30.678
- D 46.937
- We threw these plasmids away because the concentrations were too low to send in for sequencing. We remade overnight to rerun mini prep tomorrow.
- Anthony's suggestion: Use around 30 uL of elution buffer while doing mini prep to increase plasmid concentration
Thursday July 15, 2021
- Team Members: Bonnie, Elizabeth, Amogh
- Protocols Followed: Making Overnights
- Purpose: Redo overnights for plasmid purification because previous overnights were contaminated
- Results:
- The LB was contaminated so a new LB was made and autoclaved.
- The overnights made yesterday did not have AMP in them and so they were thrown away. New overnights were made using the new LB.
- The plasmids will need to be mini prepped and sent in for sequencing tomorrow afternoon.
Friday July 16, 2021
- Team Members: Bonnie, Elizabeth, Amogh, Anya
- Protocols Followed: Invitrogen MiniPrep Kit Protocol
- Purpose: Collect plasmids from overnights to send for sequencing
- Results:
- MAKE SURE TO ELUTE THE MINIPREPS IN ONLY 30ul OF ELUTION BUFFER
- Anya, Bonnie, and Elizabeth mini-prepped 6 (A-F) of the 8 overnights. All of the overnights grew about the same amount and are turbid.
- The mini prepped samples are labelled and in the -20 freezer in the main room.
Tuesday July 20, 2021
- Team Members: Bonnie, Elizabeth, Amogh, John
- Protocols Followed: Expression, Isolation, and Purification of Encapsulins
- Purpose: Prepare for Expression of the Encapsulin Protein
- Results:
- The cassie cell overnight grew to be turbid, the transformed cells did not
- Sent in samples for sequencing
- Continued expression per the protocol
- Made a small (500 uL) aliquot of 1 M IPTG to use for expression
- Made an overnight of MC1061 for both comp cells and a glycerol stock tomorrow
- Restreaked transformed cell plate to determine if cells are completely dead
- Preinduction Sample had OD of 0.837
Wednesday July 21, 2021
- Team Members: Bonnie, Elizabeth, Amogh, Alec
- Protocols Followed: Making Glycerol Stocks, Expression, Isolation, and Purification of Encapsulins
- Purpose: Begin protein expression process
- Results:
- Made new LB media since the one we had looked contaminated. Made 3 100mL aliquots of LB, using the instructions in the Making Overnights protocol.
- I labeled two of the jars "Not Used" to indicate that they are still 100% sterile from the autoclave. Try to keep using the one that is not labeled to minimize how much of the LB gets contaminated.
- Made glycerol stock of MC1061 cells as described in benchling protocol and stored in -80 freezer.
- May want to redo this if LB was contaminated?
- Expression:
- OD of induced sample: 0.579 OD, 5.79 OD/mL
- Dilution: (0.837 OD * 3 mL)/5.79 OD/mL --> 0.434*1000 --> 434 uL to PBS
- Finished the expression protocol, except for the SDS PAGE gel. The uninduced and induced tubes are both in the -20.
- Made comp cells with the MC1061 plate from Anthony
Friday July 23, 2021
- Team Members: Elizabeth, Amogh, Alec
- Protocols Followed: MA76+Alpha Factor 2-Step PCR
- Purpose: Attempt adding alpha factor using PCR in a two step process, as opposed to a single step process as before
- Results:
- qRun A PCR:
- One with primers A, B, and C as usual
- We then need to clean with a gel and run another PCR on this with just primers B and C to confirm the presence of alpha factor
- Put the PCR product in the -80C so that the dpnI doesn't do anything weird
- Ran the PCR with MA76 (T4+His+Spy)
- Ligated with quick ligase and t4 ligase buffer. We did three times the amounts listed in the protocol
- Dpn1 digest was started and put in the thermocycler to hold at 4C until monday
Monday July 26, 2021
- Team Members: Bonnie, Elizabeth, Adam
- Protocols Followed: Gel Electrophoresis
- Purpose: To confirm if new PCR process worked
- Results:
- Ran a gel from the ligated, digested PCR product from Friday
- No product :(
- Rerun same PCR and resume tomorrow
Figure 6: PCR Results
Tuesday July 27, 2021
- Team Members: Bonnie, Elizabeth, Amogh, Alec
- Protocols Followed:MA76+Alpha Factor 2-Step PCR, Ligation with T4 Ligase
- Purpose: Attempt adding alpha factor using PCR in a two step process, as opposed to a single step process as before
- Results:
- Ligated 20ul with Quick Ligase and t4 ligase buffer, following the Ligation with t4 ligase protocol.
- Possible Issues We can Already Foresee:
- Ligase expired in 2019
- Many of ligase buffers were expired but we did find one that was not
- Digested the product with dpnI.
Wednesday July 28, 2021
- Team Members: Bonnie, Elizabeth, Amogh
- Protocols Followed: Gel Electrophoresis
- Purpose: COnfirm the addition of alpha factor to our plasmid via two step PCR
- Results:
- Ligated 10ul of the PCR product from 06/28/21 following the "Ligation with T4 Ligase Protocol" and holding it in the thermocycler for 30 minutes at room temperature.
- Made a gel electrophoresis gel and stored it in a buffer on the counter.
- Started two new PCR reactions at 12pm.
- One with MA76 and undiluted primers following the "mNeon+Alpha Factor PCR" protocol
- One with ligated 06/28 PCR product and undiluted primers also following the "mNeon+Alpha Factor PCR" protocol
- Ran both PCRs in a gel.
- The ladder is in the bottom/leftmost lane. A new PCR run today with MA76 and primers A, B, and C is in the middle lane. The only PCR product from 06/28 that was ligated this morning and then PCR-ed with only primers B and C is in the top/rightmost lane.
Figure 7: PCR Results
Thursday July 29, 2021
- Team Members: Anya, Bonnie, Elizabeth
- Protocols Followed: Gel Electrophoresis
- Purpose: Determine if ligation of the plasmid occurred
- Ligation Results:
Figure 8: PCR Results
Hold at room temperature for 30 minutes, then heat inactivate as the protocol instructs
- The plasmid that was made yesterday with ABC primers was ligated. There was 55 uL of PCR product, so the amounts in the protocol were multiplied by a factor of 11 (55/5 uL PCR product). I held the ligated mixture for 30 mins rather than 10 mins as the protocol says, but heat inactivated as the protocol says. We ran out of T4 DNA ligase, so I ordered some more.
- A gel was made with the ligated product
- Gel showed positive results, so pcr was started with the ligated pcr product and only primers B and C.
Friday July 30, 2021
- Team Members: Bonnie, Elizabeth, Amogh, Anya
- Protocols Followed: MA76+Alpha Factor 2-Step PCR
- Purpose: Run PCR with only the B and C primers to complete alpha actor addition
- Results:
- Made and ran a gel of the PCR run with only primers B and C
Thursday August 5, 2021
- Team Members: Bonnie, Elizabeth, Amogh
- Protocols Followed: MA76+Alpha Factor 2-Step PCR
- Purpose: Run PCR reagent dilutions to optimize PCR
- Results:
- Started PCR reactions:
- One set of 7 for ABC primers + original plasmid
- One set of 7 for BC primers + pcr product
- Control -- no DNA
- Control -- undiluted DNA
- Dilutions:
- 1:10
- 1:100
- 1:500
- 1:1000
- 1:2000
Tuesday August 10, 2021
- Team Members: Bonnie, Elizabeth, Amogh, John
- Protocols Followed: MA76+Alpha Factor 2-Step PCR
- Purpose: Run PCR at a gradient of annealing temperatures to optimize PCR reaction
- Results:
- PCR of plasmid and alpha factor linker to add overhangs
- Annealing temperature is 56 C
- Reaction to make the insert PCR product (See Inverse Fusion PCR Cloning Paper in Literature Drive).
- Reaction is completed with only the A and B primers and on template DNA MA76 (T4 enc + his + spy).
- Annealing Temperatures: 52.5 - 66 C
- Reaction Quantities
- 0uL Reaction
- 61.6 μL nuclease free water (enough to get to 50 μL total volume per PCR tube)
- `Find in storage room freezer
- 1.6 μL of 10uM (500nM) primer A
- 1.6 μL of 10uM (500nM) primer B
- 1.6 μL mNeon plasmid DNA
- MA76 (make sure it includes his tag)
- 8 μL 10x High fidelity PCR buffer
- 3.2 μL MgSO4
- 1.6 μL dNTP mix
- 0.8 μL Platinum Taq DNA polymerase
Wednesday August 11, 2021
- Team Members: Bonnie, Elizabeth, Amogh
- Protocols Followed: Gel Electrophoresis
- Purpose: Visualize differences annealing temperature made to PCR reaction
- Results:
- Ran gels to confirm PCR results from the PCR reaction with only the A and B primers testing different annealing temperatures and a gel with the vector and plasmid PCR for Gibson assembly.
- Gel for A-G. All not big enough to be 7kB
Figure 9: PCR Results
- A-H are as follows for PCR reactions with the A and B primers
- Annealing Temperatures:
- Gel with H in lane 3, gib fragment in lane 3 and Gibson vector in lane 4.
- H should have been an amplified plasmid (7 kB) and the band is not large enough to be our encapsulin plasmid (This band is in the box below)
- The fragment pcr did not need to be run as the fragment already has overhangs
- The vector pcr should have resulted in amplification of our plasmid (7 kB) and so the band does not indicate that amplification was correct because it is too far down.
Figure 10: PCR Results
Thursday August 12, 2021
- Team Members: Bonnie, Elizabeth, Amogh
- Protocols Followed: Gel Electrophoresis, MA76+Alpha Factor 2-Step PCR
- Purpose: Re-Attempt 2-Step PCR
- Results:
- I had to change our gel electrophoresis power supply (dark blue one) and got a new submarine because the other ones were broken.
- Ran gel on yesterday's PCR product to see if the Gibson assembly plasmid successfully incorporated overhangs.
- Ran PCR on MA76 plasmid with A and B primers with an annealing temp of 55.8 C. Will run gel in the afternoon.
Figure 11: PCR Results
- Looking better today! Lane 2 is the plasmid unmodified, lane 3 is primer A, lane 4 is primer B and lane 5 is our pcr product with a and b primers
Friday August 13, 2021
- Team Members: Bonnie, Elizabeth, Amogh
- Protocols Followed: MA76+Alpha Factor 2-Step PCR, Gibson Assembly
- Purpose: Continue re-attempt of 2-step PCR and explore Gibson Assembly as a new addition method for adding alpha factor to our encapsulin
- Results:
- Inverse Fusion PCR Step 2 was started
- PCR was run on the Inverse Fusion PCR product from step one (AB primers only) with only the BC primers.
- 2ul of Gel extracted product for the AB Primer PCR and 0.5ul of the template MA76 were added to the reaction tube since we didn't know the exact concentrations of either
- Annealing temperature was set to 55.8 C, as this was the annealing temperature calculated by a Tm calculator and was the temperature most successful for the AB primer PCR.
- New gel on the top shelf of the fridge for the afternoon group. Please make sure to run control lanes of MA76, The PCR product from the Inverse Fusion with only AB Primers, Primer B, and Primer C.
- There is a label by the thermocycler for the PCR tube that is in it too!
- The results of the gel for today. Both Primers B and C in lanes 4 and 5 can be seen. The Forward Vec and Reverse Vec can also be seen in the last two lanes. The 6th lane contains the PCR product from 8/12 intended to be used for Gibson Assembly. The 3rd lane does not show any bands and holds the PCR product from the Inverse Fusion with only AB Primers. The 1st lane contains the ladder and the second lane contains the MA76 plasmid.
Figure 12: PCR Results
- Gibson Assembly was also conducted today. The standard Gibson Assembly Protocol provided by NEB was used. 3 ul in total from the fragments were used, 17 ul of water and 10 ul of the Gibson MasterMix. This was incubated in the thermocycler at 50 C for 15 min and stored at -20 C in the freezer for sequencing. The 3 ul consisted of 0.5 ul from each component in the mix. This included 0.5 ul of the vector, its respective Forward and Reverse fragments, the 1:10 diluted Alpha Linker, and its respective Forward and Reverse fragments. This totaled the 3.0 ul that was added.
Thursday September 9, 2021
- Team Members: Amogh, Adam
- Protocols Followed: Expression, Isolation, and Purification of Encapsulins
- Purpose: Confirm expression was successful in previous test
- Results:
- SDS page gel was run and stained 09/08. Was left in DI water to destine overnight.
- The lane farthest to the left is the ladder, and the labeled band should be 35.8kDa. The second lane is the pre-induced sample, followed by the induced sample. It looks like there is a band in both at about 37kDa which is what we roughly estimated the size of our encapsulin as.
Figure 13: SDS Page Gel Results
Monday September 13, 2021
- Team Members: Amogh, Adam, Bonnie, Elizabeth
- Protocols Followed: Gibson Assembly PCR
- Purpose: See if Gibson Assembly could add alpha factor to plasmid
- Results:
- Ran PCR on MA 76 plasmid to add overhangs for Gibson assembly. Added 1 uL of both forward and reverse vector sequences. Annealing temp was 57 C as calculated by NEB Tm calculator (may have been wrong - conflicting Tm is 68 C). Gel electrophoresis was run the next day and nothing showed up.
Tuesday September 14, 2021
- Team Members: Amogh, Adam, George
- Protocols Followed: Gibson Assembly
- Purpose: Continue Gibson Assembly
- Results:
- Ran gel from gibson overhang yesterday. No product seen, and the ladder is very bunched up despite being run for 45 minutes.
Tuesday September 21, 2021
- Team Members: Amogh, John, George
- Protocols Followed: N/A
- Purpose: Prepare for Lab
- Results:
- Made 1X TBE using 100 mL of 10X TBE and adding 900 mL of DI water
Monday September 27, 2021
- Team Members: Elizabeth, Amogh, Adam, George, Alec
- Protocols Followed: Invitrogen Gel Extraction Kit Protocol
- Purpose: Purify DNA and Proteins
- Results:
- Made LB and autoclaved it
- Measured gel extracted plasmid DNA with nanodrop
- 4.44 ng / ul
- high amount of protein / contaminants in DNA sample
- Anthony gave us crystallography columns for protein purification
- Made overnight cell cultures
Tuesday September 28, 2021
- Team Members: Elizabeth, Amogh, Adam
- Protocols Followed: Overnights
- Purpose: Create Overnight Cells
- Results:
- Overnights of cells for minipreps made to produce more plasmid
- MA76 glycerol stock from the -80
- Day 2 of expression protocol
Thursday October 7, 2021
- Team Members: Bonnie, Elizabeth, Amogh, Steven, Adam
- Protocols Followed: Protein Expression
- Purpose: Finish expression
- Results:
- Ran the remainder of the expression protocol up until the point of running the gel and dialysis
Tuesday, October 12, 2021
- Team Members: Adam, Amogh
- Protocols Followed: Gel Electrophoresis
- Purpose: Evaluate Gibson Assembly
- Results: Inconclusive
- Run gel for gibson pcr overhangs
Monday, October 18, 2021
- Team Members: Alec
- Protocols Followed: yeast overnights
- Purpose: created overnight yeast cultures
Wednesday, October 20, 2021
- Team Members: George, Amogh, Alec
- Protocols Followed: endocytosis into yeast
- Purpose: endocytose encapsulin without alpha factor into yeast as negative control
- Results:
- Viewed encapsulin and yeast under fluorescent microscopy
- Believe we saw encapsulin, endocytosis seems unsuccessful as expected although difficult to determine conclusively
Figure 14: Pure encapsulin (present in white circles)
Figure 15: Pure yeast
Figure 16: Encapsulin + yeast