Notebook

Lab Notebook

Week 1

6/6/18

Our first day in lab! After some training earlier in the week, we began our work in the actual lab. We learned how to make different broths and agars, and made Luria broth (LB) plates with chloramphenicol and LB plates with ampicillin.
Members: Ariel, Brittany, Emilie, Liz, Lining, Kaylee, and Elisa

Week 2

6/12/18

We compiled information about possible part sequences to use in our project and ran them through the Parts Registry to see what we can and cannot submit as our own parts. We then created several different sequences of one of our main parts, PETase until we decided upon an optimized mutant version that was recently discovered in April 2018. We created some rough sketches of what our complete plasmid would look like. In lab, we created competent cells out of E. coli strains MG1655 and BL21(DE3).
Members: Elisa and Lining

Week 3

6/19/18

We discovered that our parts were too large to fit on a single plasmid, so two were developed. One uses pSB1C3 as the backbone, and the other uses pSB3K5.We decided to have one plasmid have the PETase and MHETase enzyme sequences while the second plasmid would have sequences for glycolaldehyde dehydrogenase, glycolaldehyde reductase, glycolate oxidase, and malate synthase.
Members: Elisa and Lining

6/21/18

We emailed the researchers about the optimised PETase and did more research on previous teams who had tried to solve the plastic problem the world faces today. We then transformed the plasmids into competent 5Alpha cells to culture, but discovered that pSB3K5 does not allow the cell to grow on the plate we wanted.
Members: Elisa and Lining

Week 4

6/25/18

We decided to use pSB3K3 as the other backbone to replace pSB3K5. We then created linearized backbones by digestion. We started to develop primers so that we could amplify our parts once we received them.
Members: Elisa and Lining

6/26/18

We sent out our part sequences to be synthesized through IDT, using our two plasmid ideas. The parts all had about 30 base pairs of overlap so we could put them together in the cell. We also designed our parts so there is a ribosomal binding site between the start of each enzyme, so they can be transcribed and translated into proteins individually if need be.
Members: Elisa and Lining

Week 5

7/6/18

IDT was unable to synthesize all of our DNA sequences, so we had to break several of our sequences into smaller ones. Due to this, we had to create new primers for amplification of our new blocks. We also practiced running an SDS page, making sure that we had all the proper chemicals to do so.
Members: Elisa and Lining

Week 6

7/12/18

We rehydrated our primers that were delivered from IDT so they would be at a 10x concentration.
Members: Ariel and Elisa

7/13/18

We calibrated the plate reader using serial dilutions of fluorescein for InterLab.
Members: Brittany

Week 7

7/16/18

We diluted primers for plasmid 1 to 100x and used those primers to amplify our plasmid 1 parts. We also made more chloramphenicol plates, transformed the plasmids for InterLab into competent NEB5-alpha cells, replated cells for InterLab onto chloramphenicol plates, and created a glycerol stock for our InterLab cells.
Members: Brittany and Elisa

7/17/18

We inoculated cultures of transformed InterLab cells. We performed the InterLab Absorbance Calibration measured at 630 nm. We also performed the InterLab microsphere calibration measured at 630 nm. Finally, we transformed the part from well L4 into NEB5-alpha cells.
Members: Brittany and Liz

We ran parts 1-4 and 1-5 in a 2% gel and extracted part 1-4 (1-5 was not amplified.) We also designed new primers for the extraction and submission of parts that will allow us to put our parts in the pSB1C3 backbone for submission to the registry.
Members: Elisa

7/18/18

For InterLab, we inoculated cultures of 4L and made glycerol stocks of the other InterLab parts.
Members: Brittany

We ran parts 1-5 A,1-5 B, and 1-5 C with different reverse primers for the amplification of each on a 2% gel. We also extracted part 1-5 A. We developed the protocol we would use for the overlap PCR of our shorter parts, including combining 1-2 with 1-3 and combining 1-4 with 1-5.
Members: Elisa

7/19/18

We performed PCR overlap of parts 1-2 with 1-3 and 1-4 with 1-5. We ran the PCR products on a 1% gel and extracted the correct bands. We then followed the NEBuilder Kit protocol to create one plasmid with all of our parts using pSB1C3 as the backbone. This should be our first plasmid containing the PETase and MHETase enzymes. We plated 5 plates of our E. coli with the plasmid.
Members: Elisa

We also finished the rest of the InterLab and filled out all the forms with our data to be submitted.
Members: Brittany

7/20/18

We picked 4 colonies per plate and placed two in each of 10 tubes containing LB media and chloramphenicol. We inoculated them overnight.
Members: Elisa

7/21/18

We created glycerol stocks of the 10 samples of cells containing plasmid 1 and put them in the shaker to grow overnight.
Members: Kaylee and Elisa

7/22/18

We finished preparing the glycerol stocks of the samples and put them in the -80 Celsius freezer for storage.
Members: Kaylee and Elisa

Week 8

7/23/18

We lysed the cells and performed the plasmid miniprep of our samples to isolate their DNA and prepare them to run on a gel. We also made a 2% gel to run our digested plasmids on.
Members: Elisa and Kaylee

Week 9

Week 10

Week 11

Protocols

Making Luria Broth Plates

This is the protocol for making LB gels. The measurements given can make approximately 24 plates. If fewer plates are wanted, the measurements can be halved to make approximately 10-12 gels.

Materials

Large bottle with a screw-on lid
Stir bar & Stir plate
DI H2O
Tryptone
Yeast Extract
Agarose
NaCl
pH meter, neutral (pH 7) standard
2M NaOH
Dropper/ Pipette
Antibiotic (Ampicillin/ Cloramphenicol)
Sterile plates

Procedure

Fill a large bottle with lid with 570 mL of DI H2O. Insert a stir bar and place the bottle on the stir plate. Mix in 6g Tryptone, 3g yeast extract, 9g agar, and 6g NaCl. Ensure the solution has been mixed thoroughly and standardize the pH meter with the neutral standard solution. Add 2M NaOH to the solution dropwise until the pH reaches 7.

Check the water level in the autoclave and put autoclave tape on the bottle. Ensure the lid is on but not tightened. Put the bottle in the autoclave and tighten the door. Set it for the liquid cycle and wait until the cycle is over and the pressure reaches 0 to open the door.

Put the bottle back on the stir plate and turn it to a low setting to prevent bubbles forming. Let the mixture cool until you can touch the bottle for several seconds comfortably. Add 600 µL of the chosen antibiotic (at 50mg/mL) to the bottle. Label all plates before you begin to pour. Wearing a glove, use the aseptic technique to pour the media into the plates and leave them to solidify.

Dry Ice Baths

There are a few uses for dry ice baths in biology. They are typically made of 70% ethanol and dry ice so that the solution gets extremely cold without immediately evaporating. Dry ice baths are used for snap-freezing stock cells that are typically stored in a -80 freezer, or to cool down various things in the lab.

Materials

95% ethanol (this is what we had in stock but any percentage can be used if diluted correctly)
Pressurized CO2 container
Dewar

Procedure

Diluting 95% ethanol The basic ratio is approximately 70 mL of 95% ethanol diluted with 25 mL of water. This ratio can be multiplied for the volume necessary for the dewar, which is approximately 500-550 mL of solution. This turns out to be 350 or 420 mL of 95% ethanol mixed with 125 or 150 mL of pure water, respectively.

Carefully make dry ice or seek help from someone who can. Put it in a safe container. Not a lot of dry ice is necessary to cool down the ethanol in the dewar. Add the dry ice a LITTLE at a time, so the ethanol doesn't bubble over and make a mess. Do not handle dry ice without a glove or something to protect your skin or breathe it in. Put the lid back on the dewar to keep the solution cool and prevent evaporation.

Gel Electrophoresis

Gel electrophoresis is a quick way to determine the relative sizes of DNA pieces. When compared to a known ladder, gel electrophoresis can be used to determine if restriction enzymes cut in the predicted places or if separate DNA pieces combined together correctly during PCR.

Materials

Agarose
Flask
1X TBE Buffer
Gel Red dye (10,000x)
Loading dye
Electrophoresis apparatus
DNA
Restriction Enzymes
DI H2O

Procedure

Add DI H2O to a flask
Add Agarose
To make .5%: use .25 g Agarose to 50 mL TBE Buffer
To make 1%: use .50g Agarose to 50mL TBE Buffer
To make 1.5%: use .75 g Agarose to 50mL TBE Buffer
To make 2%: use 1g Agarose to 50mL TBE Buffer

This table was adapted from http://schepartzlab.yale.edu/intranet/protocols/AgaroseGelElectrophores.pdf

Add 1x TBE buffer and microwave until it begins to boil. If the solution is not totally clear (i.e. still has swirls in it) heat it again. Let the solution cool for a couple minutes and add Gel Red to the flask. (Add .5uL of 1000x Gel Red to 2% gel mixture.) Ensure the mold is turned so the walls are protecting the gel from running out. Pour the solution into the mold and add the well maker.
To prepare the DNA Digest Enzymes:
Add 1uL Buffer, 7uL DI H2O, 1uL of the DNA sample, and 0.5 uL of each selected restriction enzyme. The enzymes should begin cutting the DNA.

FINISH THE BELOW PROCEDURE AND EDIT AS NEEDED. NOT DONE!!

Running Gels Rotate the "submarine" (the piece holding the gel) so that the wells are closer to the black end ("run to red") Fill apparatus with 1x TBE buffer until it barely covers the top of the gel Fill wells with Ladder and desired DNA mixed with loading dye Put lid on and hook up cables, then turn on electricity to let gels run

Events

Midwestern Meetup - 6/30/18

On June 30th, Elisa and Emilie traveled to Michigan State University for the Midwestern iGEM Meetup. We spent the morning on a tour of the university’s botanical gardens learning about a range of plants and the history behind the gardens. After lunch, each team gave a short presentation and answered questions about their project. Then, we went on a tour of the Michigan State University iGEM team’s lab and facilities. Through this meetup, we made connections with other teams and have already started collaborating with the Michigan State team.

Ampacet Visit - 7/3/18

On July 3rd, Emilie and Liz spoke with a representative from Ampacet in Terre Haute about our project. As a plastics expert, Dr. Jared Tatum explained the processes that are done at Ampacet. While discussing our project, Dr. Tatum offered the team PET plastic samples for our experiments. He will be giving us pure PET bottles, PET pellets, and PET powder. After explaining our experimental protocol ideas, Dr. Tatum advised us to change our procedures. Our original idea had been to let the bacteria eat the pellets as their only carbon source and observe the change. However, Dr. Tatum advised that we use the powder instead of the pellets because of the greater surface area to volume ratio. Additionally, he informed the team about the effect that humidity plays on the characteristics of pure PET. He encouraged us to monitor the humidity during our experiments and note any change in results based on a difference in humidity. Because pure PET is so susceptible to changes based on humidity, PET used in disposable water bottles contain a great amount of additives to stabilize the plastic and ensure long shelf life. Based on this new information, we began designing an experiment that would determine the effects of these additives on the ability of the bacteria to breakdown the PET.

Skype Call with Michigan State University iGEM - 7/9/18

On July 9th, Ariel, Brittany, Kaylee, and Elisa held a Skype conference with the Michigan State University team. During the Midwest Meetup, they showed interest in collaborating with us on lab and modeling aspects. We learned from the Skype meeting that they needed help creating a mathematical model and protein visualization model for their active site mutagenesis of ACC Deaminase. As our team had skills in creating mathematical models, we offered to help them create a system of differential equations to describe their system’s kinetics and genetics. The protein visualization was not something that we were very familiar with, so they decided to pursue other venues for help on that. In return, they offered to run a couple of our experiments involving their scanning electron microscope, as the one in our lab is not as sophisticated. After the call, they followed up with research articles with information we requested as necessary for building the model. We hope to have another call to discuss our results and coordinate our needs for the scanning electron microscope.

Survey Revision with Dr. Timothy Chow - 7/19/18

On July 19th, Kaylee, Emilie, and Ariel met with Dr. Timothy Chow to discuss our community survey. He gave us some great suggestions on our survey draft, including that at the beginning, we should include why people should take the time to fill out our survey. He discussed the plausibility of reaching a large enough population to consider the sample a global representation, and we decided we will stick to a more limited population size, trying to reach staff and students from the four colleges in our community as well as other members of our local community, Terre Haute, Indiana. He also gave us advice on how we could shorten our survey while still obtaining the information we’re interested in by having respondents select one of our three implementation methods at the beginning of the survey and only answering further questions about that particular method. He presented us with some local contacts who might be able to help distribute our survey in the community. Finally, he helped us identify the questions that might be too broad or unclear to respondents. He suggested in instances where we refer to “current recycling methods,” we should provide an example for those who might feel unaware of recycling methods.

Recycling Interview with Dr. Diane Evans - 7/19/18

On July 19th, Ariel and Brittany met with Dr. Evans about her Six Sigma class and their campaigns to make the campus more sustainable. The class is geared towards teaching students through a 10-week long project, quantitatively measuring, and statistically improving a policy. Two of the class projects included decreasing the percentage of waste produced on campus by recycling plastic bottles and substituting plastic straws with biodegradable ones. Both projects have been successful, but the plastic straw project especially was a hit. She has been interviewed on local TV networks and had several articles published about the work. She worked closely with our food service provider and they have recently announced instituting biodegradable straws across all their centers. In our interview, Dr. Evans talked about her inspiration, the generally encouraging feedback about projects, challenges the class met, and what the next steps would be for the class projects. She also provided contacts for more recycling people in the community and insight based on her experience on probable responses to the three proposed implementation methods for our project.

Team:RHIT/Notebook

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Notebook

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InterLab

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Lab Notebook

Protocols

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