iGEM 2021 — Team CCA_San_Diego

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# Team:CCA San Diego/Notebook

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Our team mainly worked in the lab during the fall of 2021, with only four members physically conducting wet lab research at the J. Craig Venter Institute. We included a timeline of brief descriptions of what we did each week in the lab, as well as a more detailed lab notebook with our daily procedures, pictures, and results.

### July 25 - July 31: Linearizing our Modified Backbone Fragment

We ran PCR on our pRSII16 plasmid to linearize our desired backbone
fragment free of the lacZalpha gene. The linearized plasmid was
tested in a gel alongside a ladder and the original backbone
fragment to verify if the amplification was successful. The
following day, three 50 uL PCR aliquots were prepared with
backbones, resulting in three more linearized backbone solutions.
The three solutions were also tested on the gel electrophoresis bed
and showed successful PCRs. We performed a DpnI digest on each of
the three PCR aliquots and the initial one from the previous day to
destroy any circular backbone plasmids.

### August 1 - August 7: Collagen Gelification

Our goal for this week was to make a collagen gel out of the
collagen powder we purchased. This would then serve as a proof of
concept for our end goal: making bacterial collagen products.
Initially, our collagen powder was 2% w/v, the molarities of our
coagulation buffer were most likely inaccurate, and we also
neglected prior incubation of the buffer to 37°C. These factors may
have led to the failed coagulation when we put the acidic collagen
solution in the neutral coagulation buffer. Surprisingly, the
collagen did not congeal even after incubating it with 5 mL of
5.33%, 2%, and 1% glutaraldehyde solutions for 72 hours at 37oC.
Glutaraldehyde is one of the strongest cross-linkers used in the
literature, which led us to suspect the problem was with the
collagen powder itself. For the rest of the week, we attempted to
troubleshoot our previous experiments with different procedures and
buffers - instead of using a fiber formation buffer from research
gate, we used a PEG coagulation buffer from Cavallaro et. al, \_\_\_.
Even so, the collagen did not coagulate. These results reinforced
the notion that the collagen powder itself was the culprit. To test
this, we obtained two 2.4 mg of bovine cartilage containing
approximately 1.44 mg of Type I collagen. Since it was unable to
dissolve in the acetic acid solution after intense vortexing, we let
it dissolve in acetic acid for 72 hours. Additionally, we tested
three different high-concentration solutions of our collagen powder
so we could simultaneously test the variable of concentration.

### August 8 - August 15: Porcine Collagen Gelification

For this week, we continued our attempt at making gel. Due to
failures from previous tests of concentration and source failure (a
result of the impure collagen powder), we decided to shift to a new
procedure: porcine collagen gelification for the purpose of deriving
pure collagen. We used pig skin from a local butchery, using a
porcine extraction procedure to obtain collagen. Since the powder
that we had previously used in our gels had not worked, we hoped
this would. Another important component in our collagen gelification
attempts was determining the correct amount of crosslinking reagent
to use. We conducted gelification trials with varying glutaraldehyde
concentrations and various buffers: water, PEG coagulation buffer,
and fiber formation buffer. We placed agitating collagen samples in
petri dishes of the 3 buffers, and the collagen samples in the PEG
coagulation buffer ended up the least dispersed and the most
coagulated. This made PEG the best buffer for our purposes. We tried
a different gel procedure that utilised pepsin, which was also
unsuccessful. Therefore, we believe that the collagen powder and
porcine collagen are at fault. When our gelification attempts failed
for the porcine collagen, we ran an SDS page to verify the purity
and presence of collagen proteins from our porcine collagen
extraction. Our SDS page results do not correspond to the expected
results. The collagen powder wells had the thickest bands, with
double bands around 120kDa — the telopeptide-positive collagen had a
semi-faint band around 120kDa. Meanwhile, we began preparations for
next week's procedures. We intend to run Gibson assembly and yeast
assembly in parallel for the coming week. We successfully made
numerous E.coli-based agar plates and yeast-based agar plates. This
will be necessary for the procedures next week by providing means of
growing URA3 mutant strain yeast and E.coli.

### August 16 - August 21: 2nd Iteration of Porcine Gelification

Our initially planned Gibson and yeast assemblies were delayed
because the DNA shipment was cancelled. We maintained our focus and
approached collagen gelification through a new procedure. The new
method involved a chemical separation of pig skin mass through
numerous acid baths. When purifying the collagen protein, we
introduced samples with pepsin to compare the results of
telopeptide-rich and -poor proteins. After pelleting cycles were
completed, the collagen samples appeared to have salted out proteins
as a precipitate. To confirm this, we ran an SDS page on
telopeptide-rich, telopeptide-poor, and pure porcine samples, as
well as our old hydrolyzed collagen and bovine collagen. The results
were troubling, as they not only did not indicate any significant
trend, but also were inconsistent with our previous SDS pages on
bovine and hydrolyzed collagen. This suggests a problem with the SDS
page itself, so we will continue with the gelification procedure.

### August 23 - August 27: DNA

This week, we worked with our newly arrived DNA. On August 23rd, we
amplified the necessary primers needed. Furthermore, we worked on
back-diluting the yeast in order to prepare it. After our DNA
arrived, we amplified them via PCR and ran both Gibson and yeast
assemblies in parallel in order to create our plasmid. After running
the Gibson and the yeast assembly, we conducted an E. Coli
transformation for self-closure and ran another PCR amplification.
Afterwards, we noticed that our colonies were not growing after two
days, and this phenomenon only occurred in the Amp (-) plates. We
opted for a new yeast and Gibson assembly. These were incomplete and
will be completed on August 30.

### August 30 - September 4: DNA

After finishing both the yeast and Gibson assembly, we ran an SDS
page to see the results of the collagen gels and the bacterial
transformation. There were no bands seen for the E. coli, but there
were prominent bands visible for the collagen gels. The next day it
was determined that the E. coli from the Gibson grew colonies, and a
mini prep procedure was performed. We experimented with a new
procedure for the gels, which showed successful gelification within
30 minutes of execution. However, it did not maintain a gel-like
form for a 24 hour period. We will conduct further experimentation
with the cosolvent ratios and the collagen ratios to find the
optimal ratio for the gels. September 7 - September 12:
Troubleshooting and Collagen Gelification Experimentation We
performed a Gibson assembly transformation and implemented PCR to
amplify our gene fragments (for future use in Gibson and yeast
assemblies). To troubleshoot what may have went wrong in last week's
Gibson and yeast assemblies (the gel we did after the miniprep from
last week showed no bands), we conducted E. coli transformation on
the following: We used our PRS backbone as a positive control, last
week's Gibson assembly products and no DNA as a negative control,
and cells from last week's miniprep on a carbenicillin plate.
Colonies grew on the carbenicillin plate, so we prepared for the
miniprep by growing out cells in LB medium and amp. We then did
miniprep to purify our plasmid. We further experimented on the new
procedure for collagen gels involving a citrate buffer by changing
the ratios of glycerol to collagen protein. Changes in collagen
protein concentration showed a negligible change. Glycerol
concentration proportionally increased viscosity of the sample,
which, however, showed no indications of coagulation.

### September 7 - September 12: Troubleshooting and Collagen Gelification Experimentation

We performed a Gibson assembly transformation and implemented PCR to
amplify our gene fragments (for future use in Gibson and yeast
assemblies). To troubleshoot what may have went wrong in last week's
Gibson and yeast assemblies (the gel we did after the miniprep from
last week showed no bands), we conducted E. coli transformation on
the following: We used our PRS backbone as a positive control, last
week's Gibson assembly products and no DNA as a negative control,
and cells from last week's miniprep on a carbenicillin plate.
Colonies grew on the carbenicillin plate, so we prepared for the
miniprep by growing out cells in LB medium and amp. We then did
miniprep to purify our plasmid. We further experimented on the new
procedure for collagen gels involving a citrate buffer by changing
the ratios of glycerol to collagen protein. Changes in collagen
protein concentration showed a negligible change. Glycerol
concentration proportionally increased viscosity of the sample,
which, however, showed no indications of coagulation.

### September 13 - September 18: Yeast Assembly and Collagen Gelification with Neutral pH

The yeast grew on their plates, but were concentrated into clumps
with dead cells. As a result, we had to do a patch on fresh plates
to ensure the yeast could grow. However, the negative control
without any DNA also grew on the URA- plates, indicating that either
our plates or the yeast cells had a problem with them. After we did
the patch, we noticed similar results, despite purifying protein
lysates and DNA from the yeast. Running an SDS-PAGE on the DNA
showed nuclear bands, likely due to a low concentration or too much
chromosomal DNA. Thus, we did another yeast assembly with just the
formed plasmid from Gibson assembly, but no colonies grew on our
plates. As we got Sanger sequencing results showing faulty bp
alignment, this may indicate that our initial Gibson assembly did
not work. For collagen gelification, we took a step back in our
approach and revised our previous PBS mix gel. We tested
specifically for neutral pH's effect via NaOH balancing and received
promising results; the gels coagulated instantly in thicker, more
condensed blocks. The masses were more opaque and had a distinctive
white color. Few tiny particles broke off until the gel had been in
solution for an extended period of time. We concluded that changing
the pH to neutral was the solution for strong gelification but pH
testing on the citrate gels showed no response.

### September 20 - September 26: DNA and Implementations

DNA: This week we made new plates for our yeast assembly with
various amino acids, excluding uracil, yeast nitrogen base, agar,
and dextrose. We made new plates because yeast colonies were growing
on our URA3(-) plates, which shouldn't have been happening,
therefore we concluded that our plates were old and contaminated
with uracil. We did a Gibson assembly with both 2 fragments and 3
fragments and transformation again, and yeast assembly
transformation. After transformation, we grew yeast cells to do a
miniprep to purify our plasmid. We did PCR amplification of our gene
fragments and purified our gene fragments to do Gibson assembly
again. Small colonies grew on the plates with the yeast colonies,
but we waited an extra day for more growth of colonies to do a yeast
DNA extraction to do a gel visualization. Lastly, we ordered primers
to verify the yeast assembly. Implementation: Our best results were
from the CP-C vials with pH. However, they all lost the majority of
the coagulation after being saturated by water for an extended
period of time. Our rationale was that removing the supernatant
would prevent this degradation of coagulation. We centrifuged and
discarded the supernatant, leaving behind pancakes of pure white
collagen.

### September 27 - October 3: Gibson and Yeast Assemblies

As the last results of the Gibson Assembly were disappointing, we
decided to redo a Gibson and a Yeast transformation using fresh
stocks of reagents and the two-fragment and three-fragment assembly,
which had the highest potential for successful transformation. Upon
verification with PCR, our two-fragment Gibson Assembly was the only
fragment that contained the correct length of overlap DNA, and thus
was used in another Yeast Assembly and sent for sequencing.

### October 4 - October 8: PCR and Yeast Analysis

We first started by checking our sequencing results from our Gibson
assembly and picking colonies from the yeast plates to run PCR on
overhangs to verify if the transformation worked. As the E. coli we
believed that the assembly was successful, however the
transformation was not successful. We used genomic yeast ethanol
precipitation to extract the DNA for yeast assembly and LiOAc
transformation. We performed PCR on \_\_\_\_\_, then prepared for a yeast
miniprep and performed a gel using the pcr from the previous day. We
extracted yeast cells from the plates that grew for 2 days following
the transformation to do a yeast miniprep with in order to do a gel.
Following our gel, we did an SDS page. We did a protein
precipitation with zymolyase to break the yeast cell walls to do the
SDS page. We streaked an E. coli plate to sequence with GIB for
plasmid sequencing.

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[5](#s5)
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[8](#s8)
[9](#s9)
[10](#s10)
[11](#s11)

Lab Notebook

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# Team:CCA San Diego/Engineering

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# Team:CCA San Diego/Experiments

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With the limited space and time available to us because of COVID-19, we had just over two months to perform our experiments. Still, we developed procedures from various sources to make Collagene through all steps of our Project Design. Here, you can find them categorized into protocols for expression (of the protein), implementation (gels and threads), and general procedures for the wet lab.

# Implementation

## Gel Procedure

### Procedure

1. Powdered ECM samples were solubilized with 1.0 mg/ml pepsin in
   0.01 M HCl at a concentration of 10 mg ECM/ml
2. Stirred at room temperature for 48 h to form a pre-gel
   solution
3. To pH 7.4 with 0.1 N NaOH, isotonically balanced with 10x
   PBS(pH ~ 1.5–2.5)
4. Diluted with 1x PBS to the final concentration of 8 mg/ml,
   which allows in vivo gelation
5. Kept at 37 °C for ~120 min together with either genipin
6. Keep genipin in 50% DMSO for genipin-crosslinked hydrogel
   (ECM/G) or with EDC (Sigma)

## Porcine Skin Collagen Extraction

### Materials

* 100% Acetone (500mL)
* NaCl (40g)
* Deionized Water
* Porcine Skin Sample
* Citrate Buffer (800mL)

+ 8g Sodium Citrate
+ 800mL Deionized Water

* Pepsin

### Procedure

## Preparation of Porcine Skin

1. Thoroughly wash a skin sample obtained from a deceseased pig.
2. Use a razor or knife to slice off any muscle or fat layers
   beneath the dermis of the pig.
3. Scrape both sides of the remaining skin layer to remove hair
   and subcutaneous fat.
4. Cut the skin into tiny pieces (a blender can be used as well).
5. Prepare a 100% acetone bath in a sealable container. Put the
   skin pieces into the acetone bath, seal it, and store in -20°C
   freezer for 30 minutes.
6. Let the sealed acetone bath rest at room temperature for 1
   hour.
7. Remove the porcine skin from the container and rinse with
   deionized water.
8. Prepare a 10% NaCl bath in a sealable container. Put the skin
   pieces into the NaCl bath, seal it, and store in a 4°C
   refrigerator for 24 hours.
9. Remove the porcine skin from the container and rinse with
   deionized water.
10. Make the sodium citrate buffer with 8g sodium citrate and
    800mL distilled water. Make adjustments to reach pH 4.3.
11. Separate porcine skin into separate 50mL tubes with 5g skin
    per tube. Pour 10mL citrate buffer into each tube. Vortex the
    solution lightly.
12. Store the tubes in a 4°C refrigerator for 48 hours.

## Pepsin Digestion

13. Each tube was filled to 30mL acetic acid and half received
    600mg Pepsin too.
14. Centrifuge the tubes at 3500rpm for 6 minutes.
15. Prepare 20mL of 1.5M NaCl solution in deionized H2O.

## Tensile Strength Testing

### Materials

* Constant rate of Extension Tensile testing instrument with the
  following:
* Load cell with a maximum of 1,000N capacity
* 50mm/minute constant rate of extension
* Distance between upper and lower sets of grips is 100 mm
* Pneumatic Rubber faced grips (Rear 75 x 25mm and 25 x 25mm
  front)
* For PC operated machines, appropriate software (to BS EN ISO
  13934 Part 2;1999 )
* Printer capability
* Calibrated metal ruler

### Procedure

1. Cut 3 rectangular specimens parallel to the warp direction and
   3 rectangular specimens parallel to the weft direction.
2. Each Specimen should measure 150mm in length and 100mm in
   width
3. Place a direction line parallel to the warp on each specimen
   prior to removing from the fabric
4. Condition the specimens for a minimum of 4 hours in a
   conditioned atmosphere of 20 ± 2°C and 65 ± 2% RH before
   testing.
5. Using an Automatic Constant Rate of Extension Tensile Tester

1. The programme is automatic. The test speed is 50mm per
   minute.
2. Use the 75mm x 25mm rubber faced grip on the rear and 25mm x
   25mmm grip on the front.
3. With the correct grips in place set the distance between
   upper and lower sets of grips to 100mm, this should be
   checked using a calibrated metal ruler.
4. Select the correct programme and input the appropriate
   machine settings and fabric details etc.
5. Fix the test specimen centrally in the upper grips, so that
   the 38mm line drawn on the specimen coincides with one edge
   of the grips.
6. Close the upper grips.
7. Avoiding any pretension, adjust the specimen along the guide
   line into the lower grips.
8. Close the lower grips
9. Set the cross head in motion.
10. The maximum force at which the fabric ruptures (breaks) will
    be recorded.
11. Repeat the above procedure for the remaining specimens.

6. Any break which occurs within 5mm of the clamping line of the
   grips should be reported as a grip break
7. If the grip break falls below the lowest normal break results
   then it should be disregarded and further samples tested
8. If it falls above the lowest normal break result, then it can
   be included

# Expression

## Modified TCA/Acetone Protein Purification

### Materials

* Transformed Yeast Cells
* Eppendorf tubes
* Acetone

+ 80% acetone/li>
+ 20% TCA/Acetone
+ -20oC Acetone
+ (Supplement with 5 mM DTT)

* SDS Extraction Solution

+ 1% SDS
+ 0.1 M Tris-HCl (pH 6.8)
+ 2 mM EDTA-Na2
+ 20 mM DTT
+ 2 mM PMSF (add before use)

* All organic solvents pre-chilled at -20oC and contained 5 mM
  DTT added before use.

### Procedure

1. Prepare Crude Extract

* (Note: 0.1-0.5 g sample in 1.0-1.5 mL buffer of choice). Homogenize in a SDS extraction buffer (in a mortar on ice)
* Transfer the extract into eppendorf tubes
* Centrifuge (15000g, 5 min, 4 C)
* Pipette the supernatant to new tubes

2. Supernatant

* Add 20% TCA/acetone (1:1), fully vortex, and place on ice for 5 min
* Centrifuge (15000g, 3 min, 4 C) and discard supernatant

3. Protein Precipitate

* Briefly wash with acetone and centrifuge (15000g, 2 min, 4 C). Discard supernatant.
* Repeat Steps 8-9 at least one more time using 80% acetone
* Air-dry proteins for 3 minutes (AVOID OVER-DRYING).
* Dissolve proteins in buffer of choice.

4. Protein Determination

* Keep samples for SDS-PAGE or treat with proteolysis to remove globular domain.

## Acid Precipitation and Proteolysis

### Materials

* Lysis Buffer

+ 50 mM Tris-HCl pH 8.0
+ 1% DMSO
+ 50-200 mM NaCl
+ 1 mM EDTA
+ 1 mM PMSF
+ 1 ug/ml leupuptin
+ 1 ug/ml pepstatin A

* Equipment

+ Vortex or Bead Beater
+ Glass Beads (500 uM)

* Protease inhibitor solution

+ (Always prepare solution fresh just before using. Place on ice to prechill).
+ Pepstatin A
+ Leupeptin
+ Benzamidine
+ Aprotinin

* PMSF Stock solution (Appendix D.A; Add as necessary throughout the protocol.)

+ (Always prepare solution fresh just before using. Place on ice to prechill).
+ Dissolve 0.1742 g PMSF in 10 mL isopropanol. Wrap tube in foil and store at RT. PMSF primarily inhibits serine proteases. This is a 100x stock solution. PMSF is hazardous.

### Procedure

## Precipitation

1. Prepare the glass beads by washing them in concentrated HCl, followed by extensive rinsing (check that then pH is neutral) and drying. The dried beads should be chilled before use.
2. Resuspend cells in equal amount of chilled lysis buffer and place suspension in sturdy non-glass tube. Add PMSF (10 uL/ml of cell suspension) at this point
3. Add 1-3 g of chilled glass beads/gram of cell wet weight.
4. Vortex 3-5 times for 1 minute, keep cells on ice for 1 minute between vortexes. Use highest setting.
5. Remove glass beads.
6. Remove cell debris by ultracentrifugation at 4oC for 30 min at 45000 rpm using a 45 Ti rotor (Beckman).

## Pepsin Digestion

7. 1 g of cell paste resuspended in 20 mL of 50 mM acetate buffer at pH 2, 3, and 4.
8. Cells ruptured by sonication.
9. Lysate clarified by centrifugation (15,000 x g for 30 min, 4 C).
10. Held at 4 C for 16 h and any precipitate formed removed by centrifugation.
11. Treat supernatant with proteolysis (.01 mg/ml pepsin was added and mixture incubated for 16 h at 4 C).

## DpnI Digest

### Materials

* Dpn1 Enzyme (from -20 C: keep on ice)
* PCR Product
* 37 C Incubator

### Procedure

1. Add 2 uL of the enzyme to the completed PCR reaction. (1 uL if 20uL, 2 uL if 50 uL).
2. Use P20 set set to -5uL, and pipette up and down 7 times. (P20 to 10uL if the reaction is 20uL, P200 to 25uL if the reaction is 50uL).
3. Incubate at 37 degrees Celsius for 1-3 hours.

## E. coli transformation for self-closure.

### Materials

* Wide Orifice Tips
* "Hockey Stick" Inoculation Loops
* SOC
* High-Efficiency NEB5 Alpha Cells (stored at -80°C)

### Procedure

1. Put ice in an ice bucket.
2. Transfer a tube of E. coli competent cells (high-efficiency NEB5 alpha cells) from -80°C to ice.
3. Label a PCR strip and place it on ice for pre-cooling the PCR strip. Close the lid of the parts to be used.
4. Place the DNA sample used for transformation on ice.
5. Keep the tube of competent cells, the empty PCR strip, and the DNA sample on ice for 5 minutes.
6. Using a wide-orifice tip (to be gentle to the cells) and a pipette set to 10 ul, pipette the cells up and down 5 times to make the suspension even.
7. Add 2 ul of the E. coli cell suspension to a tube within the PCR strip.
8. Using an inoculation needle, pin-transfer the DNA sample into the cell suspension, Tap the mixture inside the tube using the inoculation needle 10 times to mix.
9. Keep the PCR strip on ice for 30 minutes.
10. Return the tube of competent cells to -80°C.
11. Use a thermocycler and set the temperature of the heat block to 42°C.

## Gibson Assembly

### Materials

* Dpn1 Enzyme (from -20 C: keep on ice)
* PCR Product
* 37 C Incubatorn

### Procedure

1. Add 2 uL of the enzyme to the completed PCR reaction. (1 uL if 20uL, 2 uL if 50 uL).
2. Use P20 set set to -5uL, and pipette up and down 7 times. (P20 to 10uL if the reaction is 20uL, P200 to 25uL if the reaction is 50uL).
3. Incubate at 37 degrees Celsius for 1-3 hours. prior to removing from the fabric

## His-Tag Purification

### Materials

* 1L Column (alternatively scale down to a syringe with small cotton bed)
* Cell Lysate
* 50% Ni-NTA Agarose Beads Slurry (50mL)
* His-Wash Buffer (750mL)

+ 50 mM Tris-HCl pH 8.0
+ 10 mM Imidazole
+ 500 mM NaCl
+ 1 mM PMSF
+ Fill to deionized H2O

* His-Elution Buffer (300 mL)

+ 500 mM Tris-Cl pH 8.0
+ 300 mM Imidazole
+ 50 mM NaCl
+ .1 mM EDTA
+ 1 mM PMSF
+ Fill to 300 mL deionized H2O

### Procedure

1. Have an ice bucket ready on your bench.
2. Prepare the wash and elution buffers with the necessary materials and molarities.
3. Resin Preparation

+ Make sure your column is stably suspended in the air (preferably use a clamp stand). Place a waste beaker underneath the column.
+ Slowly pipette 50mL of 50% Ni-NTA Agarose Beads into the column. Wait for the liquid to drip out of the column. All that is left is 25mL of agarose beads (a bllue tint means they contain nickel).
+ Slowly pipette 200mL of Wash Buffer into the column. Pipette gently onto the side of the column. Wait for the liquid to drip out of the bottom.

4. Purification

+ This step is optional: you can replace the waste beaker for a separate vial if you wish to sample the eluted E.coli particles for an SDS page.
+ Slowly pipette your cell lysate into the column (do not go over column capacity). If you are using a smaller column, you can do multiple lysate pours.
+ Slowly pipette 250mL of Wash Buffer into the column using the same gentle procedures. Wait for the liquid to drip out of the column. Do this same step a second time.
+ Remove the current collection vial from under the column. Prepare 20 labeled vials for protein collection (alternatively, you can use one vial).
+ Put the first of 20 vials under the column. Gently pipette 15mL of Elution Buffer into the column and wait for all liquid to drip out. Place the protein vial in the ice bucket.

## LiOAc/SS-DNA/PEG Transformation

### Materials

* LiOAc Mix
* PEG Mix
* YPD
li>* Salmon Sperm DNA
* ddH2O
* SD Dropout Plates
* Yeast strain
* Plasmid/DNA

### Procedure

1. Pick colony from plate into 5mL YPD for overnight growth in 30°C shaker.
2. Back dilute over night culture to OD 0.175 in 15mL growth media.
3. Grow up yeast to OD600 ~0.75 at 30°C. Takes 4-5 hours for WT in YPD.
4. Pellet at 2000 rpm for 10 min in a large centrifuge with swinging buckets.
5. Prepare lithium acetate transformation mix while harvesting cells.

* 50% w/v PEG-3350
* LiOAc 1.0 M
* 10 mg/mL ssDNA
* Plasmid DNA + H2O

6. Pour off the medium, resuspend the cells in .5x volume of sterile water and repeat step 4.
7. Pour off the water, resuspend the cells in 0.5x volume 100 mM LiAc and repeat step 4.
8. Final suspension in 0.01x volume 100 mM LiOAc (100 uL per transformation - around 300 uL for 15 mL culture)
9. Pellet at 8000 rpm for 1 min at 10 C on small tabletop centrifuge.
10. Resuspend in the lithium acetate transformation mix. I suggest resuspending with the plasmid DNA and ddH2O (54 μL) first, then mixing with the LiOAc+PEG+ssDNA mixture (306 μL) afterwards, as it is too viscous to easily disrupt the pellet.Incubate 30°C for 30 min.
11. Incubate for a further 17 min at 42°C in water bath/heat block.
12. Centrifuge 8000 rpm for 2 min and remove supernatant.
13. For a prototrpohic or auxotrophic marker, resuspend in 200 uL of H2O and incubate at RT for 10 min.
14. Plate desired volume on plates. Typically do half. Incubate 30C for 2 days - small colonies should appear on the second day.

## Yeast Plate Preparation

### Materials

* 1L Distilled Water
* 20 g Powdered Agar
* 28.4g CM Broth minus URA
* 2 L Conical Flask
* Petri Dishes

### Procedure

1. Pour 1L of distilled water into the concical flask. Measure 20g and 28.4g of Powdered Agar and CM Broth in weighboats.
2. Pour the CM Broth powder into the conical flask. Dissolve the powder by shaking the flask.
3. Shake the flask while pouring in the Agar powder. Mix the flask with a large magnet spinner for 3 minutes at 1000rpm, or until all agar clumps are dissolved.
4. Autoclave on an autoclavable tray at 121°C, S:20. Leave in autoclave for the full cycle (52 minutes).
5. Take the media out to cool down for 20-30 minutes after autoclave is done.
6. While cooling the media, set the petri dishes under the fume hood and sterilize with UV light for 15 minutes. Pipette 25 mL of media into each dish, avoiding any bubbles.
7. Leave the plate lids off until their moisture dissipates, and wait for agar to dry. Store at 4C.

## Yeast Wall Digestion for SDS-PAGE

### Materials

* Yeast Lysis Buffer

+ 20 mM HEPES, pH 7.6
+ 50 mM NaCl

* Yeast Suspension Buffer
* Zymolyase 20T
* Beta-mercaptoethanol

### Procedure

1. Spheroplast Formation

* Pellet yeast cells (bacterial culture, OD600 1.5-2.0) by centrifugation at 200-500 x g for 5-10 minutes. Suspend the cell pellet in an equal volume of Yeast Suspension Buffer. Add 1 µl of ß-mercaptoethanol per 100 µl yeast suspension.
* Gently pipet up and down until the cell suspension is homogeneous. Incubate the suspension for 5 minutes at 4°C. Gently pipet again to suspend the cells.
* Flick the vial containing Zymolyase® to mix the solution. Add 10 µl Zymolyase® for each 100 µl cell suspension in Yeast Lysis Buffer. Gently mix the content.
* Incubate the suspension at 37°C for 30-60 minutes. Note: (OPTIONAL). Lysis can be monitored by taking 25 µl of suspension and mixing with 1 ml Yeast Lysis Buffer and reading the optical density at OD 800nm.
* At the end of incubation, centrifuge the suspension at 10,000 x g for 5 minutes. Remove and discard the supernatant carefully, leaving the spheroplast pellet in the tube.

2. Spheroplast Lysis.

* For lysis, suspend the spheroplast pellet in an appropriate volume of the Yeast Lysis Buffer (2-3 times the volume of spheroplast pellet). Pipet the suspension up and down a few times. Vortex periodically and incubate on ice for 30 minutes. The lysis may be further facilitated by incubating the cells for 1-3 minutes at 37°C or a brief sonication step. Sonication is necessary for shearing genomic DNA. Please note, the higher Yeast Lysis Buffer to spheroplast pellet ratio the better the cell lysis.
* Centrifuge the lysed cells at 20,000 x g, for 30 minutes at 4°C and collect the clear lysate. Note: Additional volume of Yeast Lysis Buffer can be purchased separately for downstream applications, e.g. chromatography and dialysis, etc.

## LiOAc/Ethanol Genomic DNA Extraction

### Materials

* 0.2 M Lithium acetate 1% SDS solution
* Ethanol 96-100^ and 70%
* Heat Block

### Procedure

1. Place one yeast colony from the plate or spin down 100-200 uL of liquid yeast culture (Od600 ~0.4). Suspend cells in 100 uL of 200 mM LiOAc, 1% SDS solution.
2. Incubate for 5 minutes at 70 C.
3. Spin down DNA and cell debris at 15000 g for 3 minutes
4. Wash pellet with 70% ethanol.
5. Dissolve pellet in 100 uL of H2O or TE and spin down cell debris for 15 seconds at 15000 g.
6. Use 1 uL of supernatant for PCR.

# General

## Agarose Gel Electrophoresis

### Materials

* 0.8 g Agarose (for 0.8% agarose)
* TAE 0.5x Buffer
* 6 uL DNA Ladder (mixed w/ Loading Dye)
* 1 uL DNA samples
* Gel Casting Tray
* Gel Chamber
* UV Transilluminator
* Power Supply (15 W)

### Procedure

1. Mix 0.8g agarose with with 100 mL 0.5x TAE buffer.
2. Heat by microwave for 1 min 45 sec at ~15-30 sec intervals.
3. Pour the agarose solution into a gel tray with the well comb in place.
4. Keep newly poured gel at room temperature for 20-30 min.
5. Aliquot 1 uL of DNA samples and add 4 uL ddH2O and 1 uL 6x DNA Loading Buffer.
6. Vortex and centrifuge.
7. Remove comb once gel is solidified and pour rest of 1x TBE (75 mL into gel box). Make sure gel is fully emersed.
8. Load samples into lanes, flanked by 6 uL DNA Ladder.
9. Ensure power to the gel box is turned on. (The side where the DNA is placed is negatively charged - black- and the opposite side is positive - red).
10. Run at 120 V for 40-60 min.

## DNA Nanodrop and Purification

### Materials

* Nanodrop Machine
* Elution Buffer
* Wash Buffer
* PCR Product (DNA fragment binding buffer to sample 5:1)

### Procedure

1. Nanodrop

* Select "Nucleotide" on the machine.
* Make sure the reading is 0 ng/ul (or a negative value) meaning that the machine is clean. If not, grab a kimwipe, wipe the pedestal (top and bottom) and add 2ul of water on the pedestal.
* Click on “Measure.” Once you see the 0 ng/ul reading, proceed to wiping the water from the pedestal.
* Add 2ul of Qiagen Elution Buffer to the pedestal, this will be your “Blank.” Once you see the 0ng/ul from your blank, wipe the liquid off the pedestal. Now you are ready to measure your samples
* Load 2 ul of your sample on the pedestal, click “Measure” and write down the ng/ul value. Don’t forget to use a kimwipe to clean the pedestal after measuring each sample.
* Finally, when you are done using the nanodrop machine, load 2 ul of water, click “Measure” and leave a kimwipe on the pedestal so that the next person using it will know it is clean.
* DNA should have a 260/280 ratio between 1.8-2.0. RNA should be above 2.0. Contaminated DNA will have a low 260/280 of below 1.8.

2. DNA Elution

* In a 1.5mL microcentrifuge tube, add 2- 7 volumes of DNA binding buffer to each volume of DNA sample(Mix briefly by vortexing).
* Transfer mixture to a Zymo-Spin column in a collection tube.
* Centrifuge for 30 seconds. Discard the flow through.
* Add 200uL DNA Wash Buffer to the column. Centrifuge for 30 seconds. Repeat the wash step.
* Add >= 6uL DNA Elution Buffer directly to the column matrix and incubate at room temperature for one minute. Transfer the column for 30 seconds to elute the DNA.

## PCR Amplification

### Materials

* Thin-walled 200 uL PCR tubes

  ##### Table 2

  | Reagent | Volume |
  | --- | --- |
  | Sterile Water | 7.8 uL |
  | PrimeStar Max | 10 uL |
  | Forward Primer (100 uM Stock) | 0.6 uL |
  | Reverse Primer (100 uM Stock) | 0.6 uL |
  | Template DNA | 1 uL |
  | Final volume | 20 uL |

  * Thermocycler

### Procedure

1. In PCR tubes, add all the reagents listed in Table2.
2. Briefly spin down the PCR tubes (2-3 s) using a tabletop microcentrifuge in order to ensure that all of the reagents are in the reaction mixture
3. Place the PCR tubes into the selected thermocycler.
4. Once the lid to the thermocycler is properly closed, start the required amplification program, as in table 3.

##### Table 3

| Cycle Step | Cycling Temperature | Time | Number of Cycles |
| --- | --- | --- | --- |
| Denaturation | 98 C | 15 s | 35 |
| Annealing | 55 C | 10 s | 35 |
 Extension | 72 C | 35 s | 35 | Final Extension | 72 C | 5 min | 1 || Hold | 12 C | ∞ | 1 |

5. If the amplified DNA is NOT to be immediately used, label the tubes with your initials and date and temporarily store at 4°C or freeze at -20°C for long-term storage in assigned storage box

## SDS PAGE

### Materials

* 1x Running Buffer

+ 950 mL water
+ 50 mL MOPS SDS Running Buffer

* NuPAGE LDS Sample Buffer (4x)
* NuPAGE Reducing Agent (opt.)
* Deionized Water
* Protein Sample
* 8-15 BIS-TRIS gels from 4 C
* Coomassie Blue Stain

### Procedure

1. Preparing the Samples

* Prepare samples according to the table below.
* Heat the samples at 70 C for 10 minutes.

2. Preparing the Buffers

* Add 50 mL of 20X NuPAGETM MES or MOPS SDS Running Buffer to 950 mL of deionized water to prepare 1X SDS Running Buffer.
* For reduced samples, add 1 mL of NuPAGETM Antioxidant to 400 mL 1X SDS Running Buffer.
* Preparing the Gel

+ Remove the comb, and rinse the gel wells three times using 1X Running Buffer.
+ Remove the white tape near the bottom of the gel cassettes.
+ Place the gels in the mini gel tank.

* Loading the buffers

+ Fill the chambers with the appropriate 1X running buffer.

- Mini Tank: Add 400 mL of buffer to each chamber.
- XCell SureLockTM Mini-Cell: Add 600 mL of buffer to the lower chamber, and 200 mL to the upper chamber (for reduced samples, use running buffer with antioxidant in the upper chamber).

* Loading the Samples and Ladders

+ Load the appropriate volume of the samples in the appropriate wells.
+ Load the protein ladder in the appropriate well.

* Running the gel

+ Optimal run times vary depending on gel percentage and power supply used for electrophoresis.

- If using MES Running Buffer, run for 35 minutes at 200 V constant.
- If using MOPS Running Buffer, run for 50 minutes at 200 V constant.
- Note: If not using a Thermo Fisher ScientificTM power supply, install NovexTM Power Supply Adapters.

Protocols

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# Team:CCA San Diego/Results

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* [Home](https://2021.igem.org/Team%3ACCA_San_Diego)
* Team
  + [Team Page](https://2021.igem.org/Team%3ACCA_San_Diego/Team)
  + [Attributions](https://2021.igem.org/Team%3ACCA_San_Diego/Attributions)
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* Project
  + [Description](https://2021.igem.org/Team%3ACCA_San_Diego/Description)
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* Lab
  + [Design](https://2021.igem.org/Team%3ACCA_San_Diego/Design)
  + [Engineering](https://2021.igem.org/Team%3ACCA_San_Diego/Engineering)
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* [Modeling](https://2021.igem.org/Team%3ACCA_San_Diego/Model)
* Human Practices
  + [Inclusion](https://2021.igem.org/Team%3ACCA_San_Diego/Inclusivity)
  + [Proposed Implementation](https://2021.igem.org/Team%3ACCA_San_Diego/Implementation)
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![](https://static.igem.org/mediawiki/2021/e/e9/T--CCA_San_Diego--results.png)

### Overview

Our results consist of our DNA and protein gel verification of our Gibson assembly and lithium acetate transformation into yeast. We also present the results of extracting animal collagen in order to work on our proposed implementation of collagen hydrogels.

### Goals:

1. Have colonies grow on a selective ampicillin (+) plate and show that our plasmid is present in a gel next to our linearized backbone and correctly sequenced.
2. Grow yeast colonies on a SD dropout URA3(-) plate and show PCR verification of expression via amplifying overhangs and correct protein expression on an SDS-PAGE.
3. Obtain triple helical collagen from porcine skin for use in collagen gels.

Before diving in, we want to note that we will be mostly presenting our successful results and more documentation of our trials can be found on our [Engineering Success page.](https://2021.igem.org/Team%3ACCA_San_Diego/Engineering)

### Expression

#### DNA Preparation

To create a plasmid that could be cloned in yeast cells, several linear fragments had to be prepared, as the desired scl2 construct ([BBa\_K3956014](http://parts.igem.org/Part%3ABBa_K3956014)) by itself was too complicated to be prepared by custom synthesis. Throughout the optimization of these fragment designs with 80-bp overhangs to each other, the linearized plasmid backbone consisting of the pRS4116 plasmid (with the lacZalpha and f1ori regions removed) was kept constant. Figure 1.1 compares the lengths of the amplified, linearized backbone to the full pRS4116 plasmid, with the linearized fragment in lane 1 slightly shorter, implying successful PCR. The presence of this linear backbone fragment was further verified after a DpnI digest was conducted to remove any methylated DNA still part of a circular plasmid backbone (Figure 1.2). As the PCR product only has one visible band around the desired length, 3754 bp, in comparison to the same concentration of plasmid backbone, it can be concluded that the DpnI was successful.

![filler](https://static.igem.org/mediawiki/2021/8/8e/T--CCA_San_Diego--results1.png)
![](https://static.igem.org/mediawiki/2021/7/78/T--CCA_San_Diego--results2.png)

Throughout the experiment three construct designs were tested - two trials broke the construct into three overlapping segments, while one was able to break it into two overlapping segments. The fragments were ordered from IDT, amplified, and purified for optimal Gibson Assembly. The compiled results of the amounts of DNA used in Gibson and Yeast are listed below. All the DNA was used in assembly after using a DNA cleanup kit and measuring the concentration with a NanoDrop spectrophotometer.

#### Gibson Assembly and Transformation of E. coli

Our fourth Gibson Assembly was successful after assembling a plasmid with three fragments - the truncated pRS4116 backbone, and the scl2 construct broken up into two overlapping segments. Figure 1.3 shows that a miniprep from an efficacious colony is slightly larger than the linearized pRS4116 backbone, implying a construct the correct length of 1390 bp was inserted (band in lane GIB is slightly larger than pRS). In addition, the miniprep was amplified with primers for the inserted scl2 fragment, which showed a successful amplicon around of expected length 1396 bp.

![filler](https://static.igem.org/mediawiki/2021/thumb/b/b8/T--CCA_San_Diego--results3.png/800px-T--CCA_San_Diego--results3.png)

However, upon sequencing, the plasmid was shown to match the same base pairs as our DNA design backbone but not our construct, which prevented us from transforming our assembled 5144 bp pRS4116-His-scl2 fragment into yeast.

In our next iteration, the same DNA fragments were used and colonies grew on the appropriate Amp + plate. New primers were designed in order to amplify overlapping segments where successful ligation should occur in Gibson and yeast assembly. The two primers were designed to amplify a 678 bp fragment where our construct and backbone were ligated, and we were able to verify that in the 2-fragment Gibson Assembly, the primers did amplify the overhangs of the correct length, implying successful ligation (Figure 1.7).

![Filler - to the right of the paragraph above](https://static.igem.org/mediawiki/2021/thumb/4/48/T--CCA_San_Diego--results4v2.png/519px-T--CCA_San_Diego--results4v2.png)
![filler - to the left of the 2 paragraphs above](https://static.igem.org/mediawiki/2021/thumb/9/94/T--CCA_San_Diego--results5.png/343px-T--CCA_San_Diego--results5.png)

These colonies (shown in the last row of Figure 3.2) were sequenced with primers around the overlapping segments of the construct and backbone. Again, while the backbone was successfully sequenced, the ORF was not successfully sequenced. Although we transformed this miniprep into yeast and saw successful results, the sequencing results were not supportive of a successful assembly. Overall, as the overhangs were successfully amplified and the correct length on the gel, and the transformed bacterial and yeast colonies with the assembled plasmid grew successfully on both Amp+ and Ura- plates, we believe that the assembly was successful.

![](https://static.igem.org/mediawiki/2021/thumb/6/61/T--CCA_San_Diego--results6.png/800px-T--CCA_San_Diego--results6.png)

Throughout all of our trials, we recorded the amount of each fragment used in the Gibson Assembly, aiming to use between .2-1.0 pmol as recommended by NEB. Our most successful trial used the middle range - 0.624 pmol of DNA, with an approximate 2.5:1 ratio of inserts to backbone (Table 1).

#### Lithium Acetate Gap Repair Cloning

There were a total of eight yeast assemblies with LiOAc transformation trials. For the first seven trials, we utilized a standard LiOAc/PEG/H2O protocol, but for the eighth trial of yeast assembly, we used a new procedure with additional solvents: DMSO and TE buffer.

For our seventh iteration before we modified our protocol, we transformed the successful plasmid from Gibson assembly into yeast as well as directly transforming linear fragments for gap-repair cloning. We noticed that there weren't many colonies that grew in comparison to our pRS plasmid, but more colonies than our no DNA control plate. In order to verify that transformation was successful, we used genomic yeast extraction with ethanol precipitation before running a PCR with the same primers amplifying overhangs as we did in Gibson. However, no bands were visible when run in a gel, which upon reflection may be due an intact cell wall and failure of the genomic extraction rather than the assembly itself, as we used a different protocol for our last trial that showed successful bands.

In our last trial, we showed the most promising results in terms of the number of colonies that grew on selective media. Figure 3.2 shows that the yeast transformation of our plasmid backbone (as a positive control as it contains a URA3 gene) and Gibson Miniprep grew three colonies, while the negative control had one colony and a two-fragment yeast assembly had no large colonies. Because the miniprep plasmid from our last successful Gibson showed large colonies, we again attempted to extract the genomic DNA to run a PCR.

![filler](https://static.igem.org/mediawiki/2021/thumb/2/29/T--CCA_San_Diego--results7.png/800px-T--CCA_San_Diego--results7.png)

To isolate the DNA, we used a new protocol similar to a bacterial plasmid miniprep, and the PCR (Figure 1.8) showed one successful lane from the 2-fragment Gibson assembly of the ligated region of the plasmid around 678 bp. Interestingly, both the lane from the Gibson and the other controls from the yeast assembly, pRS4116, and no DNA transformation had strong amplicons around 1,500 bp. As the primers designed were unique in our plasmid, it may be possible, but highly unlikely, that there is a very similar genomic region within endogenous yeast chromosomes that match both primers. Alternatively, a larger sequence may have been inserted within the construct through the gap-repair process or the Taq Polymerase in the PCR reaction ran on to amplify an entire section of the plasmid due to statistical mispriming of another primer.

![filler - to the left of the paragraph above](https://static.igem.org/mediawiki/2021/thumb/b/b4/T--CCA_San_Diego--results14.png/800px-T--CCA_San_Diego--results14.png)

We again recorded the amount of DNA used in our Yeast Assembly (Table 2). As there were no definite guidelines on how much DNA to use, although protocols advise against excessive volume, the total pmol of DNA used varied more widely compared to the Gibson. Our last successful trial used a total of 1.810 pmol of DNA (second trial of the Two-Fragment Construct), which indicates that the amount of DNA does not necessarily lead to a successful assembly. As there was a successful amplicon around 678 bp, we proceeded in our attempts to verify the actual expression of the scl2 collagen protein through an SDS-PAGE.

#### Verification of scl2 expression

The verification that the DNA had been transformed into the chassis after they grew on selective media was done in several sequential steps. First, for Gibson assembly, the plasmid was extracted via miniprep and run in a gel alongside a linearized pRS4116 fragment to test if the plasmid had increased in size. To verify that the construct was correctly inserted, primers were designed to amplify the overlapping regions on both sides of the construct, with PCR products 678 bp and 713 bp in length, respectively. This was run in a gel to test if the overhangs were assembled correctly.

Finally, to verify the expression of the actual protein, several SDS-PAGE trials were run to varying degrees of success, finally obtaining proper cell wall digestion with Zymolyase to isolate protein lysates, as well as correct staining of the gel with Coomassie. The final protein gel shows many bands as expected, including the Zymolyase enzyme around 52 kDa, but not the expected construct around 42 kDa. As there is not much time left to submit results, we may have to stop here. However, we do have some promising DNA gel evidence that the construct was inserted into the yeast, and next steps with Western blotting for the His-Tag or changing promoters or chassis could verify our construct design.

![filler](https://static.igem.org/mediawiki/2021/thumb/5/59/T--CCA_San_Diego--results9.png/800px-T--CCA_San_Diego--results9.png)

### Discussion

In our lab work to craft CollaGene, we performed several Gibson assembly trials, lithium acetate transformations, and verification with both protein and DNA gels. Although we were unable to show scl2 expression within our chassis through a clear band on an SDS-PAGE, we were able to show that our designed plasmid was successfully inserted through colony PCR overhang length verification.

Prior to beginning our project, we were aware that it would be difficult to express collagen due to the repetitive nature of the collagen-like domain, with 80 Gly-Xaa-Yaa repeats. The countless amount of trials that was required to assemble the plasmid in bacteria and yeast may have been a reflection of this, as working with repetitive sequences is prone to error, especially during PCR which can form hairpin loops and dissociation of the Taq polymerase from the template during amplification, which leads to further “mega primers” that generate unwanted amplicons (Riet et. al, 2017). As our team amplified our gBlocks and genes before Gibson and Yeast assembly, it is possible that the repetitive nature of the scl2 construct prevented clear amplification of our desired sequence. To test this, we ran a gel to verify that our PCR amplicons were present on one of our three-fragment designs (Figure 1.4). The third fragment, which contained the most repetitive sequences, was around our suspected length of 445 bp, but contained other bands within the lane as expected from mispriming and repetitive sequences.

![filler - to the left of the paragraph above](https://static.igem.org/mediawiki/2021/c/c0/T--CCA_San_Diego--results10.png)

Although PCR was not done for every single trial for all three construct fragmentations, it can be inferred that mispriming and incomplete amplification may have occurred and contributed to our null sequencing and expression results.

Throughout our experimentation, the fact that yeast colonies without any DNA transformed into them still grew on selective media, albeit in lower numbers and smaller sizes, was a source of confusion. We believe that the negative controls may still contain uracil from their time spent in the YPD. However, since the colonies were noticeably smaller and lower in number, we believed that our transformation was still successful, and the positive controls and experimental groups would continue to grow in selective media.

### Implementation

#### Overview

When we were considering how to implement the results of our project in a meaningful way, we aimed to create collagen gel, felt, and threads to be used in materials such as sutures and skin grafts. Our implementation results consist of first extracting animal collagen to be then used in our successful gelification of collagen.

#### Collagen Sourcing

We were ultimately successful in isolating collagen from porcine skin. After testing two different collagen extraction procedures, we found that sodium citrate buffer was most effective in isolating collagen proteins, and we confirmed the presence of the extracted collagen with an SDS page.

![filler]()
![]()

The SDS page shows a faint band around 40 kDa in lane in the telopeptide-poor collagen digested with pepsin, indicating structural similarity with our predicted scl2 collagen after proteolysis (Figure 2.1). Further uses of our successfully extracted collagen can be found on our Proof Of Concept page.

#### Gelification

![filler]()

The engineering process for implementation was a series of gels testing the effects of different variables and reagents. After 8 iterations testing variables from glycerol to citric acid, we optimized the best gelification formula. Previous iterations had demonstrated instant coagulation when collagen gels were procured in neutral pH rather than acidic pH. Glycerol increased the viscosity of the solution while high collagen concentration predictably bolstered total coagulation, but had an observable drop off after 80% concentration.

Our results observed from citric acid and ethanol concentrations had insignificant differences, but based on their respective acidic and neutral pH, we speculated that excess of either would result in zero or lots of coagulation. Specifically, too basic or acidic of a pH would instantly dissolve all current coagulation as observed in the pH trials.

A trend we had noticed was that coagulated collagen would dissipate after prolonged time. We hypothesized that the disappearance of the coagulation was a result of the supernatant dissolving the binded collagen proteins. Thus, our final iteration added this new consideration on top of the mountains of optimization from previous iterations. We condensed the instantly coagulated collagen and then pipetted the supernatant out, replacing it with glutaraldehyde. The result was a small 1cm wide disk of solid collagen gel that acceptably resisted agitation and compression.

#### Discussion

The result of our implementation experimentation presents strong success in the physicality of making a gel. However, it is questionable whether or not the gel we synthesized is indeed made of collagen, and whether or not the same process can be recreated with Scl2. Unfortunately, we were unable to test both of these questions due to time constraints. But what we can confirm is that the collagen protein source is undeniably pure as demonstrated through SDS pages of the proteins. Thus, our current rationale is that because the collagen source we used was unequivocally collagen protein, the gel is likely to be collagen as well. This is further supported by our experimentation that showed a proportional relationship between collagen concentration and amount of coagulation. Because Scl2 proteins consist of much the same physical structure, we also believe that Scl2 will form a gel with the same gelification procedure.

### Future Steps

Our Collagene project has much to improve upon in the future. From improving efficiency of our expression method to actually implementing relevant medical situations, Collagene has a prospective future.

* ##### Inducible Promoters

  The next step for Collagene is adding inducible promoters, allowing users to toggle the collagen gene expression using various hormones, antibiotics, or even changes in temperature. Studies like Karsenty et al 91 have experimented with various promoters to alter collagen expression and we plan to do the same.

  This is especially relevant since our project used a constitutive promoter, which led to an expression of numerous protein bands in our SDS-PAGE. It was difficult to determine whether the protein was expressed with a constitutive promoter, and an inducible promoter will be much easier to regulate collagen expression.
* ##### Epidermolysis bullosa

Epidermolysis bullosa (EB) is a disorder that causes skin to become very fragile due to mutations in the production of collagen type VII in humans. The collagen VII is essential in anchoring fibrils in the dermal, which creates epidermal junction that makes our skin the malleable texture. As a possible solution, yeast or bacteria implants or other concoction can be injected into EB patients to enhance collagen production rate.

* ##### Types of Collagen

There are numerous types of collagen that are found for many different organisms, whether that be categories such as mammalian, plant, bacterial, or yeast collagen, or whether that be type I, type II, or type III collagen. Since Collagene is restricted to producing bacterial scl2 collagen in yeast, our next steps are to alter our plasmids to be able to effectively express other types of collagen. Our choice of scl2 was only for its similarity to mammalian collagen, but tackling other types of collagen is our next primary goal.

* ##### 3D printing organs

Printing organs is another possibility with collagen; since 3D printing organs have already been pondered by researchers, implementing collagen will help create a more sturdy, realistic organ because it helps create tendons and ligaments. Studies like Lee et. al 19 has already made efforts to bioprint the human heart and numerous other studies show that collage would be an effective biofuel.

### References

Riet J, Ramos LRV, Lewis RV, Marins LF. Improving the PCR protocol to amplify a repetitive DNA sequence. Genet Mol Res. 2017 Sep 21;16(3). doi: 10.4238/gmr16039796. PMID: 28973773.

Karsenty, G., & de Crombrugghe, B. (1991). Conservation of binding sites for regulatory factors in the coordinately expressed α1 (I) and α2 (I) collagen promoters. Biochemical and biophysical research communications, 177(1), 538-544.

Lee, A. R. H. A., Hudson, A. R., Shiwarski, D. J., Tashman, J. W., Hinton, T. J., Yerneni, S., ... & Feinberg, A. W. (2019). 3D bioprinting of collagen to rebuild components of the human heart. Science, 365(6452), 482-487.