Revision as of 15:06, 14 August 2018

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 re-inoculated 30μL each of our 10 samples of cells containing plasmid 1 into an LB and chloramphenicol broth and put them in the shaker to grow overnight.
Members: Kaylee and Elisa

7/22/18

We made glycerol stocks of our 10 samples by combining our media with cells and 60% glycerol in a 2:1 ratio. We then 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

For InterLab, we inoculated 2 positive control BBa_I20270 cultures and 2 negative control BBa_R0040 cultures for the CFU study in LB and chloramphenicol and put them in the shaker to incubate overnight. We also made enough LB+Chlor plates for the CFU study.
Members: Kaylee and Liz

7/24/18

For InterLab, we continued work on the procedure and used our overnight cultures from the positive and negative control samples. We diluted them 1:8 in LB+Chloramphenicol and measured their OD600 in the plate reader, along with 2 control LB+Chloramphenicol samples. We followed the protocol to dilute the rest of the samples in triplicate so they would have an OD600 of 0.1 in the plate reader. We then diluted those 12 samples according to the protocol and plated 100μL of the appropriate dilutions on LB+Chlor plates to incubate overnight so we can count colonies tomorrow.
Members: Brittany and Kaylee

We reran the digested samples of plasmid 1 on 0.7% gel.
Members: Elisa

We started amplifying plasmid 2 using the correct primers.
Members: Elisa and Liz

7/25/18

For InterLab, we counted our grown colonies on our 36 plates. We then performed the CFU/mL calculations and submitted InterLab Form IV.
Members: Kaylee and Brittany

We reran our digested plasmid 1 with the restriction enzyme BamHI on a 0.7% gel. We redigested plasmid 1 with DpnI and began making more pSB1C3 backbone in case our gel does not work correctly or give us the expected results.
Members: Elisa

7/26/18

We re-amplified and re-ran the plasmid 2 blocks.
Members: Liz

We made a new pSB1C3 backbone from 23-O in the 2018 kit 7. We also redid PCR overlap extension for 2/3 and 4/5 and ran it on a 1% gel. We extracted the correct sequences from the gel and reran the new pSB1C3 on a 1% gel.
Members: Elisa

We transformed and cultured 4 different pSB1C3 plasmid backbones to try to put our parts into. We put them in the incubator to grow overnight.
Members: Kaylee and Elisa

7/27/18

We retransformed and cultured 4 different pSB1C3 plasmid backbones.
Members: Kaylee and Elisa

7/28/18

We inoculated colonies from the plasmid 1 plates.
Members: Elisa

7/29/18

We did the plasmid miniprep on the inoculated cultures and started new inoculations for glycerol stocks of the cells.
Members: Elisa and Brittany

Week 9

7/30/18

We redid digestions of plasmid 1 using the 4 different pSB1C3 backbones several times using the restriction enzymes EcoRI, PstI, and BamHI. We also reinocculated colonies from the plate with the backbone with a red fluorescent indicator with LB+Chlor as well as IPTG. Finally, we made glycerol stocks of plasmid 1 for the four pSB1C3 backbones.
Members: Elisa

7/31/18

We did PCR overlap of p1-1 and p1-2 to create PETase. We also did PCR overlap of 2/3 and 4/5 together, ran the parts on a 0.7% gel, and extracted PETase. 2/3 and 4/5 were incorrect. We then used colony pcr on MG1655 to extract glycolate oxidase using colony PCR.
Members: Elisa and Brittany

8/1/18

We performed PCR overlap of plasmid 2 parts 3.1-3.2 & 4-5 and ran the results on a gel. We then extracted the bands.
Members: Liz

We also redid the amplification of plasmid1 part1 for NEBuilder, ran plasmid 1 part 1 and our glycolate oxidase sequence on a gel, and extracted the correct parts. We performed the NEBuilder protocol for PETase and glycolate oxidase and redid PCR overlap for plasmid 1 parts 2/3/4/5.
Members: Elisa

8/2/18

We recultured plates with PETase and glycolate oxidase, reamplified our PETase and ran it on a 1% gel, and inoculated our cultures that had glycolate oxidase and PETase.
Members: Elisa

For plasmid 2, we performed the NEBuilder protocol on the plasmid 2 parts and transformed it into competent 5-alpha cells. We are incubating the results overnight.
Members: Liz

8/3/18

We inoculated cultures of plasmid 2.
Members:Liz

Week 10

8/6/18

We found the new Glycolate Oxidase sequences because we realized we only had taken the sequence for the D subunit. We designed primers for the D, E, and F subunits for submission as well as insertion into the pETDuet vector. We made more PETase composite, made more pSB1C3 linearized backbone, and ran PETase on 0.7% gel.
Members: Elisa and Kaylee

We performed plasmid mini prep on the inoculated cells with plasmid 2 for both backbones pSB3K3 and pSB1C3. We also inoculated more colonies to make glycerol stocks of each and let them grow overnight.
Members: Liz

8/7/18

We digested more PETase, ran it on a 0.7% gel, and extracted it. We digested pSB1C3 backbone and PETase with EcoRI and PstI and digested the NEBuilder positive control. We ran the digested pSB1C3 and PETase on a 1% gel and extracted pSB1C3. The PETase sequence did not work correctly on the gel.
Members: Elisa and Kaylee

We ran restriction digest on plasmid 2 using EcoRI and PstI enzymes. We also made glycerol stocks of the plasmid 2 for each backbone.
Members: Liz

8/8/18

We made more PETase, redigested PETase with XbaI and PstI, transformed pSB1C3+Red fluorescent protein into 5Alpha cells, and plated the transformed cells on chlor. plates.
Members: Elisa and Ariel

We assembled plasmid 2 with a pSB1C3 backbone. We also transformed the plasmid into 5Alpha cells using the NEBuilder protocol. We inoculated the cells and plated them on LB+chlor plates to grow overnight.
Members: Liz and Kaylee

8/9/18

8/10/18

Week 11

8/13/18

We digested pSB1C3 backbone with EcoRI and PstI, pET-Duet backbone with BamHI and XhoI, and our isolated glycolate oxidase parts with EcoRI and PstI for the one isolated with primers to be put into pSB1C3 and with BamHI and XhoI for the one isolated with primers to be put into pET-Duet. We ran all our digested parts and uncut versions of them on a 0.7% gel, and were able to extract the pET-Duet backbone and the glycolate oxidase to go into pSB1C3.
Members: Kaylee and Ariel

We also digested our 5 plasmid 2 chosen colonies with EcoRI and PstI and ran them on a 1% gel. Unfortunately, the bands did not appear where we expected them to.
Members: Liz and Kaylee

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 6 g Tryptone, 3 g yeast extract, 9 g agar, and 6 g 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 50 mg/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.

To make liquid media stock, the same procedure can be followed without adding agar or antibiotic and then storing in a 4°C fridge.

Adapted from Dr. Irene Reizman and Prather Labs, MIT

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°C 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 Flask

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.

Adapted from Dr. Irene Reizman and Prather Labs, MIT

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
1X TBE Buffer
Gel Red dye (10,000x)
Loading dye
Electrophoresis apparatus
DNA

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 .50 g Agarose to 50 mL TBE Buffer
To make 1.5%: use .75 g Agarose to 50 mL TBE Buffer
To make 2%: use 1 g Agarose to 50 mL 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 1 µL of Gel Red to the flask. Ensure the mold is turned so the walls are keeping the gel from running out. Pour the solution into the mold and add the well maker.

Running Gels
Rotate the "submarine" (the piece holding the gel) so that the wells are closer to the black end ("run to red"). Fill the apparatus with 1x TBE buffer until it barely covers the top of the gel. Fill the wells with 10 µL of the desired ladder(s) and 10 µL of the desired DNA, mixed with 4 µL of loading dye. Put the lid on and hook up the red and black cables, then turn on the electricity to let the gels run at an appropriate voltage. Let the gels run out, but do not let the dye go all the way to the other end of the gel.

Adapted from Dr. Irene Reizman and Prather Labs, MIT

PCR Overlap

We followed the protocol from OpenWetWare for our PCR Overlap
https://openwetware.org/wiki/PCR_Overlap_Extension

Colony PCR

Materials

Taq Polymerase
DNA
Appropriate Primers
DI H2O
Plate containing colonies of bacteria

Procedure

Fill a sterile 1.5 mL centrifuge tube with 20 µL of sterile DI H2O. Take a fesh sterile pipet tip and lightly touch a colony on the plate. Pipet the tip up and down in water to create a dilute cell suspension. Run a PCR reaction by adding reagents and cell suspension to a specified volume:

10 µL Taq Polymerase (2x concentration)
0.4 µL upstream primer
0.4 µL downstream primer
7.2 µL DI H2O
2 µL colony suspension

Run PCR following these steps:

Adapted from Dr. Irene Reizman and Prather Labs, MIT

NEBuilder Kit

We followed the protocols from New England BioLabs to assemble our parts into plasmids. Our procedures for gel extraction, plasmid miniprep, transformations, and more came from New England BioLabs.
http://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2621.pdf

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.

Skype Call with University of Michigan Team - 7/25/18

Ariel and Brittany met with University of Michigan iGEM team on video call to talk with them about their project. They were working with a Cas9 system and had questions about how it could be modeled. We were able to give them initial steps on approaching the problem and explained mass-action kinetics to them. After the meeting, we followed up with an email with resources we used for our model and a presentation breaking down simple generic examples of genetics and enzyme kinetics.

Follow up with Michigan State Team - 7/31/18

On July 31st, Brittany, Ariel, and Elisa did a follow up meeting with the iGEM team from Michigan State University to talk about the model we created for them. They understood the model presentation we created and sent to them, so they had few questions. We are going to stay in contact in case they encounter questions about running the code and/or finding parameter values. We also discussed new collaboration ideas for them to help us get parts into our plasmid. As the lab work has not progressed to the point where we can use their scanning electron microscope, they are willing to try helping us get plasmid 1 created using Gibson Assembly.

Skype Call with Yale Team - 8/6/18

Kaylee, Brittany, and Ariel skyped with the Yale iGEM team to talk about our projects. Both of our teams are making cells that secrete the enzymes PETase and MHETase to break down PET plastic. They have had more success in the lab than we have, so we were able to ask some questions about how they got their plasmid working. They also suggested we try Gibson Assembly and ligation independent cloning for our plasmid. Additionally, they told us they were finding more success with the yebF secretion tag than some of the others they had tried, like our pelB secretion tag. We were able to answer some questions for them about the modeling we had done, our outreach plans, and other work on our wiki.

@@ Line 585: / Line 585: @@
     <div class = "week"> Week 11 </div>
        <div class = "date" id="81318"> 8/13/18 </div>
+         <div class = "day" id="813"> We digested pSB1C3 backbone with EcoRI and PstI, pET-Duet backbone with BamHI and XhoI, and our isolated glycolate oxidase parts with EcoRI and PstI for the one isolated with primers to be put into pSB1C3 and with BamHI and XhoI for the one isolated with primers to be put into pET-Duet. We ran all our digested parts and uncut versions of them on a 0.7% gel, and were able to extract the pET-Duet backbone and the glycolate oxidase to go into pSB1C3. <br> Members:
+Kaylee and Ariel <br><br> We also digested our 5 plasmid 2 chosen colonies with EcoRI and PstI and ran them on a 1% gel. Unfortunately, the bands did not appear where we expected them to. <br> Members: Liz and Kaylee </div>

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Description

Design

Experiments

Notebook

Results

Achievements

InterLab

Attributions

Lab Notebook

Protocols

Events