iGEM 2022 — Team michigan

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

# Notebook

---

##### TIMELINE

* [April](#april)
* [May](#may)
* [June](#june)
* [July](#july)
* [August](#august)
* [September](#september)

# 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:

![](https://static.igem.wiki/teams/4143/wiki/notebook/image12.png "Screen Shot 2022-04-11 at 3.40.56 PM.png")

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:

![](https://static.igem.wiki/teams/4143/wiki/notebook/image6.png "Screen Shot 2022-04-13 at 3.36.41 PM.png")

![](https://static.igem.wiki/teams/4143/wiki/notebook/image1.png "Screen Shot 2022-04-13 at 3.36.36 PM.png")

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](https://benchling.com/adtisch/f/lib_2XGZp2yR-notebook-entries/etr_Yr6RWwGb-0419-tuesday-april-19-2022/edit)

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

![](https://static.igem.wiki/teams/4143/wiki/notebook/image2.png "image.png")

![](https://static.igem.wiki/teams/4143/wiki/notebook/image5.png "image.png")

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

![](https://static.igem.wiki/teams/4143/wiki/notebook/image7.png "IMG_8923.jpg")

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](https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0012655_GeneJET_Plasmid_Miniprep_UG.pdf)

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

![](https://static.igem.wiki/teams/4143/wiki/notebook/image8.png "IMG_0005.JPG")

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)

![](https://static.igem.wiki/teams/4143/wiki/notebook/image21.png "Screen Shot 2022-05-03 at 11.36.24 AM.png")

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:

![](https://static.igem.wiki/teams/4143/wiki/notebook/image3.png "IMG_0008.JPG")

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

![](https://static.igem.wiki/teams/4143/wiki/notebook/image19.png "image (7).png")

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:

 ![](https://static.igem.wiki/teams/4143/wiki/notebook/image14.png "Gel 5-6 Confirmed PCR products.psd")

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:

![](https://static.igem.wiki/teams/4143/wiki/notebook/image17.png "image.png")

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:

![](https://static.igem.wiki/teams/4143/wiki/notebook/image15.png "Image from iOS.jpg")

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.

![](https://static.igem.wiki/teams/4143/wiki/notebook/image18.png "IMG_0692.jpg")

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:

![](https://static.igem.wiki/teams/4143/wiki/notebook/image20.png "image.png")

### (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](https://www.genscript.com/tools/dna-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:

![](https://static.igem.wiki/teams/4143/wiki/notebook/image22.png "image.png")

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

![](https://static.igem.wiki/teams/4143/wiki/notebook/image11.png "IMG_4849.jpg")

![](https://static.igem.wiki/teams/4143/wiki/notebook/image9.png "IMG_4850.jpg")

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:

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Time | PET Uninduced | PET Induced | PET + AMP Uninduced | PET + AMP Induced | PET + AMP + Enc Uninduced | PET + AMP + Enc Induced | T4 GALA Uninduced | T4 GALA Induced |
| 10:04 | 0.055 | 0.054 | 0.045 | 0.055 | 0.066 | 0.043 |  | 0.016 |
| 10:30 | 0.120 | 0.086 | 0.076 | 0.065 | 0.103 | 0.152 |  | 0.02 |
| 10:54 | 0.565 | 0.124 | 0.111 | 0.114 | 0.192 | 0.141 |  | 0.03 |
| 11:22 | 0.675 | 0.107 | 0.212 | 0.174 | 0.378 | 0.188 |  | 0.022 |
| 11:50 | 0.763 | 0.149 | 0.306 | 0.334 | 0.537 | 0.322 | 0.111 | 0.094 |
| 12:22 | 1.277 | 0.290 | 0.480 | 0.487 | 0.798 | 0.458 | 0.040 | 0.056 |
| 12:52 | 2.244 | 0.451 | 0.601 | 0.601 | 0.958 | 0.693 | 0.087 | 0.112 |
| 1:28 | 2.5 | 0.793 | 1.118 | 0.901 | 1.42 | 0.977 | 0.11 | 0.033 |
| 2:02 | 2.925 | 1.356 | 1.318 | 1.42 | 1.70 | 1.508 |  |  |
| 2:27 | 3.112 | 1.874 | 1.374 | 1.496 | 2.37 | 1.74 |  |  |
| 2:49 | 4.932 | 3.174 | 2.064 | 2.052 | 3.612 | 2.634 |  |  |
|  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |

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:

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Measure Start Time |  |  | PET Uninduced | PET + AMP Uninduced | Enc + PET + AMP Uninduced | PET100 uM | PET + AMP 100 uM | PET + Enc + AMP 100 uM | PET 200 uM | PET + AMP 200 uM | PET + Enc + AMP 200 uM |
| 13:40 | 23 | 23 | 0.066 | 0.082 | 0.053 | 0.08 | 0.066 | 0.052 | 0.083 | 0.066 | 0.052 |
| 14:18 | 61 | 38 | 0.187 | 0.071 | 0.09 | 0.136 | 0.062 | 0.102 | 0.191 | 0.061 | 0.151 |
| 14:49 | 92 | 31 | 0.381 | 0.142 | 0.136 | 0.283 | 0.111 | 0.19 | 0.174 | 0.123 | 0.164 |
| 15:29 | 132 | 40 | 0.881 | 0.297 | 0.313 | 0.594 | 0.27 | 0.353 | 0.251 | 0.239 | 0.399 |
| 16:10 | 173 | 41 | 1.68 | 0.455 | 0.516 | 0.943 | 0.478 | 0.572 | 0.362 | 0.385 | 0.588 |
| 16:50 | 213 | 40 | 2.618 | 0.736 | 0.844 | 1.084 | 0.774 | 0.934 | 0.578 | 0.642 | 0.85 |
| 17:25 | 248 | 35 | 3.4 | 0.932 | 1.008 | 1.176 | 1.056 | 1.168 | 0.8 | 0.748 | 1.196 |
| 18:03 | 286 | 38 | 3.975 | 1.51 | 0.9 | 1.49 | 1.665 | 1.32 | 1.475 | 1.05 | 2.085 |
| 18:44 | 327 | 41 | 2.535 | 0.975 | 1.335 | 2.39 | 1.755 | 3.06 | 1.505 | 1.57 | 1.315 |
| 19:25 | 368 | 41 | 4.06 | 2.3 | 1.715 | 2.37 | 2.08 | 1.86 | 3.005 | 1.995 | 2.88 |
| 20:10 | 413 | 45 | 4.615 | 2.345 | 2.71 | 2.38 | 2.925 | 3.38 | 2.66 | 1.935 | 3.18 |

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.

![](https://static.igem.wiki/teams/4143/wiki/notebook/image10.png "IMG_7538.jpg")

### (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:

![](https://static.igem.wiki/teams/4143/wiki/notebook/image16.png "Untitled presentation.jpg")

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:

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| time (min) | non-induced, PET | non-induced, PET + enc | non-induced, PET+AMP | non-induced: PET + AMP + enc | induced, PET | induced, PET + enc | induced, PET+AMP | induced: PET + AMP + enc |
| 0 | 0.026 | 0.057 | 0.032 | 0.043 | 0.01 | 0.054 | 0.028 | 0.026 |
| 30 | 0.003 | 0.071 | 0.006 | 0.017 | 0.103 | 0.11 | 0.016 | 0.023 |
| 60 | 0.004 | 0.132 | 0.025 | 0.024 | 0.005 | 0.129 | 0.032 | 0.019 |
| 90 | 0.004 | 0.254 | 0.034 | 0.044 | 0.002 | 0.210 | 0.067 | 0.048 |

### (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:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Percent | 10 | 20 | 30 | 40 | 50 | 60 | 70 | 80 | 90 | 100 |
| Grams A.S. | 0.0146 | 0.0246 | 0.0346 | 0.0446 | 0.0546 | 0.0646 | 0.0746 | 0.0846 | 0.0946 | 0.1046 |

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

![](https://static.igem.wiki/teams/4143/wiki/notebook/image13.png "image.png")

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](https://docs.google.com/spreadsheets/d/1PwCeEugNfEnHhW46XkqBuLXK-Z7MHSJaIJzKEz6lyGY/edit?usp=sharing)

* 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

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

# Engineering Success

---

##### CONTENTS

* [Introduction](#intro)
* [Cycle 1](#c1)
* [Cycle 2](#c2)
* [Cycle 3](#c3)

# Introduction

Throughout the course of our project, we followed the engineering design cycle of designing, building, testing,
and learning in iterative fashion. A few examples of this are highlighted below.

![](https://static.igem.wiki/teams/4143/wiki/engineering/engineering-1.png)

# Cycle 1

##### Design

To test the efficiency of the AMP + encapsulin expression system, we planned a growth assay comparing the
growth rates of bacteria with our experimental construct to relevant controls. This resulted in the following
bacterial strains:

1. Vehicle Control: pETDuet vector
2. Negative Control: T4GALA encapsulin+pETDuet vector
3. Positive Control: AMP+pETDuet vector
4. Experimental Construct: T4GALA encapsulin+AMP+pETDuet vector

We first designed our constructs for cloning via Gibson assembly, with each segment containing 20 base pair
overlaps on either end. We ensured that this included overhangs compatible with the sites created by the
restriction enzymes we planned to use for linearizing our plasmid vector. We also codon optimized our sequences
for our expression strain of BL21 E. coli.

##### Build

We used PCR to extract the encapsulin sequence from a plasmid. We ordered our AMP sequence from IDT. We
linearized our pET Duet vector using two restriction enzymes, NdeI and PacI. We then assembled all of these
sequences into plasmids using Gibson assembly. Transformation was then conducted with DH5ɑ E. coli. Then
ampicillin resistant cells were miniprepped in preparation for sequencing.

##### Test

The positive control AMP plasmid as well as the encapsulin + AMP plasmid were submitted to Eurofins Scientific
for Sanger sequencing to confirm Gibson assembly success.

##### Learn

The location-specific insertion of our sequences confirmed Gibson assembly success, although there were
mismatches throughout despite generally accurate sequencing. We attributed this difficulty to technical issues
in sequencing. In the future, we could repeat sequencing with different primers to confirm our result, sequence
the pure AMP insert gBlock to ensure accuracy, or repeat Gibson assembly and subsequent transformation to
produce the purest production of the plasmid if mutations were found. Additionally, we learned the importance of
sequencing primer choice, as when we initially sequenced the plasmid with generic T7 primers, we observed
nonspecific primer binding. Ultimately, we believe we were successful in engineering our designed plasmids.

# Cycle 2

##### Design

After cloning, we transformed these plasmids into BL21 E. coli, a gram-negative bacteria known to be
susceptible to our AMP [1]. We hypothesized that, after induction, the experimental construct would improve
bacterial growth relative to the positive control, which would demonstrate inhibited growth or cell death as the
AMP produced its toxic effects. We planned to induce protein expression and take OD600 readings to measure
bacterial growth over a period of several hours.

##### Build

In order to build the bacterial strains that we would use in our growth assay, each plasmid was successfully
transformed into BL21 E. coli bacteria, as confirmed by antibiotic selection with ampicillin. These 4 strains
were used to inoculate 8 samples of LB broth treated with ampicillin. They were then either grown in the
presence or absence of IPTG for several hours.

##### Test

OD600 readings were taken manually approximately every half hour for 7 hours. We used these results to find the
growth rates of each of the bacterial colonies. The growth rate was determined for the logarithmic phase of
growth for each sample. Then the average rate and variance of the rates were calculated.

##### Learn

This growth assay was run manually, and it was logistically challenging to take 8 accurate OD600 measurements
every half hour for 7 hours. This led to noisy data, seen below, which were hard to draw conclusions from.
Similarly, the manual sampling made it difficult to run replicates for accurate estimations of effect size. We
determined that this experiment was inconclusive, and began the planning phase over again to conduct a more
precise experiment.

![](https://static.igem.wiki/teams/4143/wiki/engineering/engineering-2.png)

Figure 1: Growth of bacteria under induced expression of indicated protein. Note high noise
caused by manual measurement.

# Cycle 3

##### Design

To address the logistical challenges of our manual experiment, we designed a protocol for an automated growth
assay. Rather than growing cultures in flasks, we grew our cultures in wells of a 96 well plate. We chose an
automated method because it allowed us to eliminate noise from human error from our measurements while
simultaneously increasing the number of replicates.

##### Build

We obtained access to a plate reader capable of running an automated growth curve experiment and built an assay
that measured bacterial concentration for 3 technical replicates of 3 biological replicates with or without
induction by IPTG for each of our bacterial strains.

##### Test

Our proposed plate was assembled and OD600 measurements were collected for each well every 5 minutes over the
course of 12 hours.

##### Learn

This assay produced high quality data that our team was able to analyze for a meaningful assessment of the
efficacy of our project. We learned definitively that the effect on bacterial growth rate caused by the
encapsulin mitigating AMP toxicity was smaller than we had expected, and this informed our analysis of the
results of our project.

![](https://static.igem.wiki/teams/4143/wiki/engineering/engineering-3.png)

Figure 2: Example curves from automated growth assay. Note the high quality of the curves.

# References

1. Lee, T. H., Carpenter, T. S., D'haeseleer, P., Savage, D. F., & Yung, M. C. (2020). Encapsulin carrier
   proteins for enhanced expression of antimicrobial peptides. Biotechnology and bioengineering, 117(3), 603–613.
   https://doi.org/10.1002/bit.27222

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# Experiments

---

##### CONTENTS

* [Universal Protocols](#universal_protocols)
* [Project-Specific Protocols](#project_specific_protocols)

# Universal Protocols

##### **Chemical Transformation**

Allowing bacterial cells to uptake desirable DNA using a
heat-shock method.

Materials:

* Competent cells
* Plasmid DNA
* Water bath at 42°C
* SOC media

  + Can use LB as backup
* Agar plate

Procedure:

1. Prewarm plates and SOC in an incubator.
2. Thaw one 50 uL aliquot of comp cells in an ice bath.

   1. Comp Cells can be found in the -80 oC freezer. They need to be kept on ice AT ALL TIMES.
3. Begin heating up a water bath to 42°C in preparation for a later step.
4. Gently mix thawed comp cells.

   1. ONLY BY TAPPING, do not pipette or vortex.
5. Add 2-5 uL of plasmid DNA. Do NOT mix.

   1. Quantity will depend on plasmid DNA concentration. If low, add on the higher side. If high, add on the
      lower side. Should nanodrop the product before this step.
6. Place on ice for 30 minutes.
7. Heat shock cells for 45 seconds at 42°C.

   1. Timing is exact here. Use the water bath to heat shock.
8. Place tubes IMMEDIATELY on ice again for 1-2 minutes.
9. Add growth media (SOC or LB) to bring the final volume to 500 uL.

   1. Should be adding about 445 uL if adding 5 uL of DNA in step 3.
10. Incubate in a shaking incubator at 37 oC for one hour.

    1. 160-225 rpm.
11. Spin down cells for 2.5 minutes at 1000 rcf (9000 rpm).
12. Carefully pour off about 80% of supernatant into a waste container, leaving 100 uL.

    1. Can gently pipet out 400ul of supernatant.
13. Resuspend cells in remaining supernatant.
14. Spread all cells onto a plate and grow at 37 oC overnight.

##### **Dpn1 Digest**

When higher amounts of plasmid template must be used in the PCR reaction, it
is recommended that the PCR product be digested with Dpn1 (NEB #R0176) in order to destroy the plasmid template
before setting up the assembly reaction. Dpn1 cleaves only E. coli Dam methylase-methylated plasmid DNA, but
does not cleave the PCR product, since it is not methylated.

Materials:

* PCR product
* 10X CutSmart™ Buffer
* Dpn1

Procedure:

1. For a 10 uL reaction, combine 5-8 uL PCR product, 1 uL 10X CutSmart Buffer and 1 uL (20 units) Dpn1. Mix by
   pipetting or inverting.

   1. Found in the main room freezer.
2. Incubate at 37°C for 1 hour.
3. Heat-inactivate Dpn1 by incubating at 80°C for 20 minutes.
4. After running, place it in the fridge to keep until ready to run a gel.

##### **Gel Electrophoresis**

Agarose gel electrophoresis or E-gels are an important validation step
during the DNA assembly process. Running an E-gel on one or more samples of DNA allows any fragments of DNA
within each sample to be separated according to molecular weight/DNA length/number of base pairs. This allows
the scientist to distinguish and isolate the desired size of DNA after it's been separated from the other
fragments in the gel. An instance where this is useful is after a PCR amplification. If the amplicon is of a
known size, then an E-gel can be used to confirm the success of the PCR if there is a large presence of the
amplicon.

Materials:

* Agarose Gel

  + 50 ml. of 0.5x TBE buffer
  + 0.5 g of agarose (to make a 1% gel)
  + 5 uL of SYBR Green
* Digested PCR product
* DNA ladder

  + HyLadder 10 kb
* Loading dye
* Gel mold & comb
* Excess 0.5x TBE buffer

Procedure:

1. Add 50 mL of 0.5x TBE buffer to a 250 mL Erlenmeyer flask.
2. Add 0.5 g of Agarose powder into the flask. Microwave flask on high for 30 second intervals until the
   agarose has dissolved (60-90 seconds total). The powder should no longer be visible in the flask. The flask
   will be hot, handle carefully. Set up gel mold with appropriate well combs in the meantime.
3. When the solution has cooled to 50 oC (use thermometer or temp. gun), but not solidified, add 5 uL of
   SYBR stain to the solution. Swirl to mix.
4. Immediately pour the solution from the flask into the gel casting tray.
5. Wait approximately 30 minutes for the gel to solidify.
6. Once the gel has solidified, carefully remove the comb, loosen the gel tray, and place the gel (in the
   casting tray) into the gel box in the orientation which allows the (negatively charged) DNA to run towards
   the positively charged (red) cathode.
7. Pour 0.5x TBE buffer into the gel rig until the gel is completely submerged.
8. Use a p20 or p10 pipettor to load ladder and DNA samples into the wells of the gel. Before starting, gather
   all PCR products, DNA ladder, and loading dye.
9. In the far left well, add 8 uL of DNA ladder.
10. From the PCR tube containing the product, remove 6 uL and place in a separate tube in order to dye the
    product.
11. Add loading 1 uL loading dye to the 6 uL product and mix by pipetting up and down.
12. Repeat steps 10 and 11 to dye each PCR product.
13. In the wells to the right of the ladder, pipette the 7 uL of dyed product making sure to not disturb the
    integrity of the gel. Each PCR product needs to go in its own well. As you load the gel, make note of
    which sample goes into which well.
14. Once all samples have been loaded, attach a lid to the gel box, and attach the lid to the power supply
    (NOTE: always make sure that the current is off or paused before inserting or removing a cords from the
    power supply). Make sure leads are color matched.
15. Set the voltage to 115V and run the gel for about 30-45 minutes. It is advisable to check up on the gel
    from time to time to make sure that it is proceeding normally and ensure the dye does not run too far
    off the gel. The desired distance for dye travel is usually about 75% of the way across the gel.
16. When the gel seems to be completed, pause/stop the voltage, disconnect and remove the lid, and take the gel
    (in its tray) to be imaged. Visualize the stained gel using a standard transilluminator (302 or 312 nm) and
    image the gel using an ethidium bromide filter.

##### **LB Broth**

LB broth is a growth media commonly used for E. coli cells.

Materials:

* Deionized Water
* 5 g. NaCl
* 5 g. Tryptone
* 2.5 g. Yeast Extract
* 1 L Pyrex Bottle

Procedure:

1. In a 1 L pyrex bottle, add 500 mL of dH2O.

   1. Use graduated cylinder for measuring.
2. Add solid chemicals to the bottle, using filter paper to measure on the scale and swirl after adding each
   component.

   1. 5 g. of NaCl
   2. 5 g. of Tryptone
   3. 2.5 g. of yeast extract
3. Lightly cover (using cap and/or aluminum foil) and label bottle and autoclave on liquid setting. Make sure
   the cap is not fully screwed on, i.e. enough that the cap stays on, but it can still move up and down and
   loosely put autoclave tape over the lid. Make sure the bottle is in a secondary container. Label bottle and
   set autoclave on liquid setting (called LIQUID 20 on the autoclave). This will take ~50 minutes.

Alternate Procedure:

1. In a 1 L pyrex bottle, add 1 L of dH2O.
2. Add solid chemicals to the bottle, using filter paper to measure on the scale and swirl after adding each
   component.

   1. 25 g of LB broth.
3. Lightly cover (using cap and/or aluminum foil) and label bottle and autoclave on liquid setting. See step 3
   above for more detailed autoclave instructions.

##### **Overnight Culture**

Based on the addgene protocol. Overnight cultures are used to grow a
small quantity of bacteria that can be used for growth assays, plate streaking, toxicity assays, etc.

Materials:

* LB broth
* Tube or Flask
* Tin foil
* Plate with desired bacteria

Procedure:

1. Add liquid LB to a tube or flask (~ 5 mL).
2. Add Ampicillin (1uL : 1mL).
3. Take a sterile pipette tip and lightly select a colony (touch the colony with the pipette tip, try not to
   touch more than one!) from the plate and inoculate the LB.
4. Cover tube/flask with tin foil, making sure it is not airtight. Label overnights clearly!
5. Incubate in a shaking incubator at 37 oC overnight (12-18 hr) at 200 rpm.
6. To store remaining overnight liquid cultures, spin down samples in the falcon tubes at 5000 rpm for 5 mins,
   decant supernatant in waste bottles, and store falcon tubes containing bacterial pellets at -20°C.

##### **Making Competent Cells**

Competent cells are used during transformation to take up new
DNA.

Materials:

* Overnight Culture
* LB Broth
* 100 mM CaCl2

  + Cold
  + Use 0.5549 g. CaCl2 per 50 mL.
* 80% glycerol

Procedure:

1. Create liquid growth culture by mixing 500 uL of overnight culture and 50 mL of LB in a 50 mL flask.
2. Incubate in a shaking incubator at 37 C. Measure OD using a spectrophotometer and a clean cuvette about
   every 20-30 minutes until it reaches about 0.5.

   1. Usually 1.5-2 hours.
   2. Growth is exponential, so be mindful that once you get close, you are very close! Check closer to every
      5-10 minutes.
3. Split growth culture into two 50 mL falcon tubes (Should have about 25mL each in them).
4. Centrifuge cells at 3000 x g for 15 minutes at 4 oC.

   1. Make sure the centrifuge is balanced and the rotor is secure.
5. Pour off supernatant.
6. Resuspend cells in 25 mL of 100 mM CaCl2, this should be done quickly with a 1 mL pipette.

   1. Gently pipette up and down
7. Combine both tubes into one new, clean 50 mL falcon tube and place on ice for 30 minutes.
8. Create a balance tube for the centrifuge.
9. Centrifuge cells at 3000 x g for 15 minutes at 4 oC.
10. Pour off supernatant.
11. Resuspend cells in 400 uL of cold 100 mM CaCl2 and 75 uL of 80% glycerol.
12. Freeze 50 uL aliquots at -80 oC.

##### **Pouring Agar Plates**

Agar plates are the medium on which bacterial colonies are grown.

Materials:

* dH2O
* NaCl
* Tryptone
* Yeast extract
* Agar
* Sterile plates

Procedure:

1. Make LB Broth

   1. In a 1 L pyrex bottle, add 500 mL of dH2O and add solid chemicals (5 g. of NaCl, 5 g. of Tryptone, 2.5
      g. of yeast extract, 7.5 g. of agar) to bottle, using filter paper to measure on the scale and swirling
      after adding each component OR in a 1L pyrex bottle, add 1L of dH2O and add solid chemicals to
      bottle (25 g. Of LB broth base and 37 g. Of agar), using filter paper to measure on the scale and
      swirling after adding each component.
2. LIGHTLY cover (make sure the cap is not fully screwed on, i.e. enough that the cap stays on, but it can
   still move up and down) and loosely put autoclave tape over the lid. Make sure the bottle is in a secondary
   container. Label bottle and set autoclave on liquid setting (called LIQUID 20 on the autoclave). This will
   take ~50 minutes.
3. After autoclaving, to add antibiotics to your LB. Take aliquots of the antibiotic from the freezer to thaw
   and add 1 mL per 1 L of agar made (instructions for how to make the antibiotic stock are below, if needed).
   So 500 mL of LB/agar solution = 500 uL of antibiotic. Add this after.
4. Once the LB container has cooled enough to hold with a heat pad, carefully take empty plates out of the
   sleeve in a stack on the bench.
5. Lift up the entire stack starting on the bottom plate's lid, and pour LB agar until the bottom surface
   of the plate is covered.

   1. You cannot set down the stack in your hand, this is to maintain sterility.
   2. Try to pour all plates evenly and avoid bubbles as much as possible.
6. Let plates solidify in stacks of 4-5 and store in the freezer.
7. Also make sure to rinse the LB container as soon as you are done, because any leftover agar will solidify
   and become very hard to clean out!

**Ampicillin Stock Procedure**:

1. To make stocks, add 100 mg (0.1 g) of ampicillin to 1 mL of dH2O and vortex well. Make 1 mL aliquots and
   store in the freezer. Working stock can be stored in a 4°C fridge. Ampicillin stock can be stored in
   2-8 oC for up to three weeks and can be stored in -20 oC for 4-6 months.
2. Add 1 mL antibiotic stock per liter of agar (or 100 uL to 100 mL of agar) AFTER autoclaving but before
   pouring plates Cool down autoclaved LB-agar in a water bath and cool to 45-50 oC before adding
   ampicillin stock. This is important because ampicillin can be inactivated by heat.

**LB Broth Base Procedure**:

1. Dissolve 15 g of Bacto agar in 1.0 L of LB medium and sterilize by autoclaving.
2. Cool to 50°C in a temperature-controlled water bath.
3. Add 0.50 mL of 100 mg/mL ampicillin. Pour into plates.
4. Note: 3.75 g Agar for 250 LB Broth Base

##### **Protein SDS Gel**

SDS Page Gels are used to visualize protein bands. They can be used to help
identify if an intended protein is in a certain solution or purification. Proteins will be separated on the
basis of molecular weight.

Materials:

* 20 mM TRIS
* 100 mM NaCl
* Lysozyme
* Yeast extract
* Agar
* Sterile plates

Procedure:

1. Keep samples cold!
2. Prepare a gel tank (use the green ones if you made your own gel; use the black ones if using a premade
   gel)

   1. Insert gel slide (facing outward if pre-made, facing inward if you made it)
   2. Fill the tank with running buffer

      1. The 10X running buffer is stored on the shelf above the gel tanks.
      2. Add 100 mL to a large flask and dilute to 1X with ddH 2 O
3. Take a 1.5 mL Eppendorf tube for each of your elution samples.
4. Add 8 L of sample to each tube, followed by 2 L of 6X dye (stored at -20 oC).
5. Heat tubes at 95 oC for 5 min.
6. Spin in a small centrifuge at 12000 rpm for 5 min.
7. Prepare to load gel plate:

   1. Remove bubbles from each of the wells by taking some of the running buffer and pipetting it into each
      well until bubbles are gone.
   2. Get a protein ladder from the -20°C  freezer.
8. Add 1-2 L protein ladder to the far left well, followed by 10 L flow-through sample (well 2).
9. Load 10 L of the remaining samples into the other wells and add another 2 L protein ladder at the end.
10. Put the lid on top of the tank, plug red and black wires into their corresponding outlets.
11. Turn on the machine, set time for 1 hour and power constant 25.
12. At the end of 1 hour, turn off the machine and remove the plate from the tank.
13. Pry plate open and trim gel to the size that you need.
14. Place gel in the bottom of a recycled pipette tip box and pour a thin layer of Coomassie dye over it.
15. Let it rock at room temperature for 1 hour.
16. After 1 hour, pour off Coomassie dye into the appropriate bottle and add a destaining solution.
17. Look for a dark band on gel between 25 and 35 kDa; this is your protein.
18. Let gel rock with a destaining solution for 2 hours.
19. After 2 hours, pour the destaining solution into a waste bottle and pour water over gels.
20. Cover the container and let it rock overnight.

##### **Protein Purification**

This protocol is used to purify proteins from
E. coli bacteria. It works on the basis that many E. coli proteins will precipitate out of
solution at a temperature of above 85C. In this protocol, dialysis was also used for further purification.

Materials:

* 20 mM TRIS
* 100 mM NaCl
* Lysozyme
* Ammonium sulfate
* Dialysis buffer
* PCR tubes

Procedure:

1. Resuspend cell pellet of ⅛ of 1L culture in 5 mL of 100 mM NaCl 20 mM TRIS at pH 7.5.
2. Add 0.1 mg/mL lysozyme and incubate on ice for 30 minutes.
3. Aliquot all 5 mLs equally into 3 eppendorf tubes.
4. Freeze thaw 6 cycles between ethanol in -80 and 37 degree water:
5. Put a plastic beaker of 200mLs of ethanol in the -80 oC freezer.
6. Put another small beaker in the 37 oC incubator.
7. Let both come to temperature.
8. Place 3 epi tubes in the freezer. Make sure to not open the door all the way, just open it a crack and
   slide the tube in and out.
9. Wait until the epi tube liquid is frozen (about 1 minute).
10. Take out and place in a beaker in the 37C incubator. Wait until it thaws completely (2 minutes or so).
11. Repeat 5 more times to lyse cells completely.

5. Spin for 10 min at 14,000 rpm, discard pellet.

   1. To discard pellet, pipette supernatant (liquid) into a fresh tube. Discard the tube with the pellet.
6. Aliquot full volume from eppendorf tubes into 20 pcr tubes and heat to 85 oF for 15 min

   1. Divide volume equally into the 20 pcr tubes
   2. Use the thermocycler at 85 oF for 15 minutes. There should be a setting called
      "purification" preloaded.
7. Spin 10 min at 14,000 rpm, discard pellets

   1. This will require taking the liquid from the pcr tubes and recombining it into eppendorf tubes so they
      fit in the centrifuge.
8. Split the supernatant into 10 tubes, each with 200 μL of solution.
9. Add ammonium sulfate in 10% fractions to each of 10 tubes and incubate on ice for 1 hr, flicking every 5
   min.

   1. This ended up being 0.014-0.14 g ammonium sulfate increasing in intervals of 0.010g.
   2. Add each mass of ammonium sulfate to each of the 10 tubes. Make sure to label appropriately
10. Spin each of these at 14,000 rpm for 10 minutes, collect pellets, resuspend in a lysis buffer (100 mM NaCl
    20 mM TRIS at pH 7.5).

    1. Do not throw out extra solutions. See step 13.

Dialyzed Solutions:

11. Pipette each of the resuspension solutions into dialysis tubing that has been presoaked in the dialysis
    buffer. Make sure to clip well.

    1. Dialysis buffer is 2 L of 150 mM NaCl and 20mM TRIS
    2. We are using one long stretch of dialysis tubing because we only have 12 clips. We are clipping in
       between each 10 samples. Make sure to label the clips with tape and sharpie to keep them apart.
12. Put the filled dialysis tube in the dialysis buffer overnight at 4 oF.

Separate from the Samples that are Being Dialyzed:

13. Concentrate the remaining supernatant from step 10 by running it through the protein concentrator tubes. 5
    PCR tubes worth of protein to 100 uL and add 0.9 uL of MES pH 6 buffer, or whatever amount and pH are
    appropriate to incubate overnight at pH 6. Add TEV to this. Save the concentrator tube to use the next day.
14. Adjust the pH of 5 PCR tubes worth of protein to pH 6 and add TEV.

# Project-Specific Protocols

##### **Encapsulin PCR**

PCR to isolate encapsulin from vectors for use in Gibson Assembly. These
steps need to be performed QUICKLY because as soon as you add the polymerase, the reaction will start.

Materials:

* Reconstitute Dry Primers To 100 uM
* Dilute Primers To 10 uM
* Template DNA
* Q5 Reaction Buffer
* dNTPS
* Thermocycler

Procedure (If Using Pre-Made Master Mix):

1. Get an eppendorf tube that has been autoclaved (in the container covered in foil).
2. Get some ice and keep everything on ice at all times.
3. Set the thermocycler.

   1. See below for details
4. Calculate the concentration of Master Mix needed to reach 50uL final volume.

   1. Will depend on DNA concentration
5. Add forward and reverse primer - 2.5 uL each
6. Add template DNA.

   1. Calculate amount using nanodrop concentration (ng/uL) and total 250 ng
7. Quickly place in the thermocycler and begin.

Procedure (If Making Master-Mix):

1. Get an eppendorf tube that has been autoclaved (in the container covered in foil).
2. Get some ice and keep everything on ice at all times.
3. Set the thermocycler.

   1. See below for details
4. Nuclease-free H2O depends on exact template DNA amount, raising the final volume to 50 uL total.
5. Add 10 uL Q5 reaction buffer
6. Add 1 uL dNTPs.
7. Add forward and reverse primer - 2.5 uL each.
8. Add template DNA.

   1. Calculate amount using nanodrop concentration (ng/uL) and total 250 ng
9. Add 0.5 uL polymerase (only if running all reactions immediately).
10. Quickly place in the thermocycler and begin.

Thermocycler Settings:

1. Initial denaturation.

   1. 98 oC for 30 seconds
2. 25 cycles.

   1. 98 oC for 10 seconds
   2. Tm
   3. 72 oC

##### **Experimental Gibson Assembly**

Use Gibson Assembly to insert AMP + Encapsulin sequence for
expression and cloning. We will be using an NEBuilder HiFi DNA Assembly Kit.

Materials:

* Cloning Vector
* AMP sequence
* Encapsulin sequence
* NEBuilder HiFi DNA Assembly Master Mix
* Deionized water

Calculations:

* Vector

  + Want 50 ng
  + Concentration = 10.49 ng/uL
  + 50/10.49= 4.8 uL vector
* AMP

  + Have 13.05 fmol of vector
  + Want 3x amount of AMP (39.15 fmol)
  + molarity = 15.13 nM (nmol/L)
  + 39.15 fmol/15.13 nM = 2.59 uL AMP
* Encapsulin

  + Have 13.05 fmol of vector
  + Want 3x amount of enc (39.15 fmol)
  + Concentration = 20.5 ng/uL
  + Convert 39.15 fmol to ng = 23.08 ng
  + 23.08 ng / (20.5 ng/uL) = 1.13 uL enc

Procedure:

1. Put everything on ice to start. Begin by adding the vector to a PCR tube. Then add AMP and encapsulin to
   the mix. Then add water and master mix.
2. Incubate at 50ºC for 15 minutes.
3. If not transforming right away, store at -20ºC.

##### **Growth Assay**

To test how the addition of various plasmids impacts the growth of cells
following induction of the lac promoter with IPTG.

Overnights:

1. Prepare eight 5 mL LB overnights, all with identical LB

   1. Uninduced: PET, PET+enc, PET+AMP, PET+enc+AMP
   2. Induced: PET, PET+enc, PET+AMP, PET+enc+AMP
2. Add 50 uL 100 mg/mL ampicillin salt to all of them EXCEPT the PET only
3. Shake overnight at 37 C at 225 rpm in shaking incubator

   1. You need to press "start/stop" to start the heating, you will hear a fan if it is on. Do NOT
      walk away until temperature has reached 37 C

Induction:

4. Innoculate 100 mL of LB+ampicillin with a 1% inoculum (1 mL sample) of the overnight growth.
   Incubate at 37 °C (need to press start to heat incubator if it is not already running, you will hear a
   fan and see the temperature rising on the indicator) while shaking at 220 rpm

   1. In other words, add 1 mL of overnight to 100 mL flask of LB+ampicillin and incubate at 37 °C (need
      to press start to heat incubator) while shaking at 220 rpm
5. Blank spectrophotometer with 1 mL LB.
6. After 1 hour of growth in the incubator at 37 °C at 225 rpm, take OD in spectrophotometer every 30 min,
   until you reach an OD between 0.45-0.55

   1. Use a new pipette for each sample you take.
   2. Use a new cuvette for each of the 8 flasks, but after each sample the contents of the cuvettes can be
      emptied into the liquid waste container and washed out with distilled water.
7. When samples reach an OD of 0.5, add 20 uL IPTG to the 4 induced samples, do NOT add any to the non-induced
   samples.

Making Growth Curves:

8. Shake at 225 rpm at 37 °C.
9. Take a 1 mL sample and measure the OD600 every 30 minutes of each sample

   1. Ensure when taking a sample to swirl beforehand, the cells can settle and alter readings.
10. Continue to shake at 225 rpm at 37 C between each sample.
11. Aim to continue reading for at least 8 hr.

    1. If the sample has an OD of  >1, dilute the volume in the cuvette with LB to ensure accurate
       readings.
    2. Record factor of dilution and be consistent across samples.
    3. For example, dilute a sample by placing 0.25 mL and 0.75 mL LB in a cuvette and then multiplying the
       measurement from the spectrophotometer by 4.

##### **Growth Assay Using CFU**

Colony Forming Unit Assays are used along with a growth assay to
analyze how a toxic protein expressed in bacteria affects bacterial growth. By counting the number of colonies
grown on a plate from a sample of bacteria, the total number of live colonies in a solution can be
determined.

Methods:

1. Create 5 mL overnight cultures in LB containing the appropriate selective agent.

   1. Incubate the liquid cultures at 37°C with shaking for 16 hours.
2. The following day, create small scale growth cultures by adding a 1% (v/v) inoculum of the overnight
   cultures to 100 mL of LB containing the appropriate selective agent.
3. When the liquid cultures reach OD600 = 0.4-0.5 , induce the expression of all

plasmids by adding  20 uL IPTG.

OD600 Growth Curve

1. Immediately upon the addition of IPTG, remove 1 mL aliquots of the growth cultures and check  their
   OD600. These will serve as T0 data points for the growth curves.
2. Continue removing 1mL samples from the growth cultures every half hour (30 min) and checking their OD600.
   For every other sample (on the hour) after the T0 sample, remove 100 uL of the aliquot (in the cuvette) and
   dilute it 1/10  in fresh LB. Multiply the resulting absorbance by the dilution factor to gain a more
   accurate measurement.
3. Aim to continue reading for at least 8 hr.

   1. If the sample has an OD of >1, dilute the volume in the cuvette with LB to ensure accurate
      readings.
   2. Record factor of dilution and be consistent across samples. For example, dilute a sample by placing
      0.25 mL and 0.75 mL LB in a cuvette and then multiplying the measurement from the spectrophotometer by
      4.

\*You will need to either take an additional sample for the CFU assay, or account for this solution when
diluting that.

CFU Growth Curve: Prepare a sterile 96 well plate for each culture in the
following manner:

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Dilution | T0 | T1 | T2 | T3 | Tn |
| 1/102 | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB |
| 1/103 | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB |
| 1/104 | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB |
| 1/105 | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB |
| 1/106 | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB |
| 1/107 | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB | 180 uL Fresh LB |

1. Place 20 uL of each time point aliquot into the first well of the plate and perform a serial dilution,
   removing 20 uL each time, and finishing on a 1/107 dilution.
2. Plate 10 uL of each dilution onto a selective plate, resulting in 6 spots. Perform this action 5 times
    to create an n=5 technical replicate for each time point.
3. After an overnight growth, count the CFU's at the lowest dilution possible and multiply it by the
   dilution factor to gain a CFU/mL measurement for each time point.

##### **Positive Control Gibson Assembly Protocol**

Use Gibson Assembly to insert AMP + Encapsulin
sequence for expression and cloning. We will be using an NEBuilder HiFi DNA Assembly Kit.

Materials:

* Cloning vector
* AMP sequence
* Encapsulin sequence
* NEBuilder HiFi DNA Assembly Master Mix
* Deionized water

Calculations:

Amount of AMP = (100 ng x 1000)/(214 bp x 650 daltons) = 0.7189 pmol

Procedure:

1. Put everything on ice to start. Begin by adding the vector to a PCR tube. Then add AMP to the mix. Then add
   water and master mix.
2. Incubate at 50ºC for 15 minutes.
3. If not transforming right away, store at -20ºC

##### **Purification Protocol for Toxicity Assays**

Toxicity assays are used to assess the effects of a
toxic protein on the bacteria they are toxic to. They can help to elucidate the strength of the toxic peptide
and determine if the potency of the peptide is retained following purification or other processing
methods.

Procedure:

1. Resuspend a cell pellet of encapsulins empty and encapsulins with an AMP in 150 mM NaCl and 20 mM pH 7.5
   TRIS. Use about 5 mL per ⅛ L culture pellet.
2. Incubate the suspension on ice for 30 min with 0.1 mg/mL lysozyme. It should say something like chicken egg
   lysozyme or smthn in the -20C freezer and be at 10 mg/mL stock.
3. At this point you should have 2 50 mL falcon tubes, each with a suspension of cells and lysozyme, and one
   of enc and one of enc + amp. Do 6 freeze thaw cycles between a cup of ethanol in the -80 and 37 degree water
   in that one incubator right there. You might want to think about aliquoting the liquid into smaller tubes so
   they freeze and thaw faster for greater efficiency and speed.

How to Freeze/Thaw:

4. Put a plastic beaker of 200mLs of ethanol in the -80C
5. Put another small beaker of water in the 37°C incubator
6. Let both the freezer and incubator come to temperature
7. Place tubes in the freezer. Make sure to not open the door all the way, just open it a crack and slide the
   tubes in and out
8. Wait until the tubes’ liquid is frozen (about 1 minute)
9. Take out and place tubes in the beaker in the 37C incubator. Wait until it thaws completely (2 minutes or
   so)
10. Repeat 5 more times to lyse cells completely
11. Spin this homogenate at 14,000 rpm in 1.5 mL eppendorf tubes in the centrifuge in the lab for 10 min
12. Collect supernatants without disturbing pellets. Discard pellets. Collect 8 uL samples for gel later.
13. Heat water to 85 degrees in a large (600mL H20) beaker. Incubate clear lysate in this bath for 15 min.
14. Spin this solution, which should have gone from clear to cloudy, at 14,000 rpm in 1.5 mL eppendorf tubes in
    the centrifuge in the lab for 10 min. It will be clear again now. Collect 8 uL samples for gel later.
15. Collect supernatants without disturbing pellets. Discard pellets.
16. Collect each of the two proteins in its own 50 mL falcon tube so that all of the protein of each kind is in
    a single tube. Add glycerol to a final concentration of 10% for each and mix thoroughly but gently. DO NOT
    VORTEX PROTEIN.
17. Freeze the protein in 1.5 mL aliquots in -80, labeled appropriately.
18. If you have time, run a protein gel of the final protein samples, and the intermediate samples you
    collected earlier.

Gel Instructions:

19. Wear gloves at all times
20. Get a protein gel out of the box in the fridge. Use the ones from last year, not the super old ones we just
    got.
21. Remove from packaging, remove plastic white tape strip from back.
22. Make 1X solution of SDS Page running buffer from deionized water from sink and 10X SDS PAGE running buffer
    from the box dispenser.
23. Put gel in the gel rig, click it in with the frame. Fill to fill line with running buffer.
24. Gently remove comb from the top of the gel
25. Mix 8 uL of each protein sample with 2 uL of the loading dye from the freezer. It is in a screw top tube
    and says something like 4X protein loading dye on the side in marker. Should smell like sulfur and be
    yellowish. Do this in PCR tubes.
26. Heat protein and load dye samples to 95 degrees for 5 min in the PCR machine. The samples should be blue
    when you take them out.
27. Spin all the samples for a min in the PCR tube mini centrifuge.
28. Load 10 uL of Invitrogen prestained protein ladder in the first well of the gel. This is just like loading
    a DNA gel except vertical
29. Load 10 uL of each protein sample in the subsequent lanes. Label samples.
30. Attach lid and supply 35 mA constant current from the power supply until the ladder and loading dye are
    ¾ - ⅞ the way down the gel.
31. Remove the gel from the rig, and use a knife or screwdriver to pop each of the little plastic rivets
    holding the front plastic piece of the gel to the back one, starting in a corner and doing 1 at a time.
32. Trim the bottom of the gel off, below where the protein is. There is a little groove in the back plastic
    piece that the gel gets stuck in, and removing this part of the gel will prevent it from tearing when you
    lift it off.
33. Gently crop gel to appropriate size so we can see the protein good after it is stained.
34. Place in the lid of a tips box and cover with coomassie blue. Put in the incubator at 50 rpm and room
    temperature for 30-45 min, or until it is hard to distinguish the ladder from the blue background.
35. Pour coomassie back into the tube it came from.
36. Cover gel in destain and return to the incubator at the same settings for an hour and a half.
37. Pour destain into the big hazardous waste jug, and cover gel in 40% destain solution and 60% water. Cover
    the gel so the liquid cannot evaporate away, and return to the incubator at the same settings overnight.
38. Image gel the next day

##### **Restriction Enzyme Double Digest (Nde1, Pac1)**

Protocol for cutting plasmid at two sites
simultaneously with restriction enzymes NdeI and PacI.

Materials:

1. DNA (1 μg)
2. 10X rCutSmart Buffer (5 μL)
3. NdeI (1.0 µl)(20 units)
4. Pacl (1.0 µl)(10 units)
5. Nuclease-free Water (Bring to 50 μL)

Procedure:

1. Set up a reaction by combining the DNA, rCutSmart Buffer, NdeI, PacI, water for 50 µl total volume.

   1. For convenience, 1.0 µl is specified; adjust as needed. In general, we recommend 5–10 units
      of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest. Enzyme volume
      should not exceed 10% of the total reaction volume to prevent[https://www.neb.com/tools-and-resources/usage-guidelines/star-activity](%3Ca%20data-fr-linked%3D)"
      style="text-decoration: none;"> star activity due to excess glycerol.
2. Incubate at 37°C for 5-15 minutes as both enzymes are Time-Saver qualified.

   1. DNA purified by standard miniprep procedures is cleaved at lower rates by NdeI. In this case,
      increasing incubation time to at least 1 hr may be necessary.
3. Heat inactivate. For heat-inactivation recommendations please refer to[https://www.neb.com/tools-and-resources/usage-guidelines/heat-inactivation](%3Ca%20data-fr-linked%3D)"
   style="text-decoration: none;"> this chart. Heat inactivation is recommended if you plan
   to continue to another step in the workflow without a DNA purification step.

##### **TEV Protease Digestion**

Cleave the linker and targeting peptide from the AMP after the
encapsulins have been destroyed.

Materials:

1. Substrate (15 μg)
2. H20 (Bring Substrate volume to 45 μL)
3. TEV Protease Reaction Buffer-10X (5 μL)
4. TEV Protease

Procedure:

1. Combine 15 μg of substrate and H2O (if necessary) to make a 45 μl total reaction volume.
2. Add 5 μl of TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.
3. Add 1 μl of TEV Protease.
4. Incubate at 30°C for 1 hour or at 4°C overnight.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# Results

---

##### CONTENTS

* [Cloning](#cloning)
* [Growth Assay](#growth_assay)
* [Protein Purification](#purification)
* [References](#ref)

Results from our project are separated into three distinct phases: cloning our plasmid constructs, evaluating
those constructs via a growth assay, and purifying our protein expression products.

# Cloning

Four plasmid constructs were designed to investigate encapsulin facilitated expression of antimicrobial
peptides (AMPs): an empty pETDuet vector (vehicle control); pET expressing a single T4GALA encapsulin monomer
(negative control); pETDuet expressing the AMP HBCM2 fused to a Tobacco Etch Virus (TEV) protease site, flexible
linker, and T4GALA targeting peptide (positive control); and pETDuet expressing HBCM2, TEV protease site,
linker, targeting peptide, and a T4GALA encapsulin monomer (experiment) (Figure 1A).

![](https://static.igem.wiki/teams/4143/wiki/results/results-1.png)

Figure 1. A) Diagram of our three protein-expressing constructs. B) Agarose gel showing PCR
amplification of the T4GALA encapsulin from its original plasmid. Lane 1: ladder, lane 3: pET vector with
T4GALA, lane 5: PCR-amplified T4GALA. C) Agarose gel for extraction of double restriction digest of pET plasmid
for linearization. From left: ladder, digested pET.

The T4GALA encapsulin construct and empty pETDuet vector containing regulatory elements and ampicillin
resistance was generously donated to our team by the Geissen Lab. Our HBCM2, TEV site, linker, and targeting
peptide insert with overhangs for Gibson assembly were ordered from Integrated DNA Technologies (IDT). PCR was
used to extract the T4GALA monomer from our donated plasmid (Figure 1B). A double restriction digest and gel
extraction were used to linearize our pET vector (Figure 1C). Gibson assembly reactions were performed to create
our pET + AMP and pET + AMP + encapsulin constructs. Successful assembly was confirmed by Sanger sequencing
(Figure 2), and all four plasmids were transformed into the same strain of BL21 *E. coli*.

Interestingly, we noted several mismatches between our AMP insert and its Sanger sequencing (Figure 2A).
Sequencing proved challenging due to nonspecific primer binding, and after trying multiple sequencing primers
early in the season, we attributed these mismatches to technical issues and proceeded with experimentation. In
the context of our later results, however, we acknowledge the possibility that there was a potentially
problematic mismatch with the sequence of our insert that could have affected the potency of the AMP.

![](https://static.igem.wiki/teams/4143/wiki/results/results-2.png)

Figure 2. A) BLAST comparison of sequencing data (Query) to expected result (Sbjct). B) A
representative chromatogram segment from our sequencing.

# Growth Assay

We reasoned that if the encapsulin was mitigating the toxicity of the AMP during expression, the growth rate of
bacteria expressing the AMP + encapsulin would be higher than the growth rate of bacteria only expressing the
AMP alone. To test this, we designed an assay in which we inoculated wells of Luria-Bertani (LB) broth
containing 100 μM ampicillin with each of our four bacterial strains. Each strain featured three biological and
three technical replicates. Protein expression was induced in half of the samples with 100 μM IPTG at time=0.
OD600 measurements were taken by a plate reader every 5 minutes for 12 hours. Logistic regressions were
performed on the growth phase of each well, and the growth rate was recorded for each. To compare these growth
rates, t-tests were performed with considerations made for the variance due to our biological and technical
replicates [1].

![](https://static.igem.wiki/teams/4143/wiki/results/results-3.png)

Figure 3: Growth assay average growth rates for 4 experimental groups: empty pET vector,
encapsulin only, AMP only, and encapsulin + AMP. Error bars represent 95% confidence intervals. A) Growth rates
for uninduced gene expression B) Growth rates for induced gene expression via IPTG.

As seen in Figure 3B, bacteria expressing our AMP and encapsulin had a higher average growth rate than bacteria
expressing the AMP without encapsulin, suggesting the mitigating effects of the encapsulin on AMP toxicity.
Although this trend was not statistically significant in our small-scale experiment, it was observed in spite of
the added metabolic burden of encapsulin expression. This suggests that even if the encapsulin failed to
increase the growth rate of the AMP + encapsulin bacteria relative to those expressing just AMP, the encapsulin
may still have mitigated toxicity and potentially improved protein yield. Further experiments are warranted to
investigate this effect, such as increasing the number of replicates or the amount of protein expressed by each
cell. Increased expression could be achieved by growing the bacteria in a richer media like terrific broth (TB)
media or waiting to induce with IPTG at a higher OD600 following initial bacterial growth.

In addition to the previously noted sequencing data, a possible explanation for the smaller-than-expected
effect of AMP toxicity on bacterial growth is the fusion of the targeting peptide and linker to the AMP-only
positive control. These domains (targeting peptide + linker) were added so they better matched the experimental
construct that also had these added to facilitate encapsulation. Additionally, previous experiments suggested
adding these domains did not affect the AMP’s toxicity [2].

# Protein Purification

In parallel with the growth assay experiments, we explored the use of our encapsulin system to express and
purify HBCM2. BL21 *E. coli* were transformed with either a pETDuet + encapsulin plasmid or a
pETDuet + encapsulin + AMP plasmid. These transformants were then grown at 37 degrees Celsius and shook at 250 rpm
in LB broth with 100 uM ampicillin. At an OD600 of 0.6, the bacteria were induced with 200 μM IPTG to promote
protein expression. The proteins were expressed for 4 hours at 37 degrees C while shaking at 250 rpm. Harvested
*E. coli* were then lysed and centrifuged to produce a clear lysate. The thermostability of our encapsulin
allowed us to then heat the clear lysate to 85 degrees C, inducing precipitation of native *E. coli*
proteins. The efficacy of these techniques was confirmed with a protein gel (Figure 4).

![](https://static.igem.wiki/teams/4143/wiki/results/results-4.png)

Figure 4: SDS-PAGE gel showing heat protein purification. Note lanes 4, 6, and 7 showing the
increasing purity of samples heated to 25, 65, and 85 degrees, respectively. From left: ladder, clear lysate
using dilute lysozyme, clear lysate using moderate lysozyme, clear lysate using high lysozyme, clear lysate with
higher concentration of cells per unit lysis buffer, clear lysate heated to 65º C, clear lysate heated to 85º C.

While this process produced a relatively pure protein sample, we investigated the use of ammonium sulfate
precipitation for further purification. Samples of heat-purified protein were incubated at 4 degrees C for 1
hour with ammonium sulfate at concentrations ranging from 10% to 100% saturation. After resuspension of
collected pellets of precipitated protein and subsequent dialysis, a protein gel showed the ability of this
process to increase the purity of our protein samples (Figure 5). We found that low concentrations of ammonium
sulfate were able to selectively precipitate our encapsulin-AMP complex, as seen by the distinct T4GALA Monomer
and AMP fusion bands at a 10% ammonium sulfate concentration.

![](https://static.igem.wiki/teams/4143/wiki/results/results-5.png)

Figure 5. SDS-PAGE gel demonstrating ammonium sulfate purification. From left: lanes show
protein pellets collected after incubating with ammonium sulfate at concentrations ranging from 10% to 100% in
10% intervals. Note the high purity of the lower concentration bands.

Next, our team began working to open the purified encapsulins, separate them from our AMP (HBCM2), and use our
TEV site to cleave the AMP from the targeting peptide. Efforts in this area are ongoing, but we are optimistic
that this can be accomplished with accessible techniques and high efficiency by first introducing the
encapsulins to a mildly acidic buffer and then exploiting the drastic differences in size and charge between the
encapsulin and AMP for definitive separation.

Finally, we designed plasmids for experiments that explore the addition of multiple AMPs to each targeting
peptide for a maximized yield of AMP from each encapsulin. Each encapsulin produced represents a fixed energetic
cost and a fixed number of encapsulated targeting peptides, regardless of the number of AMPs attached to each
targeting peptide. We hypothesize that, to a threshold, there will not be a significant increase in toxicity
from adding additional AMPs to each targeting peptide. Our computational modeling team estimated the maximum
number of AMPs that could be chained together per targeting peptide, and this will inform the length of the
chains we plan to test. More information on the completed modeling that continues to guide our design can be
seen on the model page.

# References

1. Blainey, P., Krzywinski, M. & Altman, N. Replication. Nat Methods 11, 879–880 (2014).
   https://doi.org/10.1038/nmeth.3091
2. Lee, T. H., Carpenter, T. S., D'haeseleer, P., Savage, D. F., & Yung, M. C. (2020). Encapsulin carrier
   proteins for enhanced expression of antimicrobial peptides. Biotechnology and bioengineering, 117(3), 603–613.
   https://doi.org/10.1002/bit.27222

{% endblock %}