UNL 2017

Helping reduce methane emissions from livestock

Notebook

Detailed protocols can be found on our experiments page.

Week 1

Training week 1:
All team members participated in a two week training program. We learned how to create LB Broth, and agar and how to create and store agar plates. Training was done on how to inoculate an LB culture and how to perform chemical and electro transformations. PCR (Polymerase Chain Reaction) was performed on a mCherry gene for a preliminary lesson on how the process is done.

PCR of mCherry:

Week 2

Training week 2:
The team training was continued this week. We worked on restriction enzyme digestion on the mCherry gene in preparation for molecular cloning of the gene into a different vector. Training was done on setting up agarose gel and using the electrophoresis equipment to isolate the mCherry gene.

Restriction enzyme product:

PCR temperature gradient product:

Colony PCR product of mCherry gene:

Amplification PCR product:

PCR amplification for sequencing:

Week 3

We spent this week waiting for our DNA parts to be shipped. In the meantime we had deliberations and meetings with Dr. Samodha C. Fernando to gather details about the implementation of our project. Our project was researched in more detail. We started to implement Dr. Fernando’s feedback into our project. We also used this week to look more into possible human practice events.

Week 4

While we were waiting for our parts to be shipped and continuing research we had a meeting with Quantified Ag (cattle based startup) to get more information about how the cattle industry is ran and how our project could fit in. We started our visits to a local museum and set up a booth with information on global warming and our iGEM project. We had a few skype calls with other iGEM teams from Pakistan, Mexico and Boston, to talk about our projects and possible collaborations.

Week 5

Nic and Tyler prepared the plasmid pSB1C3 by culturing and miniprep-ing to isolate the plasmid. The plasmid was digested at the XbaI and SpeI sites in preparation for SLIC ligation. SLIC was performed after the three gene inserts were cut at the same positions. H (Nitrite Reductase nrfAH) and B(Bromoperoxidase) were transformed while nrfA had to go through the SLIC process again.

Restriction Digest of pSB1C3:

Week 6

Tyler and Nic prepared glycerol stock of the inoculated colonies from the “B” and the “H” transformed cultures. Cultures were then created from the stock and the plasmid was purified. The B plasmid was digested at the AatII and HindIII binding locations, whereas the H plasmid was digested at the BpvcI and SalI binding sites. Gel electrophoresis showed that the size of the cut gene was not at the correct locations. PCR on the gene blocks was tried anyway in preparation for sequencing. The A (Nitrite Reductase nrfA) gene was transformed and a glycerol stock was made after culturing.

Restriction Enzyme Digest to test for successful transformation:

Week 7

Jessica performed PCR on A, B, and H samples so that the team could get a higher concentration for sequencing. The team learned from sequencing that A was possibly correct so we set it aside for further sequencing. B and H were likely incorrect so we went through steps to perform SLIC again. Nic digested the B and H vectors and Jessica digested the RFP to make an empty vector. After that Nic performed the SLIC procedure and then transformed our newly cloned B and H plasmids while Jessica learned how to do protein purifications and western blots. This week Jessica also performed colony PCR on the newly cloned B colonies. One promising match was found and was further sequenced.

PCR amplification of each gene insert: A, B, H:

Documentation of which sections were extracted for DNA recovery and sequence preparation:

Justin's training protein purification:

Restriction digest on vectors B and H using XbaI and HindIII:

Restriction Digest of pSB1C3-RFP using XbaI and SpeI:

Electrophoresis performed on whole vector from glycerol stocks: B1,B2, H1, and H2:

Week 8

More restriction enzyme digests and sequencing were done by Nic and Jessica to gather more information to decide if B and H were cloned correctly. While this was being done more empty vector was prepared by Nick Flaxbeard just incase we had to do SLIC again. Sequencing results for A were promising so Jessica performed a protein purification on the A1 colony while continuing to send in sequencing samples for the B8 colony.

Restriction digest using...B10:

PCR amplification on B8 and on the H insert:

PCR Amplification of A1, B1, H2:

Protein Purification A1:

Failed H insert PCR amplification:

Week 9

Jessica performed multiple gradient PCRs on H to test if it could be amplified. These were not working so our team felt safe to assume that H was not cloned properly and Nic began working on recloning H using a different method. While this was being done Nick Flaxbeard started characterizing the A1 colony using the Nessler's test.

Another failed PCR amplification of H:

B8 PCR Amplification for sequencing:

Week 10

More sequencing samples for A and B were sent out by Jessica. Nic performed a restriction digest of the H insert for restriction digest cloning. The sequencing samples for the B8 colony proved to be very promising so Jessica performed a protein purification and western blot on the B8 colony that did not prove successful. This was deemed to be because the protein was producing at its natural level of expression which was very low. Our team decided that it needed to be cloned into an inducible vector.

Failed couple digestion using XbaI and SpeI*:

Protein purification of B8:

Western Blot of B8 protein purification:

Same Western Blot after washing in nanopure water overnight:

Week 11

This week marked the start of our school year. A week long break from the lab was taken during this transition.

Late August/September/October

Jessica performed protein purifications and an enzyme assay for B8 while Nick Flaxbeard worked on characterizing A using the Nessler’s test. Jessica finished sequencing A. The results showed a deletion in the sequence. Our team decided to no longer pursue the characterization of A and instead we worked on completing the interlab study. Nic worked on using restriction digest cloning to reclone H. Nic’s restriction digest cloning was successful so Nick Flaxbeard began characterization of H using the Nessler’s test.

Protein Purification of bromoperoxidase using an inducible promoter: