April

(04/11) Monday April 11, 2022

Names: Bonnie, Adam, Elizabeth, Amogh

Date: Monday April 11, 2022

Goal: 

To test polymerase effectiveness using donated template DNA from a university class

Names of Protocols Used: 

Encapsulin PCR, Protocol from Ammerlaan, Gel Electrophoresis

Calculations: 

Notes/Modifications: 

Reactant Amounts:

1: Test DNA with Test Polymerase

10ul Go Taq green

10.65 ul NFW

2ul E. Coli DNA

0.177ul Forward Primer

0.177ul Reverse Primer

2: Our DNA with Test Polymerase

10ul Go Taq green

8.7ul NFW

2ul E. Coli DNA

1.15 Forward Primer

1.15ul Reverse Primer

3: Test DNA with our Polymerase

10ul Q5 High Fidelity

10.65 ul NFW

2ul E. Coli DNA

0.177ul Forward Primer

0.177ul Reverse Primer

4: Our DNA with our Polymerase

10ul Q5 High Fidelity

8.7ul NFW

2ul E. Coli DNA

1.15 Forward Primer

1.15ul Reverse Primer

Thermocycler Settings:

MT for test polymerase:

95C 10min

30 cycles of 

95C 30sec

46C 30sec

72C 1.5 min

Hold 72 for 5 min

Hold 4C 

Our Polymerase:

95C 10min

30 cycles of 

98C 30sec

53C 30sec

72C 1.5 min

Hold 72 for 5 min

Hold 4C 

Q5 MasterMix had precipitate in the bottom, not a good indicator it will work. 

Results/Data:

Images indicate polymerase is not ruined!

Interpretation of Results:

Bands in lanes 2 and 3 were the ones for our polymerase, indicating that is likely was not ruined. Lanes 4 and 5 had polymerase given to us by another teacher as a test, and the master mix may not have had polymerase in it. 

Summary and Next Steps: 

Run our next PCR

 

(04/12) Tuesday April 12, 2022

Names: Bonnie, Kimi, Jaclyn, Sophia, Adam, Jack

Date: Tuesday April 12, 2022

Goal: 

Prep things for lab

• Sterile LB for overnights

• Agar Plates

• Sterile Glycerol

• Find PCR tubes

• Check miniprep kits/centrifuge needs

Names of Protocols Used: 

Making LB, Making glycerol Stocks, Pouring Plates

Calculations: 

N/A

Notes/Modifications: 

N/A

Results/Data:

Made LB, Plates, and Glycerol

Interpretation of Results:

N/A

Summary and Next Steps: 

Make overnights for plasmid miniprep

 

(04/13) Wednesday April 13, 2022

Names: Bonnie

Date: Wednesday April 13, 2022

Goal: 

Restreak plates to get single colonies

Prep for overnights in stock room tomorrow

Names of Protocols Used: 

Making Overnight Cultures

Calculations: 

Added 5ul of the 100mg/ml ampicillin to the LB, based on pouring agar plates protocol

Notes/Modifications: 

2 Culture tubes are in the top shelf of the door in the fridge in the storage room (on the right right when you walk in). The sterile loops were put in there too. The two restreaked plates are in the incubator in the classroom, one for the empty vector and one for the encapsulin vector. 

Results/Data:

Interpretation of Results:

N/A

Summary and Next Steps: 

Make overnight cultures tomorrow for minipreps Friday

 

(04/14) Thursday April 14, 2022

Names:  Bonnie, Kimi, Jaclyn

Date: Thursday April 14, 2022

Goal: Start overnights

Names of Protocols Used: N/A

Calculations: 

Notes/Modifications: 

I added one colony from each of the plates to an appropriately labeled culture tube. These tubes were prepared with bonnie handwriting and they said they had AMP already. 37 degrees and 210 RPM overnight. Plates parafilmed and moved to fridge. 

Results/Data:

 To be recorded the day after due to the material prepared being overnights

Interpretation of Results:

  To be interpreted the day after due to the material prepared being overnights

Summary and Next Steps: 

Next step: observe and interpret the quality of overnight samples

(04/15) Friday April 15, 2022

Names: Sophia, Anya, Steven, Sashider, John, Jack, Thomas, Amruta

(04/19) Tuesday April 19, 2022

Date: Friday, April 15, 2022

Goal: 

Make glycerol stocks of the overnights

Mini prep the plasmids out of the bacteria

Names of Protocols Used: 

Invitrogen miniprep kit protocol (with modified step in between centrifuge and adding of the resuspension buffer). 

Calculations: 

To dilute 80% glycerol to 50% with the overnight and get 1 mL of the 50%, take 0.625 mL of the 80% glycerol and add 0.375 mL of the overnight. 

Notes/Modifications: 

• For the glycerol stocks: gently mixed the bacteria and glycerol with the micropipette.

• To centrifuge the PET Duet T-4 Gala and the empty PET Duet, we made 4 Eppendorf tubes of each overnight (with 1 mL of the overnight in each tube). This was done because we didn't have one tube big enough to put in the centrifuge. After dumping the supernatant, we resuspended the bacteria in 1 tube in 250 mL buffer, then transferred the buffer with the resuspended bacteria into the next tube and resuspended the bacteria in that. We continued this until the bacteria in all 4 tubes were resuspended. (leaving one tube of T-4 Gala and one tube of empty) 

• Note: the resuspension buffer is very old/not refrigerated in 4C

• Note: Before Elution, the Elution Buffer was heated in order to increase the yield

Results/Data:

• We stored the finished product in -20 in the fridge. 

Interpretation of Results:

• N/A

• the results still need to be intepreted using gel eletrophresis maybe? 

Summary and Next Steps: 

• We don't know if the miniprep worked because the kit was from 2019 and there were precepitation in the solution. 

• We need to nanadrop/run a gel for our miniprep result. 

• if the results from nanodrop and gel are good, we can do a snager sequence to confirm our data. 

 

(04/19) Tuesday April 19, 2022

Names: Elizabeth, Bonnie, Sophia

Date: 4/19/22

Goal: 

Test restriction enzyme function on the PetDuet empty vector, both individually and together. We will run a gel to confirm both presence of plasmid and enzyme function. 

Names of Protocols Used: 

Restriction enzyme double digest

Gel electrophoresis

Calculations: 

Estimated that there were max 20 ug of DNA from the miniprep eluted in 75 uL of elution buffer. So, we are going to use 3.75 uL of DNA in the restriction enzyme protocol. 

39.25 uL of water.

40.25 if only one enzyme is used. 

Notes/Modifications: 

No nanodrop was available for use, thus the estimation for amount of DNA couldn’t be calculated. 

Heat inactivated enzymes at 65 C. 

Results/Data:

Order of gel:

• Lane 1: skipped

• Lane 2: ladder

• Lane 3: T4

• Lane 4: PetDuet

• Lane 5: NdeI

• Lane 6: PacI

• Lane 7: NdeI+PacI

Interpretation of Results:

Ladder looks very blurred, voltage/current may have been too high when run. 

Not strong bands for any of the other lanes, weak bands could be a result of residual dye or extremely low concentration of DNA. The placement and number in each lane also do not make a ton of sense. 

Summary and Next Steps: 

Nanodrop plasmids just to see if anything is there. 

Rerun gel with ladder and pUC19 to ensure gel formula and ladder is ok. 

Will make new overnights and miniprep with a new kit at the end of the week. 

 

(4/20) Wednesday April 20, 2022

Names: Adam Tisch, George Rabadi, Xuan Le

Date: 4/20

Goal: 

Run gel on pUC19 to try to fix the issue of blurry gels.

Names of Protocols Used: 

Gel Electrophoresis 

Calculations: 

50 mL TBE 1X

0.5 g Agarose

5 uL sybr

7 uL ladder and pUC19, 1 uL loading dye for lanes 2 and 4, respectively

8 uL for lane 3 prestained 10 kB ladder

Notes/Modifications: 

Running gel at 90V rather than 120V at Robert's suggestion to fix smearing

Also made sure to give it time to actually set rather than rushing it

Wells were loaded correctly with care

Results/Data:

Order of gel:

• Lane 1: Skipped

• Lane 2: 1kb Ladder

• Lane 3: 10kB Ladder

• Lane 4: pUC19 (2.6 kB)

 

(04/22) Friday April 22, 2022

Names: Elizabeth, Bonnie

Date: 04/22/22

Goal: 

Figure out why our ladder is smearing when we run gels

Names of Protocols Used: 

Gel electrophoresis

Notes/Modifications: 

Used a series of ladder dilutions

Run at 110V constant voltage

Lanes:

1:6 10x ladder to nuclease free water

2:5 10x ladder to nuclease free water

3:4 10x ladder to nuclease free water

1:6 1x ladder to nuclease free water

2:5 1x ladder to nuclease free water

3:5 1x ladder to nuclease free water

50 pg/ul Puc19

100 pg/ul Puc 19

Results/Data:

Nanodropped the miniprepped plasmids from Friday 04/15/22.

Empty petduet 1.2 ng/ml

T4 enc 0.919 ng/ml

Interpretation of Results:

The nanodropped plasmids showed that there was no DNA in the eluent. 

Bands of ladder still look streaky and weird. Lower concentration does seem to be better

Summary and Next Steps: 

Retry the mini-preps with a new kit and make sure the overnights are sufficiently turbid.

Explore whether or not the ampicillin not being amp salt could be an issue. 

 

(04/25) Monday April 25, 2022

Names: Bonnie, Elizabeth

Date: Monday April 25, 2022

Goal: 

Make overnights for tomorrow's minipreps

Names of Protocols Used: 

Making Overnight Cultures

Calculations: 

Added 5ul of the 100mg/ml ampicillin to the LB, based on pouring agar plates protocol

Notes/Modifications: 

2 culture tubes are in the incubator in the corner of the lab space. 

Summary and Next Steps: 

Miniprep tomorrow

 

(04/26) Tuesday April 26, 2022

Names: Amogh, Sophia, Sriram, Jaclyn

Date: 4-26-22

Goal: 

Purify plasmid for sequencing

Names of Protocols Used: 

GeneJET Plasmid Miniprep

Notes/Modifications: 

The old centrifuge only went at 5400 rpm for 2 min when we needed 8000 rpm. The pellet was slightly runny, so we ran the tubes again for 5400 rpm for 2 min. 

Results/Data:

Pure DNA was eluted in 50 uL

Interpretation of Results:

N/A

Summary and Next Steps: 

Pure DNA was put into -20 C

 

(04/28) Thursday April 28, 2022

Names: Carolyn, Amogh, George

Date: 4-28-2022

Goal

Use gel electrophoresis to analyze miniprepped PET duet vector and T4 encapsulin, and using NanoDrop to determine the concentration of each of these samples

Name of Protocol Used

Gel Electrophoresis

Calculations

1% agarose 

50 mL 1X TBE Buffer

0.5 g agarose

5 uL SYBR 

Loading of Gel:

Lane 1: 1 kb DNA Ladder (1 uL ladder + 1uL loading dye + 4 uL water)

Lane 2: T4 enc (5 uL DNA + 1 uL loading dye)

Lane 3: T4 enc dilution (1 uL DNA + 1 uL loading dye + 4 uL water)

Lane 4: PET duet vector (5 uL DNA + 1 uL loading dye)

Lane 5: PET duet vector dilution (1 uL DNA + 1 uL loading dye + 4 uL water)

Gel was run for ~1 hour at 90V until the dye ran 75% of the way down the gel

Notes/Modifications

• Loading lane 5 made hole in the well, so there should not be any results for that sample

• We did not have access to a UV visualizer at the time, so the gel is stored in the fridge with 1X TBE buffer

• Also used nuclease-free water to make up 1X TBE to make gel 

Results/Data

NanoDrop:

• T4 enc = 275 ng/uL

260/280: 1.84

260/230: 2.16

• pET duet vector = 636 ng/uL

260/280: 1.88

260/230: 2.24

Gel: imaged on 5/2/2022

Interpretation of Results

• Gel looks okay, DNA might appear bigger than it should be a LOT of DNA was added (ran the gel before running nanodrop), but each lane shows one band which is good

Summary and Next Steps

• Run PCR to get the encapsulin out of the vector and restriction digest the empty vector, then can add encapsulin to pet duet vector

 

May

(05/03) Tuesday May 3, 2022

Names: Carolyn, Adam, Sashider, Amruta, Sophia

Date: 5-3-2022

Goal: 

Use PCR to get the T4 encapsulin DNA out of the vector it is currently in to use for cloning. Use (and test) restriction enzymes to cut empty pETDuet vector to insert PCR encapsulin later.

Names of Protocols Used: 

Restriction Enzyme double digest (protocol shown below)

PCR

Calculations: 

PCR (per NEB protocol for Q5 HF 2x master mix)

• 25 uL of 2x master mix

• 2.5 uL of 10 uM forward primer

• 2.5 uL of 10 uM reverse primer

• 1 uL of a 1:30 T4 encapsulin DNA:water dilution (in vector)

• 19 uL of nuclease-free water

10 ng x (uL/275 ng) = 0.03636 uL DNA --> too small, so I will make a 1:30 dilution and take out a microliter to get ~9 ng of DNA

50 uL - (25+2.5+2.5+1) = 19 uL nuclease-free water

Relevant Dilutions

• 1:2 dilution of Enc:water (dilute down to 92 ng/uL)

• 1:10 dilution of each primer

• For each primer, resuspend in water (take ng x 10 and add that amount of nuclease-free water) to get a 100 uM stock --> make a 1:10 dilution for each so you can add 2.5 uL of a 10uM primer solution (as per NEB instructions)

Cycle Conditions

Initial Denaturation: 98 C for 3 min

Run for 25 cycles

Denature: 98 C for 10 seconds

Anneal: 67 C for 30 seconds

Extension: 72 C for 30 seconds

Final Extension: 72 C for 2 minutes

Hold at 4C 

Restriction Enzyme Digest (per NEB protocol)

1 ug = 1000 ng x (uL/636 ng) = 1.57 uL pet duet vector

Results/Data:

Order of Loading: all lanes have 1 uL DNA + 1 uL loading dye + 4 uL water

• Lane 1: 1 kb ladder

• Lane 2: Nde1 + PET duet

• Lane 3: Pac1 + PET duet

• Lane 4: Nde1/Pac1 + PET duet

• Lane 5: Negative control (put in <10 ng DNA)

• Lane 6: PCR product with T4 enc

• Lane 7: PET duet vector (no restriction enzymes)

Interpretation of Results:

 Results will be recorded the day after

Summary and Next Steps: 

  Results will be recorded the day after, and interpretations will be made accordingly.

(05/04) Wednesday May 4, 2022

Names: Adam, Carolyn, Sashider, Sriram, Amruta

Date: 5/4/22

Goal: Cleanup pcr and rerun gel to try to see better

Names of Protocols Used: 

Gel and pcr cleanup from kit

Calculations: 

We used the DNA purification for less than 2000 bp protocol on the card for pcr cleanup. This was confirmed and we will use the same protocol later. 

Notes/Modifications: 

• Microwave broke, so the agarose was not 100% dissolved before pouring. This could explain why the bands are a little messy.

Results/Data:

Lane 1: 1kb ladder

Lane 2: 10kb ladder

Lane 3: NdeI

Lane 4: PacI

Lane 5: double digest

Lane 6: petduet vector

Lane 7: PCR product

Interpretation of Results:

Experiment was inconclusive since we didn’t perform Dpnl digestion before cleanup.

We decided that we had inconclusive evidence for a successful restriction digest because we do not see the 100 bp cutout. 

Summary and Next Steps: 

We are repurchasing restriction enzymes and will redo the experiment.

(05/05) Thursday May 5, 2022

Names: Adam, Carolyn, Sashider, Cameron, Sophia, Amruta

Date: 5/5/22

Goal: PCR a better sample of enc and confirm result

Names of Protocols Used: 

PCR 

Calculations: 

25 uL MasterMix

Dilute 0.73 to 2000 of 275 ng/uL template and add 1 uL to pcr mix for 0.1 ng template

1:10 dilution and then 2.5 uL of each to get 0.5 uM

19 uL nuclease free water

Cycle:

32 cycles

1 min 98 degrees

Repeat:

10 s at 98 degrees (first 6 cycles were for 30s, my fault)

30 s at 67 degrees

30 s at 72 degrees

2 min final extension and then hold at 4 degrees

Notes/Modifications: 

Results/Data:

No PCR products obtained at the time

Interpretation of Results:

 It seems that DNA wasn’t loaded. 

Summary and Next Steps: 

Repeating the experiment and training members on PCR protocol is necessary. 

 

(05/06) Friday May 6, 2022

Names: Adam, Sabine

Date: 5/6/22

Goal: Figure out what to do about the failed pcr from yesterday

I nano dropped with Anthony, we were at 27.2 ng/uL. Anthony's advice was that if we cleaned it up again after DpnI we would not have enough for stuff, and so we should run the original pcr back, and that's what we did. 

Names of Protocols Used: 

 Gel Electrophoresis 

Calculations: 

275 ng/uL of T4 was diluted 1:30 in the high concentration sample for a total of 9.17 ng template, and 1:600 for 0.45 ng template in the low condition. 

Otherwise, the reactions were 25uL Master Mix

2.5 uL 10 uM each primer

19 uL nuclease free h2o

3 min 98 degrees

30 cycles:

10 s 98 degrees

30 s 67 degrees

30 s 72 degrees

then 2 min 72 and hold 4

Run gel quick, 150 V, 

lanes edge to in: Ladder, Low, High

Notes/Modifications: 

Results/Data:

 

Qualitatively, the gels were satisfactory. 

Interpretation of Results:

 Why the gel looks good: 2019 Nuclease free water in buffers, we really cooked it until it was good and clear and hot and dissolved, and we used an infrared thermometer to add Sybr at exactly 50 C. The liquid was 100 degrees when we pulled it off the hotplate. The gel was run for ~30 min at 150 V, and felt warm to the touch when removed from the rig.

Summary and Next Steps: 

 Re-do PCR. 

 

(05/09) Monday May 9, 2022

Names: Bonnie, George, Amogh

Date: Monday May 9, 2022

Goal: 

Digest Encapsulin PCR from Friday 5/6/22

Do PCR Cleanup on these digested samples

Run PetDuet with new restriction Enzymes

Names of Protocols Used: 

Restriction Enzyme Double Digest

Dpn1 Digest

Calculations: 

636 ng/uL --> using 1.6ul of DNA for restriction enzyme digest

Notes/Modifications: 

DpnI: 8ul PCR product, both low and high concentration, 1ul Cutsmart, 1ul Dpn1

Results/Data:

PCR cleanup product and enzyme digested products are labelled and in the freezer

Interpretation of Results:

N/A

Summary and Next Steps: 

Gibson assembly and gel on that result tomorrow

 

(05/10) Tuesday May 10, 2022

Names: George Rabadi, Zoe Jackson

Date: 5/10/2022

Goal: 

After discussion with Anthony, we need to rework our process before we run Gibson.

When we used the nanodrop on the sample from yesterday, the amount was still too low to work with, and that included both pieces of the vector along with some junk. Anthony recommended that we run a gel with this sample to confirm that our digestion and everything was successful. If the gel turns out as we expect, we will need to do a gel extraction on the larger piece of the vector (the part that we want to insert the AMP into). At that point, we would again nanodrop that sample and use that concentration for our Gibson calculations.

Names of Protocols Used: 

Gel Electrophoresis

Calculations: 

Notes/Modifications: 

1 uL of Ladder used. The gel was run for ~35 min.

Results/Data:

The ladder was run in the well one from the right and the sample was run one to the left of that.

We didn't have time to visualize the gel, so it is sitting in the gel tray to be visualized.

Interpretation of Results:

 The gel will be visualized the day after 

Summary and Next Steps: 

  The gel will be visualized the day after 

 

(05/11) Wednesday May 11, 2022

Names: Elizabeth, Bonnie

Date: May 11, 2022

Goal: 

Run a gel of the newly digested product, start Gibson assembly if successful

Names of Protocols Used: 

Gel Electrophoresis

Calculations: 

 N/A

Notes/Modifications: 

Lane 1: Skipped

Lane 2: Ladder (0.5 ul ladder, 6.5 ul water)

Lane 3: 1:10 diluted PET, undigested with restriction enzymes

Lane 4: Restriction enzyme product from 5/10/22

Results/Data:

Interpretation of Results:

Digested product is present, per Robert we will probably not see the smaller band.

Summary and Next Steps: 

Continue with gibson assembly

 

(05/16) Monday, May 16, 2022

Names: Adam (morning, Gibson), Amogh, Amruta, Sophia, Jack (evening, gel electrophoresis and chemical transformation)

Date: 05-16-2022

Goal: 

Gibson of positive control

Names of Protocols Used: 

Gibson assembly

Gel electrophoresis

Chemical transformation

Calculations: 

6 uL vector = 50 ng vector = 14.93 fmol of vector. It was nanodropped at 8.36 ng/uL. Moles calculated here https://nebiocalculator.neb.com/#!/dsdnaamt with length of pet and 50 ng plugged in

The insert (not B) is small so I'm adding 3x moles to get ~44 fmol of positive control insert which happens to be at 15.136 nM (don't ask why). So I'm adding 2.95 uL of insert

10 uL Gibson Mastermix

Add nuc free h2o to get to 20 uL reaction

Then 15 min in thermocylcler at 50 degrees and place in -20 for later

Notes/Modifications: 

In the gel, lane 1 = ladder, lane 2 = positive control gibson assembly, lane 3 = the NEB gibson positive control

Results/Data:

 The reactions will be left to proceed. Results will be quantified and interpreted the day after

Interpretation of Results:

  The reactions will be left to proceed. Results will be quantified and interpreted the day after

Summary and Next Steps: 

 The reactions will be left to proceed. Results will be quantified and interpreted the day after

(05/17) Tuesday, May 17, 2022

Adam: I went in to check on the transformation from the night before, there were no colonies on either the pUC19 plate or the Gibson product plate.

(05/18) Wednesday May 18th, 2022

Names: Sashider, Sriram

Date: 05/18/22

Goal: 

Run a gel of Gibson product, digested vector, and AMP

Names of Protocols Used: 

Gel Electrophoresis

Calculations: 

 N/A

Notes/Modifications: 

Add 1ul of each to 4 ul of H2O and 1 ul of dye

Results/Data:

Bottom to Top:

Ladder

AMP

Vector

Gibson Product

Interpretation of Results:

AMP and Vector is visible, but the ladder is not visible, so we need to look at why the gel didn't run properly and what is causing the intense green color at the top of the gel

Summary and Next Steps: 

Reconsider how gels are run and determine why there is so much green at the top of the gel.

 

(05/19) Thursday May 19, 2022

Names: Bonnie, Sophia

Date: 05/19/22

Goal: 

Make more digested PET so that we can do Gibson tomorrow (double digest with restriction enzymes, gel extract)

Names of Protocols Used: 

Restriction Enzyme Double Digest, Gel Electrophoresis, Gel Extraction

Calculations: 

For RE:

Using 1.6ul PET based on calculations done on Monday May 9th

Adding 1ul each of Nde1, Pac1 (both old and new)

40.4ul of nuclease free water

5ul CutSmart

digested for 1 hour

For Gel:

Using new TBE

Washed all equipment first

5ul sybr

Waited until able to hold for 10 seconds before adding syber

Notes/Modifications: 

For the gel: 

Skipped lane 1, lane 2 = ladder (1 ul 1kb ladder, 6 ul nuc free water, 1 ul loading dye), lane 3, 4, 5, 6, 7, 8 = digested PET

Gel was run at 110 V

Gel that was cut to be extracted, first chunk 1.108 g, second chunk 0.849 g, total = 1.957 g 

Results/Data:

Digested 1000ng of PET (1.6ul)

Ran gel in 6 lanes of 15 lane gel

Digested 1.8g in gel extraction (~10 tubes)

Resulted in 60ul of gel extracted PET (eluted gel extract in 6ul/tube)

Interpretation of Results:

N/A

Summary and Next Steps: 

Nanodrop gel extract tomorrow

 

(05/20) Friday, May 20, 2022

Names: Adam

Date:  Friday, May 20, 2022

Goal: Evaluate gel extraction from before, and move forward. Due to a bad sample this meant redoing gel extraction of digested PET

Names of Protocols Used: 

Robert recomended 3 ug digest, so I repeated the digestion from Thursday three times and then extracted.

I made three digestion tubes. Each one contained:

1.6 uL PET Cleanup for 1 ug DNA

1.6 uL of NdeI, old PacI, new PacI (this is because I meant to add 1 uL but messed up and added 1.6 instead. This is still below the 10% restriction enzyme threshold recomended by NEB so I think it's ok. Also, the whole Pac mixup situation lol)

40.4 uL Nuclease free H2O

5 uL cutsmart buffer

These samples were placed in the thermocycler for 49 min at 37 degrees. Then I added 10 uL of loading dye to each of them, mixed, and let them sit at room temp for ~15 min to let the SDS do its thing because there was no heat inactivation and I was concerned about DNA binding to the protein

Ok, now for the gel: I did 0.75% agarose (0.375g in 50 mL) on Anthony's suggestion. 50uL of 0.5X TBE made with 2019 nuclease free water, running buffer was also nuclease free but 1X. Heated to 88 degrees C in microwave, 2x 30s, add 5 uL sybr and pour at 53 C per infared thermometer.

Ladder was 1 uL ladder, 4 uL H2O nuc free, and 1 uL loading dye. 4 wells were taped together for load, ladder in single well with a spacer between.

Run at 90V for a while, then slowly turned up to 120 V because it was already 8 pm. Loading dye was 9/10 the length of the gel when I pulled it.

I cut out the single defined band in a smiley type shape. Trimmed off excess bits vertically, horizontally, and by depth as well but it still came out to be about 0.93 g of gel. Minimal UV exposure time for the DNA. I dissolved in 4 mL dissolving buffer, washed twice with 200 mL wash buffer, and eluted in 15 uL per protocol in extraction kit. 

Calculations: 

 N/A 

Notes/Modifications: 

 N/A

Results/Data:

 Gel Extraction was completed

Interpretation of Results:

 N/A

Summary and Next Steps:

Start testing Gibson Assembly 

(05/24) Tuesday May 24, 2022

Names: Elizabeth

Date: 05/24/22

Goal: 

Nanodrop restriction enzyme product from last week

Start Gibson or redo restriction depending on results

Make non-ampicillin plates for testing comp cell viability

Names of Protocols Used: 

Pouring agar plates

Positive Control Gibson Assembly

Experimental Gibson Assembly

Calculations: 

 N/A

Notes/Modifications: 

Using protocol from 5/16 --> 13.05 fmol

50 ng (ideal amount of vector) /10.49 ng/uL = 4.8 uL

Want 2-3x vector to AMP ratio, using 3 b/c AMP is small

Vector = 13.05 fmol --> want 39.15 fmol insert (AMP)

Concentration of 15.136 nM

2.56 uL of insert

Total rxn (20 uL):

4.8 uL restriction digested PET Vector

2.56 uL AMP insert

10 uL Gibson assembly master mix 2X

2.64 nuclease free water

Results/Data:

Nanodrop of restriction digested PET Vector = 10.49 ng/uL

Interpretation of Results:

 Concentration too small for accurate 280/260 & 260/230

Summary and Next Steps: 

2 Plates of comp cells were plated without transformation onto non-amp plates, check for growth tomorrow

 

(05/25) Wednesday May 25, 2022

Names: Bonnie, Elizabeth, Sashider, Amruta, Jaclyn

Date: Wednesday May 25, 2022

Goal: 

To chemically transform our Gibson positive control construct into our competent cells. 

Names of Protocols Used: 

Chemical Transformation

Calculations: 

N/A

Notes/Modifications: 

Puc19, NEB Gibson Positive Control, our Gibson Positive Control 

All plated on both an ampicillin and a non-ampicillin plate (25ul on the non-amp, 75ul on the ampicillin plates)

Non-ampicillin plates had very little DNA on them, especially the one for the NEB positive control

5ul of DNA Used in each transformation

We were very carefully to follow protocol exactly. Soc was not pre-warmed because the one set at room temp was non the most recent bottle. 

Results/Data:

N/A

Interpretation of Results:

N/A

Summary and Next Steps: 

Check for growth on the comp cell plate tomorrow

 

(05/26) Thursday May 26, 2022

Names: Elizabeth, Bonnie, Sashider, Sophia

Date: 5/26/22

Goal: 

Make an overnight

Names of Protocols Used: 

Making overnight cultures

Calculations: 

Notes/Modifications: 

Results/Data:

(05/27) Friday May 27, 2022

Adam and Jack

Miniprep of the two cultures started on Thursday the 26th. Each was from a different colony on the our construct amp plate. We used 4 mL for the miniprep, divided into four tubes to spin down the cells in the microcenterfuge. These were combined into one tube on resuspension and the protocol was followed as specified in manufacturer instructions. Spin at 13,000 rpm on our microcenterfuge for indicated steps. Elution buffer heated with 65 degree waterbath briefly before elution. Some lysis buffer spilled, this experiement was unaffected but may affect supply for future. 

Note this was performed after overnights had been incubating for ~24 hr. This may be an issue if we have a bad yield. Samples will be sent for sequencing asap.

I also streaked these cultures onto amp plates, but since it is the weekend these were left at room temp adjacent to the incubator, and will be collected Monday. For backup purposes, I also made glycerol stocks of each with 70 uL of overnight combined with 70 uL of 80% gylcerol. 

Link to protocol used: https://tools.thermofisher.com/content/sfs/manuals/MAN0012655_GeneJET_Plasmid_Miniprep_UG.pdf

 

(05/31) Tuesday May 31, 2022

Names: Adam, Amruta, Cameron, Sashider

Date: 5/31/22

Goal: 

Do a Nanodrop of Mini-Prep done on Friday May 27 and determine if the concentration is good enough to send in for sequencing

Names of Protocols Used: 

Making Overnights

Calculations: 

10 mL of LB broth, 10 ul penicillin and then the two strains from cultures were used.

Notes/Modifications: 

Results/Data:

Nanodrop concentrations were too low (about 20 ng/ul?), meaning that sequencing could not happen (need about 60). 

Interpretation of Results:

It was determined that the mini-prep would be redone with cultures that had already been streaked onto plates. 

Summary and Next Steps: 

Overnights were set up today to allow for the bacteria to grow. Mini-prep will be done of these overnights tomorrow where we will hopefully get higher concentrations of DNA to send in for sequencing.

 

June

(06/1) Wednesday June 1, 2022

Names: Elizabeth, Bonnie, Adam, Jack, Emily & George

Date: 6/1/22

Goal: 

Make new overnights to prep for more miniprepping in an effort to improve concentration

Names of Protocols Used: 

 Overnight prep

Calculations: N/A

Notes/Modifications: N/A

Results/Data:

Tubes were very turbid the next morning

Interpretation of Results:

Turbidity in tubes is indicative of growth

Summary and Next Steps: 

Miniprep these overnights 

(06/2) Thursday June 2, 2022

Names: Elizabeth & Adam

Date: 6/2/22

Goal: 

Mini-prep new overnights from 6/1

Names of Protocols Used: 

https://tools.thermofisher.com/content/sfs/manuals/MAN0012655_GeneJET_Plasmid_Miniprep_UG.pdf

READ THE ACTUAL PROTOCOL BEFORE STARTING, NOT JUST THIS ONE!

overnight lids fell off

4.5 uL culture start with, 2 min at 8,000 rpm to pellet

all other steps carried out at 13,000 rpm

Resuspend the pelleted cells in 250 µL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain. GET AS MUCH OF THE LB OUT AS YOU CAN WITHOUT DISTURBING THE PELLET, RESUSPEND WELL

IF THERE IS PRECIPITATE IN LYSIS SOLUTION, WARM THE STOCK TO 37 DEGREES AND THEN VORTEX GENTLY.

Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA. INVERT 6 TIMES, THEN WAIT 2 MIN.

Add 350 µL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy. MIX IMMEDIATELY AND THOROUGHLY

Centrifuge for 5 min to pellet cell debris and chromosomal DNA.

Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. Note. Close the bag with GeneJET Spin Columns tightly after each use!

Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. Note. Do not add bleach to the flow-through, see p.8 for Safety Information.

Add 500 µL of the Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.

Repeat the wash procedure (step 8) using 500 µL of the Wash Solution.

Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.

Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 µL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min. WE DID 30 uL

Discard the column and store the purified plasmid DNA at -20°C.

Calculations: N/A

Notes/Modifications: 

Had aerobic bacterial growth

Heated the lysis buffer to 37 C and vortexed before use to get rid of any precipitate

Results/Data:

Nanodrop success!

Strain 1: 77.74 ng/uL

Strain 2: 71.80 ng/uL

Interpretation of Results:

Ready to sequence

Summary and Next Steps: 

Send to eurofins Friday

(06/3) Friday June 3, 2022

Names: Elizabeth

Date: 6/3/22

Goal: 

Submit minipreps for sequencing

Names of Protocols Used: 

Calculations: 

Notes/Modifications: 

Results/Data:

Interpretation of Results:

Summary and Next Steps: 

Submitted 10 uL of each sent to eurofins using T7 primers

 

(06/06) Monday June 6, 2022

Names: An

Date: Monday June 6, 2022

Goal: Start overnights for growth assay tomorrow

Names of Protocols Used: 

Preparing overnights in given conditions

7 mL clear LB from the fridge added to each of 4 falcon tubes. Then we added 10 uL of ampicillin 100 mg/mL to each, but they each had a white dot suctioned to the pipette so who knows really.

Then we added strain 1, strain 2, empty pet, T4 enc construct. 

caps tapped but not screwed on the tubes and then 225 rpm 37 degrees overnight.

Calculations: N/A 

Notes/Modifications: N/A

Results/Data: Overnights prepared

Interpretation of Results: Overnights will be checked for quality the morning after 

Summary and Next Steps: 

Growth Assay to be done next

(06/07) Tuesday June 7, 2022

Names: Adam, Jack, Vamsi

MSBTGrowthData672022.xlsx

Date: 

Goal: Test growth assay 

Step 1, evenly divide remaining good LB broth into 8 Falcon tubes, because these are probably clean. The more broth in each the better, and save 3 mL for a blank. ~50 mL per one.

Add 1 uL amp @ 100 mg/mL per mL of LB broth. 

Initiate samples with 5 mL, but don't be afraid to audible this lower if it seems like too much. 

I'd like to test 1 IPTG concentration 0.2 mM and 0.2 mM, and we will induce one of the samples at OD = 0.1. 

Record OD when it is measurable at 0.1, and then every 30 min thereafter. 

Note: if OD is over one, dilute in half and double the result.

Names of Protocols Used: 

We actually induced one set at ~0.1 and didn't induce the other set. The lids and oxygen needs better controlling. 

Initial values of overnights:

.975, .890 strain1

.864, .908 strain2

.360, .355, empty pet

enc .701, 0.590

Calculations: N/A 

Notes/Modifications: 

Results/Data: Samples were left to grow

Interpretation of Results: N/A

Summary and Next Steps: 

Make LB broth

 

(06/08) Wednesday, June 8, 2022

Names: Amogh, Elizabeth, Emily, Jaclyn, Sashider, Sriram

Date: 6/8/22

Goal: 

Make LB Broth

Names of Protocols Used: 

Calculations: 

Notes/Modifications: 

10 250 mL Erlenmeyer Flasks with 50 mL Deionized Water Each

1 g LB Broth Mix into Each

Autoclave 121C for 15 min

Add 50 ul Ampicillin to 2 Erlenmeyer flasks (each)

Results/Data: LB broth made

Interpretation of Results: LB broth made 

Summary and Next Steps: 

 

(06/13) Monday June 13, 2022

Names: Elizabeth, amogh, Jaclyn, George

Date: 6/13/22

Goal: 

Make more LB

Make overnights to resequence positive control

Names of Protocols Used: 

Making overnight cultures

Calculations: N/A 

Notes/Modifications: N/A 

Results/Data:

3 overnights from each of the 2 plates

Interpretation of Results: Qualitatively overnights are satisfactory

Summary and Next Steps: 

 

(06/14) Tuesday June 14, 2022

Names: Elizabeth

Date: 6/14/22

Goal: 

Remake overnights

Gibson assembly

Names of Protocols Used: 

Calculations: 

Nanodropped PCR cleanup (labelled H and L in freezer in eppendorf tubes)

Low: 16

Notes/Modifications: 

Overnights need to be remade due to incubator not being at the correct temperature when I came to check today, therefore nothing grew. 

Results/Data: N/A

Interpretation of Results: N/A

Summary and Next Steps: 

Miniprep overnight samples the next lab session

(06/15) Wednesday June 15, 2022

Adam 

Miniprep:

6 overnights were started last night with three colonies from each of the two positive control pet + amp strains. Miniprepped with standard protocol:

4.5 mL of overnight grown 225 rpm 37 degrees with lids taped but not screwed. Each sample pelleted in 3 1.5 mL Eppendorf tubes 2 min at 8,000 rpm. Supernatent dumped and sleft upside down on paper towel with top open to remove excess liquid. 

Resuspend in 250 uL of resuspension buffer, add lysis buffer, which had no precipitate, invert 6 times, let sit for ~2 min, add 350 uL neutralization buffer vigerously with pipette submerged, rapid 6 shakes immediately. 

pellet 5 min 13,000 rpm. Careful decanting with pipette, spin 1 min, add 500 uL wash times two cycles, add 30 uL elution buffer carefully, directly to bottom without touching membrane, let sit 3 min, spin 2 min at 13000 rpm.

nanodrop results:

Sequencing primer design and order:

Eurofins primer design site was confusing and gave primers within the requested target range. This was sketchy so I found a different tool: GenScript Sequencing Primer Design. Divide plasmid into 10 bp sections, and then select 2 forward primers that are ~200 bp upstream of what we were expecting 

 

(6/15) Wednesday June 15, 2022

Names: Emily, Jack, Sriram, Amruta, Sashider

Date: Wednesday June 15, 2022

Goal: 

Redo encapsulin PCR to replace lost PCR product.

Names of Protocols Used: 

Encapsulin PCR 

Gele Electrophoresis

Notes/Modifications: 

Reaction recipe:

• 25uL master mix

• 2.5uL Fwd primer (10 uM)

• 2.5uL Rev primer (10 uM)

• 19uL nuclease free water

• 1uL template DNA (1:600)

Thermocycler settings:

• 3 min at 98 degrees

• 30 cycles of:

• 10 sec at 98 degrees

• 30 sec at 67 degree

• 30 sec at 72 degrees

• 2 min at 72 degrees

• Hold at 4 degrees

Gel Electrophoresis:

• Followed protocol to make gel

• Dilution:

• 1 uL ladder + 6 uL water

• 1 uL T4 + 1 uL loading dye + 5 uL water

• 1 uL PCR product + 1 uL loading dye + 5 uL water

• Run in gel at 110 V for 35 min

• Image (ladder, T4, PCR)

Results/Data: 

Interpretation of Results:

 Gel electrophoresis conducted successfully

Summary and Next Steps: 

 Send positive control Gibson for sequencing ASAP 

 

(06/16) Thursday June 16, 2022

Names: Elizabeth, Adam

Date: 6/16/22

Goal: 

Send positive control Gibson for sequencing

DpnI digest PCR product

PCR Cleanup

Names of Protocols Used: 

Dpn1 Digest

2uL of Dpn1 incubated at 37 for 1 hr, heat inactivate 80 degrees for 20 min. Will transform this monday and see what's up

Calculations: 

Notes/Modifications: 

Of 1.1, 1.2, 1.3, 2.1, 2.2, 2.3, all were good concentration except 1.1

Results/Data:

Calculations for Gibson Assembly (Will be Done Tomorrow?) -> Could Potentially Be Generalized For Future Gibson Assembly Calculations, Sort of Like a Template

Interpretation of Results:

 N/A 

Summary and Next Steps: 

 Perform Gibson Assembly 

 

(06/17) Friday June 17, 2022

Names: Adam

Date: Friday June 17, 2022

Goal: 

Gibson Assembly

Names of Protocols Used: 

add 5.5 uL of vector 

Dilute AMP B in 31.2 uL H20

Dilute AMPB 1:5 and add 2.6 uL

Dilute Enc 1:10 and add 2 uL

Add 20 uL HiFi mastermix

Pipette mix gently and centrifuge 

20 min at 50 degrees, then cool to 4 degrees and pop in fridge

67716549287__A623A1DD-44E8-4100-B249-8F4140776678.HEIC

Calculations: N/A

Notes/Modifications: N/A

Results/Data: Samples prepared 

Interpretation of Results:Samples prepared 

Summary and Next Steps: 

 Transform product next 

 

(06/20) Monday June 20, 2022

Names: Elizabeth, George

Date: 6/20/22

Goal: 

Transform gibson product

Prepare overnights for growth assay tomorrow

Names of Protocols Used: 

Chemical Transformation

Making overnights

Calculations: 

Added 3 uL of PCR product (not very much left)

Added 5 of Gibson assembly product

Notes/Modifications: N/A

Results/Data: Samples prepared, quantified after.

Interpretation of Results:N/A

Summary and Next Steps: 

For overnights:

2 each of empty petduet, T4Gala+petduet, and positive control gibson (from strain AT-05-27-22-2)

 

(06/21) Tuesday June 21, 2022

Names: Jaclyn Carrannanto, Cameron Haynes

Date: Tuesday June 21, 2022

Goal: Growth Assay and start overnights of transformation colonies if there are any. Also look into measurement thing and then getting free miniprep and sending chem department a budget

Names of Protocols Used: 

Goal: To show the toxicity of AMP expression.

Prepare overnights of empty PET, PET + ENC, Strain 1, and Strain 2 (Whichever showed good sequencing results. Prepare 8 250 mL erlenmeyer flasks with 50 mL of LB broth with ampicillin 0.1 mg/mL.

Inoculate each of the 50mL classes to an initial OD of 0.03 (Do this by measuring the OD of the overnights, and then doing a dilution. Dilute the overnights 1:4 or 1:5 and multiply by 4 or 5 to get the actual value (because the spectrophotometer is less accurate at higher OD). Make 2 of each bacteria.

Induce one culture of each type of bacteria immediately with 0.5 mM IPTG. Grow at 37 degrees and 280 RPM. Record OD measurements for all samples every 15 min for 5 hours.

Calculations: 

Notes/Modifications: 

empty pet1: 1.68 OD ; empty pet 2: 1.30 OD

positive control 1: 2.200 OD; positive control 2: 

WHAT WAS DONE:

50 microliters of 100 mg/mL ampicillin was added to each of 2 250 mL Erlenmeyer flasks containing ~50 mL LB Broth. These flasks may have already had ampicillin, but we had no way of really knowing since they were just labeled "Amp" with blue tape. Thus, it was decided that 2x Amp wouldn't hurt anything that we actually wanted to grow: these two flasks will be used for the two replicates of a single kind of transformed cell (i.e one of empty PET, PET + ENC, Strain 1, Strain 2) that will be used in today's experiment.

Results/Data: N/A 

Interpretation of Results: Results inconclusive 

Summary and Next Steps: 

Redo Growth Assay 

(06/22) Wednesday June 22, 2022

Names: Adam Tisch, Cameron Haynes

Date: 06/22

Goal: Growth Assay!

Names of Protocols Used: 

Goal: To show the toxicity of AMP expression.

Prepare overnights of empty PET, PET + ENC, Strain 1, and Strain 2 (Whichever showed good sequencing results. Prepare 8 250 mL erlenmeyer flasks with 50 mL of LB broth with ampicillin 0.1 mg/mL.

Inoculate each of the 50mL classes to an initial OD of 0.03 (Do this by measuring the OD of the overnights, and then doing a dilution. Dilute the overnights 1:4 or 1:5 and multiply by 4 or 5 to get the actual value because the spectrophotometer is less accurate at higher OD.) Make 2 of each bacteria.

Induce one culture of each type of bacteria immediately with 0.5 mM IPTG. Grow at 37 degrees and 280 RPM. Record OD measurements for all samples every 15 min for 5 hours.

Calculations: 

Notes/Modifications: 

LB Agar from LB Broth Protocol:

https://www.protocols.io/view/lb-luria-bertani-agar-q26g72b3lwz1/v1

Results/Data:MSBT 06_22_22 IPTG Induced Plasmid Expression Growth Assay - Sheet2.csv

Interpretation of Results: Results inconclusive as to rate of growth 

Summary and Next Steps: 

Redo Growth assay

 

(06/22) Wednesday, June 22, 2022

Names: Amruta, Jack, John, Sashider, Sriram

Date: 6/22/22

Goal: 

Make Overnights of Gibson Product from 6/21

Names of Protocols Used: 

Making Overnights

Calculations: 

Notes/Modifications: 

5 ml of LB Broth

1 ul to 1 mL of Ampcillin (5 ul)

1 ul Gibson Product overnights to innoculate

5 Overnights

Results/Data: N/A

Interpretation of Results: N/A 

Summary and Next Steps: 

Miniprep and send Overnights for Sequencing Tomorrow (06/23)

 

(06/23) Thursday, June 23, 2022

Names: Adam, George, John, Sashider

Date: 06/23/22

Goal: 

Miniprep the Gibson Product Overnights (Made on 06/22)

Names of Protocols Used: 

Miniprep 

Calculations: N/A

Notes/Modifications: 

5 Gibson Product Overnights Made on 06/22 = 5 Mini-Prep Eppendorf Tubes Made (1 for Each Gibson Product Overnight

Results/Data:N/A (Overnight) 

Interpretation of Results:N/A 

Summary and Next Steps: 

Send Mini-Prepped DNA for Sequencing

 

(06/29) Wednesday June 29, 2022

Names: Adam, Sriram, Sashider, Amruta, George

Date: Wednesday June 29, 2022

Goal: Growth Assay Trial 2

Names of Protocols Used: 

The day before I had prepared 50 mL LB aliquots, all with identical LB. I added 50 uL 100 mg/mL ampicillin salt to 9 of them. Before innoculation, I added 5 uL IPTG to 3 and 10 uL IPTG to another 3. There were only 3 because one of the overnights, the one with just the T4 enc did not grow. We attributed this to an old plate, it was about 3 months old, and made a new amp plate of it from our glycerol stock. The overnights that grew were diluted 1:4 and then measured. The appropriate amound of liquid overnight was initialize our samples at an OD600 of 0.03 was added. There were three bacteria conditions, PET Empty, PET + AMP, and unconfirmed PET + AMP + ENC (it was CH strain 2), and three IPTG conditions, 0 uM, 100 uM, and 200 uM. These samples were shaken at 225 rmp and grown at 37 degrees for several hours, and samples were taken about every 40 min to measure OD600. OD600 was the absorbance of the sample compared to sterile LB broth at 600 nm.

The collected data are as follows:

I'm pretty sure that something is up with the measurement at 18:44, we switched who was taking the measurement here, and I think the bacteria settled, so the values are either way high or way low depending on whether they were taken at the top or bottom of the flask. Without this one measurement, we actually have really nice looking graphs:

 

July

(07/07) Thursday July 7, 2022

Names: Jack, Adam, Sashider, Sriram, Steven

Date: 7/7/22

Goal: Make lb broth and glycerol stock overnights (enc) and streak the encapsulin. 

Names of Protocols Used: How to make Lb and overnights

Calculations: 

Ampicilin made 10mg/ml, both are in the fridge in the team room. 

5 µl ampicilin for 1µl/ml per overnight. 

Also, we changed the trash. This means autoclaving the old bag and then putting it in the normal trash and then grabbing a new biohazard one. 45 min grav cycle

(07/8) Friday July 8th, 2022

Names: John Yin Adam Tisch, Ahmed

Date: Friday, July 8th, 2022

Goal: 

Pellet cells for protein expression

Notes/Modifications:

5 mL AMP+Enc cells added to 1L of broth with 100 mg of Ampicillin added. Grown to OD 0.6 at 225 rpm and 37 degrees and then induced with 200 uM IPTG. Grown for 3-4 more hours at 37 degrees and then spun and froze.

Due to the large amount of culture, the cells were pelleted by pipetting 50 mL of culture into 8 conical tubes. These tubes were centrifuged approximately 3 times at 5000 RPM for 15 minute intervals. The tubes were then stored at -80 degrees Celsius for future use.

(07/11) Monday July 11, 2022

Names: George Rabadi, Sabine Meurs, Jaclyn Carrannanto, Amruta Venkatesh, Amogh Angadi

Date: Monday July 11, 2022

Goals: 

FROM ADAM: 

1) figure out what we need to buy 

2) see if we can show any encapsulins were produced by finding an encapsulin band in the protein gel 

3) figure out if we need to alter the lysis buffer and lysis protocol to have more efficient lysis.

First, try to locate lysozyme in the freezer and a protein gel rig from a USB person. Put ethanol in a container in the -80 so it gets good and cold by the time you need it.

Then, come up with a lysis buffer for cell lysis. This will probably be smthn along the lines of 20 mM TRIS ph 7.5 and 150 mM NaCl. Then take one of the cell pellets out of the -80. Keep the cells and everything on ice the whole time. Resuspend in your buffer, just add buffer until it is no longer thick and sticky. Take 1 mL of these resuspended cells and put it in a 1.5 mL Eppendorf tube. Add lysozyme if you have it. Get a 37 degree waterbath going next to the -80, and quick freeze thaw cycles (long enough in each temp to freeze and then thaw the liquid). Repeat this 6-10 times depending on whether you have lysozyme and then spin at max speed in the eppendorf centrifuge for long enough to pellet all the cell fragments. The liquid should be clear when you are done, start with about 10 min spinning and reevaluate.

Then, if you were able to find a protein rig, follow the sds-page gel protocol from last year to the best of your ability. If you can stain it too (This takes a while, the reagents are in the cabinet I think) that would be awesome, but if not just leave the gel in water and cover overnight in the fridge not freezer. Put any left over resuspended cells from the pellet in the -20, and any left over lysed cells or lysed and pelleted cells in the fridge at 4degrees.

Names of Protocols Used: 

Calculations: 

Notes/Modifications: 

Results/Data:

WHAT WE ACTUALLY ACCOMPLISHED:

Made Lysis Buffer

0.121g Tris

0.4383g NaCl

Add to 40mL of DH2O

Bring to a pH of 7.5 using HCl/NaOH

Bring final colume to 50mL

Lysis Procedure

Cycle between hot water (37C) and cold ethanol (store in -80C while waiting) 6-10 times

Storing in -80C freezer because we need to leave

Interpretation of Results:

Summary and Next Steps: 

Finish/Redo lysis procedure and run SDS gel

 

(07/13) Wednesday July 13, 2022

Names: Adam

Date: 7/13/22 

Goal: Repeat Lysis, test gel, try heat precipitation purification step

Protocols Used: 

Resuspend pellet of 1/8 L cells in 150 mM NaCl, 20 mM TRIS pH 7.5 from last year in my handwriting

Place plastic beaker ethanol in -80, and normal one h20 in 37 degree incubator in store room upstairs. Add 50 uL lysozyme 10 mg/mL to 500 uL resuspended cells, and 5 uL to a second 500 uL, and 0 to a third incubate on ice for a half hour.

6 freeze thaw cycles for each tube

Keep everything on ice all the time

Spin at 14,000 RPM for 10 min. Take samples from 0.1 lysozyme condition and heat to 65 or 85 degrees in thermocycler. Then trim tubes with scissors, put them in 1.5 mL tubes, and spin at 14,000 rpm for 10 min.

Transfer all supernatant into different tubes away from pellets. 

Gel was run with 8uL protien sample mixed with 2uL of 4x protein sds gel loading dye from anthony in freezer. I used 3 uL ladder. Run at 45 mA, constant amps. pop casing, stain, destain. I stained way too long, like 2 hr in max concentration coomassie stain. 

Results/Data:

from left: Ladder, lysis with no lysozyme, lysis with 0.1mg/mL lysozyme, lysis with 1mg/mL lysozyme, lysis from two days ago, cell pellet resuspended in 1/10th the volume as today, after heating to 65 degrees, after heating to 85 degrees.

Interpretation of Results:

Protein expressed successfully, 85 degree heat purification worked well, continue lysis with 0.1 mg/mL lysozyme incubating for half hour on ice. 

 

(07/18) Monday July 18, 2022

Names: George Rabadi, Sabine Meurs, Amogh Angadi, Sophia Tesic, Kimi Lillios, Rafee Mirza, Amruta Venkatesh, Jaclyn Carrannanto

Date: 7/18/2022

Goal: 

• Nanodrop gel extract (for next time)

• plan out next Gibson

• make 500 mL pH 7.5 1 M tris

• 78.8g Tris-HCl for 500mL 

• start overnights for enc, enc + amp, amp, and blank pet

 

(7/21) Thursday July 21, 2022

Names: George Rabadi, Amogh Agandi, Sabine Meurs, Kimi Lillios

Date: 7/21/22

Goal: 

• Make LB for Growth Assay

• Prep overnights for Growth Assay

• Prep Ampicillin Stock

• Re-streak plates

Names of Protocols Used: 

•  LB Broth

  

Notes/Modifications: 

Made 8 100mL beakers of LB Broth

• 2.5g of LB mix in 100mL DI water

Prepped overnight

• Two 5mL tubes of Empty Pet

• Two 5mL tubes of Pet+Encap

• Two 5mL tubes of Pet+AMP

• Two 5mL tubes of Pet+AMP+Encap

Prepped Ampiccilin Stock

• 0.1g Ampicillin salt to 1mL DI Water

Restreaked Plates

• Restreaked Pet Deut Empty BL21 D3E3 from 4-14-22

• Restreaked AMP+Encap+Pet from 6-22-22

• Restreaked Strain AT-5-27-22-2

(07/22) Friday Jul 22, 2022

Names: Steven Gong, Elizabeth, Jaclyn

Date: 07/22/2022

Goal: Growth assay

Names of Protocols Used: Growth assay protocal 

Calculations: N/A 

Notes/Modifications: 

Added IPTG after an hour of incubation in the LB broth at room temperature, the OD value did not reach 0.5 yet. 

For the first 2 readings, samples were not in shaking incubator, were stationary on counter at room temp

Results/Data:

 

(07/27) Wednesday July 27, 2022

Names: Adam, Bonnie, Emily, Sriram, Amruta, Jack

Date: Wednesday July 27, 2022

Goal: 

To purify the AMP out of the encapsulin

Names of Protocols Used: 

Resuspend 1/8th L pellet in 5 mL 150 mM NaCl, 20 mM pH 7.5 tris. Add 0.1 mg/mL lysozyme, 6 Freeze thaw cycles between -80 and 37 degrees, spin 10 min at 14,000 rpm in eppendorf centrifuge. Heat to 85 degrees for 15 min in pcr machine. Spin 10 min at 14,000 rpm. Add 200 uL of this to each of the ammonium sulfate conditions. Incubate on ice for 1 hr. Spin 10 min at 14,000 rpm, resusupend in 200 uL of lysis buffer. Place in dialysis with lysis buffer 10 kDa overnight, using serial clipping method. 

Remaining 0.75 mL following heat purification step concentrated to 0.1 mL and then added 900 uL of pH 5.5 mes. This was then incubated overnight in 4 degrees with 10 uL of TEV.

Calculations: 

Ammonium Sulfate Quantities for 10% Dilutions: 

  

Notes/Modifications: 

We used MES buffer, but it is all we have of that. We need to make another buffer at pH 6 at some point.

Results/Data:

Interpretation of Results: Samples left for evaluation 

Summary and Next Steps: 

Evaluate purification from 27th

 

(07/28) Thursday July 28, 2022

Names: Adam, Sophia

Date:  Thursday July 28, 2022

Goal: Evaluate purification from 27th

Names of Protocols Used: 

Remove ammonium sulfate samples from dialysis after ~20 hrs. No stiring while dialyzing in 4 degrees. Take 8 uL of each sample, mix with 2 of 4x protiein gel loading dye, run gel at ~35 mA. Coomassie stain 40 min, destain and image. Benchmark invetrogen prestain protein ladder.

  

  

Results/Data: Imaged stain

Interpretation of Results: Verified that protocol works 

Summary and Next Steps:

Enc T4 Gala Empty Expression 

 

August

(08/05) Friday Aug 5, 2022

Names: Amogh

Date: 8/5/22

Goal: Enc T4 Gala Empty Expression

Names of Protocols Used: 

Enc T4 Gala Empty Expression. 

  

Results/Data:Spun down cultures and then put 8 50 mL Falcon tubes with pellets in -80

Summary and Next Steps:

Redo buffer prep 

 

(08/08) Monday August 8, 2022

Names: Sophia, An, Jaclyn

Date: 08/08/22

Goal: 

Prepare for this week's experiments

Names of Protocols Used: 

• Making buffers (steps below under calculations)

Instructions for the day:

You will need to do the buffers in the upstairs storage room because the pH meter is in there

You can autoclave all of these solutions at the same time because they will all be about 1 L in volume

To make a buffer at pH 6: 

• Prepare 800 mL of dH2O in a suitable container. --> Get a 2 L jug from the glassware cabinet. Measure liquid with a graduated cylinder though.

• Add 209.24 g of Bis-Tris to the solution (more basic than bis, add less tris, half amount, test pH, then add in increments to 7.5-9 (8), then use acid to bring back to 7.5)

• Starting pH without HCl is 9.90. Concentrated HCl is used to adjust the pH. Autoclave before use or storage.

• Add water to bring to 1L and check pH again (ideally shouldn't change but adjust if necessary)

To make a buffer at pH 7: 

• 100 mM NaCl and 20 mM TRIS at pH 7.5

• 5.84g NaCl

• 2.42g Tris

• 1L water

• You may need to adjust the exact amounts to get to pH 7.5. Add in small increments, because once you get close the pH will change FAST. Also we don't want to ruin the concentrations too much, so keep track of how much additional you add. 

• You can use small amounts of HCl too if you need it to be slightly more acidic (lower pH)

• Autoclave as well

To Make Plates 

• Follow steps 3-12 of the "Pouring Agar Plates" Protocol. Please only add ampicillin to 3/4 of the agar mixture. The other 1/4 do not add ampicillin. When pouring the plates, make sure to label which contain ampicillin

Notes/Modifications: 

• HCl located under vent hood of third floor prep room

• Undershot pH of pH 6 buffer and added HCl to raise to 6 

• Both autoclaved buffers left in their labeled 2 L jugs in the autoclave to sit after the cycle finished

Summary and Next Steps: 

• We currently have a stock of agar plates split into 3s, but did not get around to making the unsectioned plates, so that needss to be done. 

 

(08/09) Tuesday August 9, 2022

Names: Elizabeth, Bonnie

Date: 8/9/22

Goal: 

Purification and Release of AMP

Names of Protocols Used: 

https://docs.google.com/document/d/1-ZU1RNEAk0BuEKHKn_T_EkYI14sqwSc6aJChfvFoXz8/edit

Calculations: 

50 uL of lysozyme

Notes/Modifications: 

Buffer was at a pH of 7 as opposed to pH of 7.5

We used 2 tubes of cell pellet form the -80C, 3 remain

Results/Data:

Concentrator tube was ineffective due to an error in the protocol. Ammonium sulfate was added when it should not have been.

Summary and Next Steps: 

The protocol was sorted out, and the experiment will be redone tomorrow

 

(08/10) Wednesday August 10, 2022

Names: Elizabeth, Bonnie, Sashider, Amruta, Sriram

Date: 8/10/22

Goal: 

Make new plates/overnights of frequently used plasmids, redo purification for toxicity assays

Names of Protocols Used: 

Purification Protocol for Toxicity Assays

Calculations: 

Notes/Modifications: 

Some stored at 10% protein and 90% glycerol and some stored at 90% protein and 10% glycerol (thus former sample is not viable)

3-4 tubes of each type for both AMP and enc (12-16 tubes total)

Results/Data:

Stored in -80C, labeled pure encapsulin and pure AMP

Interpretation of Results: N/A 

Summary and Next Steps: 

Disconnect AMP via TEV Protease following pH opening of encapsulin

Run SDS PAGE gel

 

(08/11) Thursday August 11, 2022

Names: Bonnie, Elizabeth, Jaclyn, Jack, John

Date: Thursday August 11, 2022

Goal: 

To conduct a growth assay and CFU assay to assess whether or not the addition of an AMP to a factory bacteria causes increased cell death and if the further addition of an encapsulin helps to protect the factory bacteria

Names of Protocols Used: 

Making Ampicillin Plates, New Growth Assay (Using CFU)

Calculations: 

1ml of overnight culture added per 100ml of LB (1% inoculation)

Notes/Modifications: 

Link to Growth Assay Spreadsheet

• empty induced in column 1, empty in column 2 (for hour 5 reading)

Results/Data: Growth assay to be quantified 

Summary and Next Steps: 

Revisit growth assay 

 

(08/16) Tuesday August 16, 2022

Names: Adam

Date: 8/16

Goal: Open encapsulins and test toxicity assay. Also start overnights for miniprep for growth assay and analyze last growth assay data. 

Names of Protocols Used: 

Toxicity assay 

Thaw 90% protein 10% glycerol from -80. Dispose of 10% protein 90% glycerol.

Reduce volume by 90% in protein concentrator, refill with pH 6.0 buffer to original volume.

Combine with an aliquot of test cells in various percentages, incubate at room temp for 1 hr. Plate and place plates in the incubator.

1:1 protein, 1:10 protein, 1:100 protein

dilute cells and protein 1:1, 1:10, 1:100

Times two, one for enc without amp and one for enc + amp

how much protein do we need?

10 uL + 1 uL + .1 uL per protein condition

For incubation at rt: 20 uL protein + 20 uL bacteria, 36 bacteria + 4 protein, 39.6 bacteria + 0.4 protein. Replicate with each protein at each pH.

Start overnights of enc, enc + amp, amp, and pet, 5 mL aerobic growth and shake overnight for miniprep tmr.

Ok... Below is what actually happened:

Concentrate 1 mL of enc and enc+amp to ~100 uL. Add ~450 uL of buffer at either 6 and 7.5 to half of protein for each. Incubate 1 hr at rt. Dilute mixture 1:10, 1:100, 1:1000 and plate 10 uL on 1/3 of the third plates. Leave in the incubator overnight. Hopefully the results will be drastic. 

Preserve protein in the fridge for potential TEV digestion tomorrow and repeat growing with more replicates. 

Results/Data:

Nothing grew, not even the overnights

Interpretation of Results:

Incubator error or something weird like that, will repeat

Summary and Next Steps: 

Redo overnights and identify sources of error 

 

(08/17) Wednesday August 17, 2022

Names: Adam

Date: Wednesday August 17, 2022

Goal: Toxicity assay 

Names of Protocols Used: 

gel goes ladder, 6+, 8+, 6-, 8-

Calculations: N/A

Notes/Modifications: N/A

Results/Data:Protein samples from yesterday were kept in fridge overnight, will be used again today

Run gel of protein samples and blank plates as control

Summary and Next Steps: 

Make more overnights for further experimentation

(08/31) Wednesday August 31, 2022

Names: Adam, Sophia

Date: Wednesday August 31, 2022

Goal: Start overnights

Names of Protocols Used: Make LB broth, start overnights

Results/Data:7 mL of LB plus 7 uL Ampicillin

37 degrees overnight 150 rpm

Interpretation of Results:N/A

Summary and Next Steps: 

Carry on with transformations 

 

September

(09/02) Friday September 02, 2022

Names: Bonnie Spence, Elizabeth Snider

Date: Friday September 02, 2022

Goal: 

To transform the 

• Empty pet duet

• pet duet with encapsulin only

• pet duet with AMP only

• Pet Duet with Encapsulin and AMP 

plasmids into the same strain of competent cells (BL21)

Names of Protocols Used: 

Chemical Transformation

Calculations: 

Adding 5ul plasmid DNA to each 50 ul comp cell aliquot, so will add 445ul Soc media

Notes/Modifications: 

• Using BL21 comp cells aliquoted 06/24/221

• The plasmid concentrations were not nano-dropped beforehand

Results/Data:

• Resulting solutions will be spread on plates and left in an incubator to be checked in the morning 

Nanodrop Results:

• Pet Duet: 38.8ng/ul, 260/230: 1.732, 260/280: 1.986

• Encapsulin: 46.212ng/ul, 260/230: 1.922, 260/280: 1.929

• AMP: 38.428ng/ul, 260/230: 1.567, 260/280: 1.941

• AMP+Encap: 26.588ng/ul, 260/230: 1.423, 260/280: 2.006

Interpretation of Results:

• N/A

Summary and Next Steps: 

Spread cells on plates overnight and check for growth in the morning

(09/27) Friday September 27, 2022

Names: Jack, Robert

Date: 9/27/22

Goal:

Re-run growth assay in 96 well plate

Notes/Modifications

Plates were shaked so proper growth was ensured. 4 different samples: empty pET, encapsulin, AMP, encapsulin + AMP 3x replicates each

Results:

Inconclusive