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Notebook

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

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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.

 

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.

 

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.

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.

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.

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.

 

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.

 

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.

 

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!

 

 

 

 

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.

Kyra and Rachel

Ran 12 hour ethanol Soxhlet extraction on composite ion gel.

 

October 15th, 2020

Allie, Kyra, Melody, Rachel

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

 

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.

 

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.

 

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.