iGEM 2020 — Team UCSC

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# Team:UCSC/Notebook

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# Notebook

A collection of our team's wet-lab and dry-lab work broken down by day.

[![](https://static.igem.org/mediawiki/2020/e/e1/T--UCSC--down-arrow.png)](#Member-Training)

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July

S

M

T

W

T

F

S

1

2

3

4

5

[6](#jul-6)

[7](#jul-7)

[8](#jul-8)

[9](#jul-9)

[10](#jul-10)

11

12

[13](#jul-13)

14

15

16

[17](#jul-17)

[18](#jul-18)

19

[20](#jul-20)

21

[22](#jul-22)

[23](#jul-23)

24

25

[26](#jul-26)

[27](#jul-27)

28

29

[30](#jul-30)

31

August

S

M

T

W

T

F

S

[1](#aug-1)

[2](#aug-2)

[3](#aug-3)

[4](#aug-4)

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

September

S

M

T

W

T

F

S

1

2

3

[4](#sep-4)

[5](#sep-5)

[6](#sep-6)

[7](#sep-7)

[8](#sep-8)

[9](#sep-9)

[10](#sep-10)

[11](#sep-11)

[12](#sep-12)

[13](#sep-13)

[14](#sep-14)

[15](#sep-15)

[16](#sep-16)

[17](#sep-17)

[18](#sep-18)

19

20

[21](#sep-21)

[22](#sep-22)

[23](#sep-23)

[24](#sep-24)

[25](#sep-25)

26

[27](#sep-27)

[28](#sep-28)

[29](#sep-29)

[30](#sep-30)

October

S

M

T

W

T

F

S

[1](#oct-1)

[2](#oct-2)

3

4

[5](#oct-5)

[6](#oct-6)

[7](#oct-7)

[8](#oct-8)

[9](#oct-9)

10

11

[12](#oct-12)

[13](#oct-13)

[14](#oct-14)

[15](#oct-15)

[16](#oct-16)

[17](#oct-17)

[18](#oct-18)

[19](#oct-19)

[20](#oct-20)

[21](#oct-21)

[22](#oct-22)

[23](#oct-23)

24

25

26

[27](#oct-27)

28

29

30

[31](#oct-31)

## Week of July 6th, 2020 - July 12th, 2020

## July 6th, 2020

### Allie and Melody

Made Hestrin–Schramm (HS) Media, inoculated media with *K. rhaeticus* cultures
from Imperial College received in the mail in an agar stab.

### Josh and Sophia

Sorbitol was tested as a plasticizer for bacterial cellulose.

## July 7th, 2020

### Conor and Neil

* NaOH as a decrystallizing and swelling agent for the production of
  amorphous gelatinous MCC was tested.
* Cellulose did not fully dissolve.

## July 8th, 2020

### Faith, Josh, Sophia, Taylor

Urea and sodium hydroxide were tested as solvents for MCC.

1. Sample was placed in the fridge to thaw and vortexed every 5-10
   minutes a total of nine times.
2. The 10 mL sample was split into 10 1 mL samples and centrifuged at
   8000 rpm for 30 minutes.
3. The collective supernatant was transferred back into a 15 mL falcon
   tube and stored in a fridge.
4. The resulting supernatant was translucent,
   non-viscous and composed about 8 mL of the greater 10 mL sample.

### Kyra and Rachel

1. Made 4 different plasticizer mixes (glycerol, glycerol/Milli-Q H20,
   citric acid, citric acid/Milli-Q H20) and made 4 different mixtures
   with these mixes and ZnCl2 + MCC to test for plasticizer qualities.
2. Prepared plasticizer mix (glycerol and citric acid) for amorphous cellulose protocol.
3. Prepared amorphous cellulose (MCC) with NaOH and left overnight stirring in
   a 5°C cold room.

## July 9th, 2020

### Kyra and Rachel

1. Prepared plasticizer mix (glycerol and citric acid) for amorphous cellulose protocol.
2. Prepared amorphous cellulose (MCC) with NaOH and left overnight stirring in
   a 5°C cold room.

### Claudia and Conor

1. Prepared aqueous glycerol solution at three different concentrations.
2. BC split into five pieces, performed purification procedure on 2 BC samples
   and plasticized three BC samples with aqueous mixture skipping purification.
3. Plasticized samples were dried in oven and stored in fridge overnight.

## July 10th, 2020

### Human Practices Team

The Human practices team met with Husein Ajwa to learn more about the strawberry production industry and acceptable agricultural films.

### Allie and Melody

Mixed Glycerol, ZnCl2, and MCC to see if it forms a gel like substance to
understand if glycerol is a promising plasticizer. The glycerol mixture formed a gel-like
substance, convincing us it could be a good plasticizer.
This experiment demonstrated that the concentration of MCC is what creates the gel,
not the amount of glycerol.

### Claudia and Conor

1. Purified BC samples submerged in aqueous acetic acid solution for pH neutralization.
2. BC plasticized with same aqueous glycerol solution at three different concentrations.
3. Samples centrifuged, dried in oven, and stored in the fridge overnight.

### Kyra and Rachel

Finished titrating amorphous cellulose to a pH of 10 and further baked
ZnCl2/MCC with glycerol and citric acid plasticizers.

##

###

## Week of July 13th, 2020 - July 19th, 2020

## July 13th, 2020

### Claudia and Conor

1. Dried samples assessed for plasticity through melting on hot plate.
2. Samples heated at different intervals to obtain melting point.
3. 50% glycerol concentration rendered to be the least burnt of the five samples.

## July 17th, 2020

### Faith and Taylor

Yamanaka media was created. The pH of Flask A (diH20 and glucose)
was supposed to be 5.0, but could not be reached. Flask A ended up at pH 3.75.

## July 18th, 2020

### Allie, Melody, Claudia

1. BC inoculated in HS and Yamanaka media in 4 separate tubes.
2. Split BC into small pieces by vortexing and pipetting, then placed in media.
3. Cultured *E. coli* in both medias to test growth within two separate tubes.

##

###

## Week of July 20th, 2020 - July 26th, 2020

## July 20th, 2020

### Claudia and Taylor

1. Prepared NaOH aqueous solution for BC purification.
2. Drained media to dry BC sample.
3. Placed BC with aqueous solution in a falcon tube, left to sit overnight for 48 hours.

## July 22nd, 2020

### Claudia and Taylor

1. Purified BC dried with filter paper and neutralized with distilled water.
2. Prepared 1% PEG aqueous solution, BC submerged in solution for two hours.
3. Needed to be lyophilized but was never granted access to the proper equipment.

## July 23rd, 2020

### Human Practices Team

The Human practices team met with Dr. Lisa DeVetter to learn more about biodegradable film production and ASTM standards to consider.

### Josh and Sophia

Sorbitol and glycerol at different concentrations were
tested as potential plasticizers with MCC.

## July 26th, 2020

### Human Practices Team

HP found a USDA bill that tells us that we may not use biodegradable cellulose mulch in organic farming because it is considered a GMO.

## Week of July 27th, 2020 - August 2nd, 2020

## July 27th, 2020

### Conor

Bacterial cellulose was vacuum filtered then dehydrated using
a food dehydrator producing a thin, circular piece of cellulose.

## July 30th, 2020

### Human Practices Team

Members of the Human practices team visited Ajwa Lab to learn about fumigation testing of agricultural films.

## August 1st, 2020

### Allie and Faith

Luria Broth/Kanamycin Media was made for Agar Plates.
The agar plates were made with the media, but they did not solidify.

## August 2nd, 2020

### Allie and Faith

Luria Broth/Chloramphenicol Media was made for Agar Plates.
The agar plates were made with the media and they solidified successfully.

### Claudia and Kyra

Aliquots of glutaraldehyde were collected in 38 falcon tubes for proper storage
and stored in the freezer overnight.

## Week of August 3rd, 2020 - August 9th, 2020

## August 3rd, 2020

### Allie and Claudia

1. Tyrptone, yeast extract, and NaCl mixed together until dissolved and then autoclaved.
2. Prepared MgCl2 aqueous solution simultaneously and autoclaved.
3. Both solutions covered in foil and stored in fridge.

## August 4th - August 17th, 2020

### Quarantine 😷🦠🤧

Lab work was suspended during an outbreak in Baskin Engineering.

## Week of August 17th, 2020 - August 23rd, 2020

## August 19th - September 3rd, 2020

### CZU Complex Fires 🔥🌲🏫

Lab work was suspended during the CZU Complex Fires that threatened the UCSC campus.

##

###

## Week of August 31st, 2020 - September 6th, 2020

## September 4th, 2020

### Human Practices and Plasticizers Teams

Met with Dr. Oded Shoseyov and Dr. Doug Hayes to gain insight about CBM incorporation/choice and plasticizer protocols, respectively.

## September 5th, 2020

### Conor, Gabriel, Taylor

1. Growth of previous liquid cultures was checked.
2. LB/chloramphenicol liquid cultures were created and inoculated with
   cells from previous liquid cultures.
3. New cultures were incubated at 37°C and shaken at 250 rpm.

## September 6th, 2020

### Gabriel and Kyra

* Glycerol stocks for long-term storage of Imperial College transformed
  *E. coli* were made, and placed in a -80°C freezer.

##

###

## Week of September 7th, 2020 - September 13th, 2020

## September 7th, 2020

### Allie, Kyra, Melody

1. Amplified CBM3a and pET-28a backbone #1 (25 µL reactions).
2. Ran a gel to confirm sizes of the pET-28a backbone #1 and CBM3a
   gene block from IDT before amplification.
3. Gel told us that our CBM3a looks weird with 3 bands instead of 1;
   we suspected that our CBM3a IDT order may have been wrong.

## September 8th, 2020

### Allie, Kyra, Melody

1. Ran a 50ul amplification of the pET-28a backbone #1
   and another 50 µL amplification of the CBM3a gene block
   to attach Bsa1 recognition sites for Golden Gate Assembly.
2. Ran a gel of amplified pET-28a backbone #1 and CBM3a to confirm sizes.
3. Did DPN1 selection on the backbone to digest methylated template DNA
   and followed up with a Zymo Clean kit to remove dNTPs.
4. Finished with Golden Gate Assembly of the CBM3a gene block.

### Nav and Neil

Cleaned bacterial cellulose in order to prepare for room drying technique.
Dried samples were tested in the XRD machine.

## September 9th, 2020

### Allie, Kyra, Melody

Transformed our golden gate product into Mix-and-Go DH5ɑ.

### Claudia, Gabriel, Nav, Rachel

1. Attempted to dry out BC pellets but pH was too high. Postponed.
2. Made 250 mL of HS media for Plasticizers work.

## September 10th, 2020

### Claudia, Nav, Rachel

1. Incubated overnight cultures to warm up.
2. Inoculated 100 µL of culture with SOB media in two
   flasks containing 50 µL of media.
3. Took OD600 values of culture to assess for bacteria growth.
4. Incubated culture and took OD600 value intermittently.
5. Once threshold was reached, cells were suspended in glycerol
   and stored in the freezer.

## September 11th, 2020

### Allie, Kyra, Melody

1. Grew colonies for 13 hours. Selected 12 colonies for Colony PCR.
2. Made liquid cultures of the colonies of interest.

## September 12th, 2020

### Allie, Kyra, Melody

1. Miniprepped 2 of the colonies that showed slightly varying bands
   (a little above and below 0.9 kb). We then sent these two products for sequencing.
2. Nano-dropped the miniprep results to get ready for Golden Gate Assembly.

### Conor and Josh

1. Bacterial cellulose was vacuum filtered then dehydrated
   using a food dehydrator producing a thin, circular piece of cellulose.
2. Dried bacterial cellulose was added to ZnCl2
   at various concentrations to test hypothesis of whether BC dissolution was
   dependent on the concentration of ZnCl2.

## September 13th, 2020

### Allie, Kyra, Melody

1. After getting consistent bands at 0.9 kb from Golden Gate products
   when it was supposed to be at 1.3kb, we linearized the un-amplified pET-28a backbone #1
   using XbaI and ran it alongside our un-amplified CBM3a gene block.
2. Gel results showed us 3 bands for the CBM3a but a good band for the backbone.
   We decided to try going forward with making liquid cultures from our Golden Gate plates.

##

###

## Week of September 14th, 2020 - September 20th, 2020

## September 14th, 2020

### Allie, Kyra, Melody

We ran 20 liquid cultures of our Golden Gate product on a gel to see where the band was at.
The gel showed bands around 5.4 kb, with some variation (visible below).
To make sure, we decided to get samples ready for sequencing.

### Nav and Taylor

1. Bacterial cellulose samples were placed between
   chromatography paper in an accordion set-up.
2. Two glass slides were put on the very top and very bottom
   of the set-up with string tying it all together to press water out.
   Samples were left to dry for 4 days.

![](https://static.igem.org/mediawiki/2020/4/49/T--UCSC--Notebook-September-14.jpeg)

## September 15th, 2020

### Allie, Kyra, Melody

Miniprepped 2 samples (lanes 7 and 8 from the gel on September 14th)
of the plasmids containing our pET-28a backbone #1 and CBM3a.

### Claudia, Nav, Sophia

1. Electrocompetent cells and DNA were mixed together in separate tubes
   then transferred to a cuvette for electroporation.
2. Cells were then pipetted into SOC recovery media and incubated for an hour,
   plated, and stored in the fridge overnight.

## September 16th, 2020

### Allie, Kyra, Melody

Sent 2 miniprep samples to SequeTech to get our plasmids sequenced.

### Claudia, Nav, Sophia

1. Rehydrated gene blocks and prepared working stock for both CBM2a and mRFP primers.
2. Amplified primers with pET-28a backbone and ran samples on a gel twice.
3. Ran PCR using backbone.
4. Made another gel to assess gene blocks, primers, and backbone length.
5. Backbone would not show up in gel electrophoresis.

### Nav and Taylor

1. Prepared bed mulch plastic samples given to us by Ajwa Lab to undergo XRD analysis (XRD machine visible below).
2. XRD analysis was performed to set a standard for crystallinity that
   the Komaplastic formulation should strive to duplicate.

![](https://static.igem.org/mediawiki/2020/8/89/T--UCSC--Notebook-September-16.jpeg)

## September 17th, 2020

### Allie, Kyra, Melody

1. Received and interpreted sequencing results using Geneious.
   The Golden Gate products contained only the mRFP and not our CBM3a.
2. Due to failure of golden gate confirmed by sequencing results: restarted experiment again.

1. Amplification of pET-28a backbone #1 and CBM3a
2. Ran a gel to confirm results.
3. Did DPN1 selection and Zymo Clean.
4. Nanodrop results showed very bad contamination and extremely low concentrations,
   leading us to restart amplification again.
   We had too low of a concentration to continue with Golden Gate Assembly.

3. We selected more colonies from our previous Golden Gate plates
   to run colony PCR again, this time using T7 primers.

### Claudia, Nav, Sophia

1. Ran PCR reaction again but with Manny’s backbone instead.
2. Ran CBM2a PCR product with pET-28a backbone.
3. Ran 50 microliter PCR reaction to amplify mRFP gene block.
4. Ran 27 PCR cycles for Manny’s backbone and gene block.

## September 18th, 2020

### Allie, Kyra, Melody

1. Miniprepped an aliquot of our pET-28a backbone #1 then re-amplified our pET-28a
   backbone using T7 primers, expecting to see a band at around 0.3kb and allowing us
   to confirm that the backbone is correct.
2. Ran the pET-28a backbone on a gel alongside our colony PCR reactions.

### Claudia, Nav, Sophia

1. Performed a 25 microliter PCR reaction of CBM2a gene block
   from IDT and our previous CBM2a PCR product, ran on gel.
2. PCR products showed two bands, whereas we only wanted the top band.

### Nav and Taylor

1. Checked the room-dried bacterial cellulose samples, removed them from the
   chromatography backing paper.
2. Prepared samples and delivered to Oliver Lab for XRD analysis.

##

###

## Week of September 21st, 2020 - September 27th, 2020

## September 21st, 2020

### Allie, Kyra, Melody

1. Ordered a new CBM3a gene block from IDT after they confirmed that
   our CBM3a gene block was truncated, explaining the results of the amplification
   of our CBM3a gene block containing multiple bands when run on a gel.
2. While waiting for the IDT order to come in, ran an amplification
   of CBM3a gene block currently in the lab, one more time.

### Claudia, Nav, Sophia

1. Ran gel with 2 lanes containing CBM2a PCR product.
2. Prestained gel with 4 µL of SybrSafe.
3. Performed gel extraction for desired band.

## September 22nd, 2020

### Allie, Kyra, Melody

1. We amplified 6 reactions of our CBM3a gene block
   and ran it alongside our original CBM3a IDT order:
2. Ran another gel with our CBM3a gene block
   and did a gel extraction to isolate the 1.3 kb band.

![](https://static.igem.org/mediawiki/2020/2/2d/T--UCSC--Notebook-September-22.png)

### Claudia, Nav, Sophia

1. Ran two 25 µL PCR reaction of gel extraction.
2. Ran on gel, several bands appeared along with desired band
   and nanodropped PCR products.
3. Set up 50 µL touch down PCR reaction for CBM2a gel extraction,
   left running overnight.

### Kyra and Rachel

Prepared xanthan gum, MCC and diisopropylimidazolium composite ion gel.

## September 23rd, 2020

### Allie, Kyra, Melody

1. Ran our gel extraction of CBM3a on a gel alongside the amplified CBM3a
   to determine that only the 1.3 kb band was present in our gel extraction.
2. Re-amplified our CBM3a isolated from gel extraction using CBM3a primers with BsaI sites attached.
3. Miniprepped pET-28a backbone #1, treated it with DPN1
   and then cleaned it up using Monarch DNA Clean-up Kit.
4. We split the amplified pET-28a backbone #1 into 3 PCR tubes:

1. Positive Control: No DPN1
2. Negative Control: DPN1
3. Golden Gate: DPN1

5. We then nano-dropped the pET-28a backbone and CBM3a.
6. We linearized our pET-28a, negative and positive controls using XbaI
   and ran these samples on a gel alongside CBM3a gene block. Curiously,
   the CBM3a gene block did not show up on a gel and the 3 samples
   containing pET-28a all showed up at 1.3 kb rather than 5.4 kb.
7. We made liquid cultures of the pET-28a backbone #1 to figure out
   if amplification of the backbone causes the problem
   or if there is a problem with the backbone itself.

![](https://static.igem.org/mediawiki/2020/f/fe/T--UCSC--Notebook-September-23.png)

### Claudia, Nav, Sophia

1. Used Zymogen DCC kit to clean mRFP PCR products and nanodropped.
2. Clean up and nanodropped 50 µL CBM2a reaction.
3. Made LB/Kanamyacin plates.
4. Calculated femtomol conversions for mRFP and CBM2a sequence length.

## September 24th, 2020

### Allie, Kyra, Melody

1. Miniprepped pET-28a backbone #1.
2. Used XbaI to linearize aliquots of liquid cultures made of pET-28a backbone #1.

1. We ran it on a gel and saw a band showed up in our desired region (5.4 kb).
   This told us that there was something wrong with pET-28a backbone #1
   and annealing of the pET-28a primers during amplification causing the 1.3 kb band
   to show up instead of the 5.4 kb band.

## September 25th, 2020

### Allie, Kyra, Melody

1. New IDT order arrived: ran IDT order on a gel.
![](https://static.igem.org/mediawiki/2020/f/f5/T--UCSC--Notebook-September-25.png)2. IDT order is bright, indicating that IDT did in fact mess up our order the first time.
3. Amplified new CBM3a gene block from IDT.
4. Decided to amplify a new backbone because backbone #1 was causing us problems
   (which we call pET-28a backbone #2).
5. DPN1 selection on the backbone and did a Zymo Clean Up.

1. Ran a gel to make sure clean up was performed correctly.

6. Went with lane 2 for the pET28 backbone #2 and lane 5 for the CBM3a for Golden Gate Assembly.
7. Ran Golden Gate Assembly for CBM3a.
![](https://static.igem.org/mediawiki/2020/3/30/T--UCSC--Notebook-September-25-1.jpeg)

## September 27th, 2020

### Allie, Kyra, Melody

Transformed golden gate product for CBM3a.

## Week of September 28th, 2020 - October 4th, 2020

## September 28th, 2020

### Allie, Kyra, Melody

1. Nothing grew on the plates indicating GGA didn’t work.
2. Amplified multiple pET-28a backbone #2 and ran a gel to confirm size.
3. DPN1 selection and Zymoclean up. Ran another GGA.

![](https://static.igem.org/mediawiki/2020/b/b1/T--UCSC--Notebook-September-27.jpeg)

## September 29th, 2020

### Allie, Kyra, Melody

Transformed golden gate product using Mix-and-Go DH5ɑ cells.

### Claudia, Nav, Sophia

PCR amplified PET28a plasmid, ran product on gel, and performed gel extraction
of linear band and 50 µL PCR of extraction.

## September 30th, 2020

### Allie, Kyra, Melody

1. Nothing grew on the GGA for CBM3a.
2. Tried GGA one more time with pET-28a backbone #2 and CBM3a

### Claudia, Nav, Sophia

1. Cleaned up plasmid gel extraction PCR with NEB kit.
2. While gel was running, nanodropped and cleaned up PCR product.

1. 260/280: 1.83
2. 260/230: 1.93

3. Unsuccessfully imaged gel.

## October 1st, 2020

### Allie, Kyra, Melody

1. Transformed Golden Gate product with Mix-and-Go DH5ɑ cells.
2. Did a negative control plate containing pET-28a backbone #2
   with DPN1 selection and water as the insert.

### Claudia, Nav, Sophia

1. Used Zymogen DCC kit to clean mRFP PCR products.
2. Enhanced the CG content in the PCR products, and nanodropped products.

## October 2nd, 2020

### Allie, Kyra, Melody

Nothing grew on the golden gate plates except on the negative control plate.

##

###

## Week of October 5th, 2020 - October 11th, 2020

## October 5th, 2020

### Allie, Kyra, Melody

Transform pET28a backbone #2 into Mix-and-Go DH5ɑ cells to make copies
of the backbone from a methylating bacteria to help solve the DPN1 issue

## October 6th, 2020

### Allie, Kyra, Melody, Rachel

1. Amplification of CBM3a and pET-28a backbone #2.
2. Went with pET28a backbone #2 and CBM3a #1.
3. DPN1 testing with 3 different tubes of DPN1
   (DPN1 #1, DPN1 #2, DPN1 #3) and our backbone #2.

![](https://static.igem.org/mediawiki/2020/a/a1/T--UCSC--Notebook-October-6.jpeg)

## October 7th, 2020

### Allie, Kyra, Melody, Rachel

1. Plate #1 grew colonies with DPN1 #1 (the one we had been using the whole time).
2. Plate #2 didn’t grow colonies with DPN1 #2.
3. Plate #3 grew colonies with DPN1 #3.
4. This experiment proved that our DPN1 #1 wasn’t working causing issues during our golden gate.
   We decided to use DPN1 #2 from now on.
5. Made liquid cultures of colonies that grew on our backbone #2 plate.

### Nav and Taylor

Prepared samples for lyophilization (freeze-drying) and delivered them to Millhauser Lab.

### Claudia, Nav, Sophia

Amplified backbone again using mini prepped backbone and diluted primers
for two 50 µL solutions.

## October 8th, 2020

### Allie, Kyra, Melody, Rachel

1. Miniprepped pET-28a backbone #2 liquid cultures.
   Using these minipreps, ran amplification of backbone,
   performed DPN1 selection and Zymo Clean Up.
2. Ran GGA with negative controls, transformed GGA product into
   Mix-and-Go DH5ɑ cells and chemi-competent cells.

## October 9th, 2020

### Allie, Kyra, Melody

GGA plates have grown colonies, demonstrating success!

![](https://static.igem.org/mediawiki/2020/4/4e/T--UCSC--Notebook-October-9.jpeg)

##

###

## Week of October 12th, 2020 - October 18th, 2020

## October 12th, 2020

### Allie, Kyra, Melody

Colonies overgrew a little,
so diluted colonies from GGA plates (10/9) and spread them on LB/Kan plates.

## October 13th, 2020

### Allie, Kyra, Melody

1. Plates from Golden Gate restreak for CBM3a came out successful.
2. Made liquid cultures from restreaked GGA plates (10/12).

## October 14th, 2020

### Allie, Kyra, Melody, Rachel

1. Ran colony PCR using colonies from restreaked golden gate plates (10/12) and T7 primers.
2. Ran colony PCR products on a gel.

![](https://static.igem.org/mediawiki/2020/b/b2/T--UCSC--Notebook-October-14-1.jpeg)

### Kyra and Rachel

Ran 12 hour ethanol Soxhlet extraction on composite ion gel.

![](https://static.igem.org/mediawiki/2020/5/5e/T--UCSC--Notebook-October-14.jpeg)

## October 15th, 2020

### Allie, Kyra, Melody, Rachel

Made index plates from golden gate plates (10/12) in preparation for colony PCR.

![](https://static.igem.org/mediawiki/2020/6/60/T--UCSC--Notebook-October-15.jpeg)

## October 16th, 2020

### Allie, Kyra, Melody, Rachel

1. Selected colonies from the index plates (10/15) and ran colony PCR.
2. Ran a gel to determine size of colony PCR products.
3. Lanes 1-3 and 5-9 had bands at around 0.3 kb. Lane 4 had a band at 1.3 kb indicating
   our GGA reaction was successful.

### Kyra and Rachel

Retrieved sample from Soxhlet extraction and pressed between
2 glass plates to dry at room temperature for 4 days.

![](https://static.igem.org/mediawiki/2020/2/27/T--UCSC--Notebook-October-16.jpeg)

## October 17th, 2020

### Allie, Kyra, Melody, Rachel

1. Selected more colonies from our index plates (10/15) to run more colony PCR reactions
   to reaffirm results from previous day.
2. Ran colony PCR reactions on a gel. Lanes associated with 2 different colonies had
   a band at 1.3 kb indicating the CBM3a gene block was present.
3. Made liquid cultures of these 2 colonies from index plates made (10/15).

## October 18th, 2020

### Allie, Kyra, Melody

1. Miniprepped liquid cultures that were made previous day (10/17).
2. Linearized miniprepped products using XbaI and ran on a gel
   to confirm the complete size of GGA product containing the CBM3a gene block.
   The gel had bands both at near 6 kb (our desired region) and at around 3.5 kb,
   indicating that our miniprepped samples contained 2 colonies.
3. Restreaked new plates from index plates (10/15).

##

###

## Week of October 19th, 2020 - October 25th, 2020

## October 19th, 2020

### Allie, Kyra, Melody

Selected colonies from new index plates (10/18) and ran colony PCR.

![](https://static.igem.org/mediawiki/2020/0/04/T--UCSC--Notebook-October-19.jpeg)

## October 20th, 2020

### Allie, Kyra

Made liquid cultures from index plates (10/18).

## October 21st, 2020

### Allie, Kyra, Rachel

Miniprepped liquid cultures (10/20).

## October 22nd, 2020

### Kyra, Melody, Rachel

Nano-dropped miniprep GGA products and selected 4 samples to be sent out for sequencing.

## October 23rd, 2020

### Kyra, Rachel

Transformed the 4 samples selected for sequencing into electrocompetent BL21
in preparation for protein production.

##

###

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# Team:UCSC/Engineering

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# Engineering Success

Step by step, to success!

[![](https://static.igem.org/mediawiki/2020/e/e1/T--UCSC--down-arrow.png)](#Engineering-Process)

* [Engineering Process](#Engineering-Process)
* [Discover](#Discover)
* [Research and Design](#Research-and-Design)
* [Build](#Build)
* [Assay](#Assays-and-Future-Work)
* [Learn](#Learn)
* [Improve](#Improve)

## Engineering Process

With COVID-19 restricting lab access and creating other limitations,
it is more important than ever to thoroughly plan and design each piece of our project
so that it may come together smoothly despite time restrictions.
As we progress in our research, the design of our project will evolve and become
more advanced. We have achieved engineering success through the following the cycle:

#### Discover → Research and Design → Build → Assay → Learn → Improve

## Discover

Plastic waste is a widely discussed issue. We specifically focused on learning about
the huge plastic waste problem in the agricultural industry. Although plastic mulches
are a necessity in farming practices, they produce acres of plastic waste that have little
chance of making it to a recycling facility due to soil contamination on the plastic and
expensive shipping costs.

As our team learned more about this issue, a light bulb went off: we should engineer
a biodegradable thermoplastic using a cellulose producing microorganism that can save
growers money and protect the earth! With this solution in mind, we began designing our
research plan and our product goals.

## Research and Design

Upon starting our research, our team planned to test a range of chemical plasticizers
and copolymers. We began by modeling amorphous (non-crystalline) cellulose by testing
microcrystalline cellulose with various ionic liquids, producing cellulose that is similar
to the expected results of CBM binding. This model of our bacterial cellulose was used
in order to select plasticizers and copolymers in a timely fashion, since bacterial cellulose
can take up to ten days to form. Thanks to this work, once CBM expression begins we will
have a better idea of the plasticizer, copolymers, and cross-linking agents to incorporate.
We tested various chemical plasticizers such as sorbitol, glycerol, and more.
Additionally we have experimented with the inclusion of cross-linking agents
such as citric acid and glutaraldehyde in concurrence with chemical plasticizers.
These concepts are described in our
[Results page](https://2020.igem.org/Team%3AUCSC/Results).
Our team also met with professional chemists and polymer scientists, namely Dr. Peter Weiss,
Dr. Rebecca Braslau, and Dr. Doug Hayes, for advice on finding effective plasticizing
and decrystallizing agents.
Due to COVID-19 and the fires in California, our ability to continually do lab work was
hindered so saving time was a top priority. Using ionic liquids that mimic the effects
of CBM binding allowed us to understand the importance of the decrystallization of cellulose,
even without stable lab access.

Carbohydrate binding modules (CBMs) are regions on protein complexes such as cellulase
that attach to the fibrils of cellulose and induce structural modifications
[[1]](#ref-1).
CBMs have been shown to increase cellulose affinity to dyes, amplify tensile strength,
and produce other qualities like filtration abilities [[1]](#ref-1).
There are 80 families of CBMs, with multiple members of specific
CBMs in each family [[2]](#ref-2).
There is an impressive range of CBMs available to experiment with,
and previous work has shown that individual CBMs can add very different qualities to cellulose.
There is little published work on using a CBM to create a plasticized cellulose,
making our initial experimentation novel.

Despite the overwhelming variety of CBMs, we decided to test just two CBMs to begin with:
CBM3a and CBM2a. CBM3a was chosen since it is part of the
[2018 Toulouse iGEM team’s](https://2018.igem.org/Team%3AToulouse-INSA-UPS) Sirius
fusion protein, part [BBa\_K2668020](http://parts.igem.org/Part%3ABBa_K2668020).
We also selected Carbohydrate Binding Modules of Type 2a (CBM2a), because it has a slightly lower
binding affinity to cellulose, which we theorize will allow greater plasticizer incorporation
[[3]](#ref-3). CBM2a binds to both amorphous and
crystalline cellulose irreversibly, but its low binding affinity allows it to traverse
along the strands while never fully dissociating from the cellulose matrix.

| Part Name | Part Number | Part Type | Description | Designer | Length |
| --- | --- | --- | --- | --- | --- |
| Sirius: CBM3a - mRFP1 fusion (Sirius) | [BBa\_K2668020](http://parts.igem.org/Part%3ABBa_K2668020) | Coding region | CBM3a, mRFP, N- and C-Terminus linkers. | Younes Bouchiba | 1,349 bp |
| Carbohydrate Binding Modules of Type 2a (CBM2a) Fused to mRFP1 (CBM2a) | [BBa\_K3426000](http://parts.igem.org/Part%3ABBa_K3426000) | Coding region | CBM2a, mRFP, N- and C-Terminus linkers. | Kyra Eyerman | 2,371 bp |

It is vital that we not only account for the production of our plasticizer,
but also its integration with bacterial cellulose. Cellulose is a highly crystalline compound
which is composed of a dense fibril network that is held together by intra and intermolecular
hydrogen bonding. CBMs can interrupt these bonds and allow for the incorporation of our
plasticizing molecules. This is why it is important for us to find a CBM that can facilitate
the attachment of our plasticizing molecules.

### Design for the Future

Though we weren’t able to get to our desired final step, our team designed an experimental
process that will get us to our goal: a simple co-culture between our bacterial cellulose
producer and the organism producing our plasticizing chemical and CBM. Together these
organisms will synthesize our thermoplastic. *E. coli* will produce our CBM as
well as the plasticizing molecule of interest while *K. rhaeticus* produces the
bacterial cellulose.

We decided on creating a co-culture because the plasticizing agent can attach to nascent
cellulose strands as *K. rhaeticus* synthesizes them, effectively creating a plastic factory
in the media without the need for external interference. We theorize that the attachment
of a CBM and plasticizer right as cellulose strands are extruded will result in more
thorough incorporation throughout the cellulose matrix, in comparison with adding CBMs
in solution with fully formed crystalline cellulose.

While designing this co-culture, we understood the possible roadblocks such as competition
between organisms for nutrients in the media. The co-culture would function using quorum
sensing between the two organisms. Quorum sensing is the ability of one organism to detect
and respond to cell population density of the other organism by gene regulation.
A signaling molecule is used to send and receive signals in the culture, allowing the two
strains to live harmoniously without using up eachother’s carbon sources. This ensures one
organism doesn’t outcompete the other.

A co-culture is ideal for our project because it creates a sustainable biofactory of our
desired product. Our two strains can live harmoniously and actively produce our plastic bed
mulch without exhausting their resources in the media. Once formed, we can extract our plastic
and cure it without killing our culture in the process. This idea was inspired by the
[2016 Imperial College iGEM team](https://2016.igem.org/Team%3AImperial_College),
since they were able to create a co-culture of two engineered E.coli.
Their project included a quorum sensing, circuit-like interaction between their two strains.
The Imperial College of London was also able to create a co-culture between two engineered
*K. rhaeticus* strains that communicated using pSender and pReceiver plasmids,
which our team received from the college as a stepping stone to eventually create
communication between our bacteria. Imperial College’s contribution to iGEM also helped us
build our co-culture design.

## Build

After the design was outlined, we transitioned into heavily researching carbohydrate binding
modules as a method of decrystallizing cellulose and attaching our plasticizer molecule.
Bacterial cellulose is extremely rigid, so decrystallizing it is necessary in order to do the
proper modifications to create a thermoplastic.

At this point, our team branched into two main areas of interest: plasticizing molecules
and CBMs. We first explored the field of biopolymer additives including plasticizers,
cross-linking agents, and copolymers. Plasticizers such as glycerol and sorbitol are known
to increase tensile strength in cellulose-based polymers. Cross linking agents, such as
citric acid and glutaraldehyde are molecules that contain two or more reactive ends capable
of chemically attaching to specific functional groups. This would serve as a method to
increase linkage of a plasticizing molecule to cellulose. Additionally, citric acid has been
proven to reduce water vapor permeability, a characteristic we strive for in bed mulch films
[[4]](#ref-4) [[5]](#ref-5). Copolymers are produced by the copolymerization
of two or more different monomers. This allows for them to take on properties of their
constituent parts. So far, we have explored xanthan gum and PHAs.
Xanthan gum has been proven to increase tensile strength which will help to counteract
the brittleness of cellulose [[5]](#ref-5) [[6]](#ref-6).

In order to produce CBMs, we built off of the work of the
[2018 Toulouse iGEM team](https://2018.igem.org/Team%3AToulouse-INSA-UPS),
Cerberus. We designed a pET28a plasmid containing their fusion protein, Sirius,
or in the registry, [BBa\_K2668020](http://parts.igem.org/Part%3ABBa_K2668020).
This part contains a gene for CBM3a as well as a red fluorescent protein (mRFP).
Our plasmid was designed for Golden Gate Assembly using BsaI recognition sites to
construct our plasmid. We ordered our gene blocks from IDT and devised a plan to use
CBMs as a method of cellulose strand separation, allowing for incorporation of a plasticizing
molecule.

Our team also created a new part containing CBM2a fused to mRFP1,
[part number BBa\_K3426000](http://parts.igem.org/Part%3ABBa_K3426000).
We designed this protein part based on the Sirius part, since the
2018 Toulouse iGEM team blueprinted an assaying method utilizing the mRFP.
Our part contains the genes for CBM2a, an mRFP, and N- and C-terminus linkers.
We designed this part to be assembled into a pET28a plasmid using Golden Gate Assembly
to achieve scarless cloning with BsaI recognition sites. Our research design accounts for
comparison of CBM3a and CBM2a incorporation into cellulose using colorimetric assaying
of the mRFP with plain bacterial cellulose (BC) as our control. We plan on incorporating our
plasticizing molecules as well and studying how this may affect the CBM3a or CBM2a binding
affinities. We have been successful in cloning the Sirius part we ordered, but are still in
the process of cloning our new CBM2a part.

## Assays and Future Work

Since we weren’t able to assay the binding affinity of our CBMs and potential plasticizers
to cellulose, we designed what these experiments may have looked like.
Once the CBM has been expressed in BL21 cells, we plan to begin large scale protein production
and purification.

We will perform a binding assay using the
[cellulose pull down assay method](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Experiments)
the 2018 Toulouse iGEM team used on their CBMs to illustrate CBM binding to cellulose.
Fluorescence will be analyzed to understand the presence of molecular interaction between
cellulose and the CBM.

In addition, our team explored the use of bacterial polyesters, polyhydroxyalkanoates (PHA),
as a potential plasticizer for our future work. PHAs are a large family of biodegradable
polymers produced by bacteria. They possess the capability of acting as a plasticizing agent
to bacterial cellulose and are considered to be “microbial plastics”. PHAs can be synthesized
from *E. coli*, thus making it a good contender for the plasticizing molecule
in co-culture design.
[The 2018 Edinburgh iGEM team](https://2018.igem.org/Team%3AEdinburgh_OG), Valeris.ED,
utilized PHAs for their project to make a “biobased plastic” by constructing PHA synthesis pathways
by adding specific genes to *E. coli* to facilitate PHA synthesis.
Edinburgh also constructed a thermoplastic as an alternative to petroleum based plastic,
but through the use of by-products from whisky distilleries and pot ale (containing dead yeast,
proteins from barley, and mainly water), to produce PHA plastic in *E. coli*.
Opening an avenue of potential future collaboration between our team and Edinburgh
to understand PHA synthesis mechanisms within E.coli, but most importantly tackle plastic pollution,
is helpful to the creation of our own “biobased” thermoplastic.

## Learn

Our team was able to interview
[Dr. Oded Shoseyov](https://2020.igem.org/Team%3AUCSC/Human_Practices#oded-shoseyov),
an expert in CBM attachment and cellulose film production. When telling him about the
gene blocks we made for our CBMs, he told us to think twice about adding an mRFP gene
because they can hinder binding affinity of CBMs to cellulose. He told us it is because
the mRFP gene is a large protein that interferes with binding. He additionally pointed out
that the fluorescence will not allow us to quantify the amount of CBM binding, but rather
can only be used as a baseline detection method. He instructed us to remove the mRFP and
follow a different selection and quantification, which we have laid out in the next section,
Improvement.

Our cellulose-based film will be installed in farms around growing crops, so it is necessary
that the bacteria we are using in the production of the film do not contaminate crops or soil.
Due to our biology and engineering backgrounds, we know all the recombinant DNA used in
our project is not harmful to soil or humans because CBM and bacterial cellulose DNA already
exist in nature. However, we understand that GMO production is a sensitive topic and many
growers feel uncomfortable knowing our product could contain live bacteria and recombinant DNA.
With this in mind, we have developed a proposed curing process that involves dehydration of
the samples and conditions that should not allow the bacteria to survive. We will also ensure
that all recombinant DNA present in the film is denatured via UV radiation, enzymatic degradation,
or temperature exposure.

## Improve

Although we were not able to conduct the experiments related to the improvement
of the Sirius part, we did design the Golden Gate construct without the mRFP using Geneious.

| Part Name | Part Number | Part Type | Description | Designer | Length |
| --- | --- | --- | --- | --- | --- |
| Carbohydrate Binding Modules of Type 3a (CBM3a) - Improved | [BBa\_K3426001](http://parts.igem.org/Part%3ABBa_K3426001) | Coding region | CBM3a Sirius part from the 2018 Toulouse Team improved to remove the mRFP, N- and C-Terminus linkers. | Kyra Eyerman | 480 bp |

We also designed the protocols required for testing this plasmid design against bacterial
cellulose. The following is the method of selection and quantification Dr. Shoseyov
instructed us to go forward with instead of using the mRFP, starting with protein production:

[Selection and Quantification Protocol](https://static.igem.org/mediawiki/2020/5/5e/T--UCSC--Protocol-Protein_Production_Purification.pdf)

The advantage of this selection and quantification assay is it allows for accurate determination
of the maximum amount of CBM saturation of cellulose. Through this, we will be able to experiment
with varying amounts of CBMs and plasticizing molecules to achieve the optimum ratio between the two.
Additionally, this assay will help us understand the differences in affinity between CBM2a and CBM3a
that wouldn’t be observable simply using the cellulose pull down method used by the Toulouse 2018 team.
This is why it was also necessary for us to remove the mRFP, since it will not offer benefits to our
assays and will possibly hinder plasticizer incorporation.

## References

[[1] Carbohydrate Binding Modules: Biochemical Properties and Novel Applications](https://mmbr.asm.org/content/70/2/283.full)

[[2] Carbohydrate Binding Module Families - CAZypedia](https://www.cazypedia.org/index.php/Carbohydrate_Binding_Module_Families)

[[3] Measurements of single molecular affinity interactions between
carbohydrate-binding modules and crystalline cellulose fibrils.](https://pubmed.ncbi.nlm.nih.gov/23532050/)

[[4] Plasticizers Derived from Biomass Resources: A Short Review](https://www.mdpi.com/2073-4360/10/12/1303)

[[5] Natural-based plasticizers and biopolymer films: A review](https://www.sciencedirect.com/science/article/pii/S0014305710004763)

[[6] Structural, Thermal, Physical, Mechanical, and Barrier Properties of Chitosan Films with the Addition of Xanthan Gum](https://pubmed.ncbi.nlm.nih.gov/28218968/)

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# Team:UCSC/Experiments

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# Experiments & Protocols

All the work we have done!

[![](https://static.igem.org/mediawiki/2020/e/e1/T--UCSC--down-arrow.png)](#Member-Training)

### Throughout the summer we have worked on experiments to get us to our goal. Check out what we have done!

[Inoculation of *K. rhaeticus*](https://static.igem.org/mediawiki/2020/e/ef/T--UCSC--Protocol-Inoculation_of_Komagataeibacter_rhaeticus.pdf)

[Gelation of MCC with NaOH](https://static.igem.org/mediawiki/2020/6/6e/T--UCSC--Protocol-Gelation_MCC_NaOH.pdf)

[Preparation of Amorphous Cellulose](https://static.igem.org/mediawiki/2020/e/ef/T--UCSC--Protocol-Amorphous_Cellulose_Preparation.pdf)

[MCC Dissolution in NaOH and Urea](https://static.igem.org/mediawiki/2020/2/2e/T--UCSC--Protocol-MCC_Dissolution_NaOH_Urea.pdf)

[Plasticization with Glycerol](https://static.igem.org/mediawiki/2020/0/02/T--UCSC--Protocol-Plasticization_with_Glycerol.pdf)

[Yamanaka Media](https://static.igem.org/mediawiki/2020/6/68/T--UCSC--Protocol-Preparation_of_Yamanaka_Media.pdf)

[BC Dehydration](https://static.igem.org/mediawiki/2020/5/59/T--UCSC--Protocol-BC_Dehydration.pdf)

[LB/Cloramphenicol Media](https://static.igem.org/mediawiki/2020/0/0e/T--UCSC--Protocol-LB_Chloramphenicol_Media.pdf)

[LB/Kanamycin Media](https://static.igem.org/mediawiki/2020/b/b3/T--UCSC--Protocol-LB_Kanamycin_Media.pdf)

[Imperial Plasmid Reinoculation](https://static.igem.org/mediawiki/2020/3/3c/T--UCSC--Protocol-Imperial_Plasmid_Reinoculation.pdf)

[Glycerol Stocks for Plasmid Storage](https://static.igem.org/mediawiki/2020/a/a8/T--UCSC--Protocol-Plasmid_Glycerol_Stocks.pdf)

[Gel Electrophoresis](https://static.igem.org/mediawiki/2020/e/ec/T--UCSC--Protocol-Agarose_Gel_Electrophoresis.pdf)

[CBM3a Amplification](https://static.igem.org/mediawiki/2020/9/9f/T--UCSC--Protocol-CBM3a_Amplification.pdf)

[pET28a Amplification](https://static.igem.org/mediawiki/2020/e/e5/T--UCSC--Protocol-pET28a_Amplification.pdf)

[BC Cleaning](https://static.igem.org/mediawiki/2020/7/7c/T--UCSC--Protocol-BC_Cleaning.pdf)

[Dpn1 Selection](https://static.igem.org/mediawiki/2020/c/c3/T--UCSC--Protocol-Dpn1_Selection.pdf)

[DH5-alpha Transformation](https://static.igem.org/mediawiki/2020/0/0f/T--UCSC--Protocol-Mix_and_Go.pdf)

[HS Media](https://static.igem.org/mediawiki/2020/8/85/T--UCSC--Protocol-HS_Media.pdf)

[Colony PCR](https://static.igem.org/mediawiki/2020/1/1c/T--UCSC--Protocol-Colony_PCR.pdf)

[Liquid Cultures](https://static.igem.org/mediawiki/2020/5/58/T--UCSC--Protocol-Liquid_Cultures.pdf)

[Xba1 Restriction Digest](https://static.igem.org/mediawiki/2020/f/fd/T--UCSC--Protocol-XbaI_Restriction_Digest.pdf)

[X-ray Diffration](https://static.igem.org/mediawiki/2020/a/ab/T--UCSC--Protocol-BC_Xray_Diffraction.pdf)

[Electrocompetent Cell Transformation](https://static.igem.org/mediawiki/2020/b/b4/T--UCSC--Protocol-Electrocompetent_Cell_Transformation.pdf)

[CBM2a Assembly](https://static.igem.org/mediawiki/2020/1/1f/T--UCSC--Protocol-CBM2a_Assembly.pdf)

[Xanthan Gum](https://static.igem.org/mediawiki/2020/8/88/T--UCSC--Protocol-MCC_Xanthan_Gum.pdf)

[Lyophilization](https://static.igem.org/mediawiki/2020/c/c0/T--UCSC--Protocol-BC_Lyophilization.pdf)

[Chemicompetent Cell Transformation](https://static.igem.org/mediawiki/2020/4/45/T--UCSC--Protocol-Chemicompetent_Cell_Transformation.pdf)

[BL21 Cell Transformation](https://static.igem.org/mediawiki/2020/4/4f/T--UCSC--Protocol-BL21_Cell_Transformation.pdf)

[Protein Production Purification](https://static.igem.org/mediawiki/2020/5/5e/T--UCSC--Protocol-Protein_Production_Purification.pdf)

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# Team:UCSC/Results

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[Home](/Team%3AUCSC)

Project

[Description](/Team%3AUCSC/Description)
[Implementation](/Team%3AUCSC/Implementation)
[Model](/Team%3AUCSC/Model)
[Engineering](/Team%3AUCSC/Engineering)
[Proof of Concept](/Team%3AUCSC/Proof_Of_Concept)
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[Results](/Team%3AUCSC/Results)

Human Practices

[Integrated Human Practices](/Team%3AUCSC/Human_Practices)
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[Notebook](/Team%3AUCSC/Notebook)
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Team

[Members](/Team%3AUCSC/Team)
[Attributions](/Team%3AUCSC/Attributions)

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# Results

The milestones we made in our wet-lab work!

[![](https://static.igem.org/mediawiki/2020/e/e1/T--UCSC--down-arrow.png)](#Genetics)

* [CBM Results](#Genetics)
* [Plasticizer Results](#Plasticizers)
* [Assay Results](#Assays)

## CBM Results

### CBM3a Experiments

### Amplification of Gene Block and Backbone:

**Goal:** To PCR amplify pET28a(+) backbone #2 and the new CBM3a gene block we
received from IDT to attach Bsa1 recognition sites for construction of our plasmid through
Golden Gate Assembly.

**Result:** Long range amplification of the pET28a(+) backbone required
troubleshooting. We were working with two different pET28a(+) backbones (denoted as #1 and #2).
Throughout our amplification process, we learned pET28a(+) backbone #1 might have some mutation
or off target annealing causing issues with sizing of the amplification, which would come out
to be around a 1.3 kb band, shown in Figure 1.

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