6/17-6/21

Tour Greenhouse Space

Who? Allison, Jacob, Jason

Protocol: N/A

Notes: We met with Dr. Gschwend, a professor in the Dept. of Horticulture and Crop Science, about the possibility of using greenhouse space for testing and growth purposes if we ended up needing a space for controlled growth of transfected plant species.

Results: N/A

Meeting with Justin North

Who? Allison, Chris, Jason, Jacob, Prajwal

Protocol: N/A

Notes: Dr. Justin North, a post-doc researcher in the Dept. of Microbiology, gave the team an in depth lecture about the structure and function of nitrogenase, as well as challenges and considerations with cloning it.

Results: N/A

Primer Design

Who? Jason, Jacob, Prajwal

Protocol: N/A

Notes: We started the process of designing primers for our experiments. This included extensive research on the genome of our donor strain (R. palustris) and how to properly piece together the desired parts.

Results: N/A

Meeting with Erin Gloag

Who? Jacob, Prajwal, Jason

Protocol: N/A

Notes: We met with Dr. Erin Gloag, a post-doc researcher in the Dept. of Microbial Infection and Immunity, about the use of Pseudomonas vectors and recipient strains for our project. She also gave some pointers and tips for Gibson Assembly and some helpful resources to look into.

Results: N/A

6/24-6/28

Streak Out/Culture Initial Strains

Who? Prajwal, Jacob, Jason

Protocol: Bacterial Liquid Culturing, 3-Phase Streak

Notes: We grew up P. protegens PF-5 and R. palustris CGA010 for convenient use later on in the summer

Results: We got healthy cultures and colonies of our desired bacteria.

Parts Discussion

Who? Prajwal, Jacob, Jason

Protocol: N/A

Notes: We sat in a room and specifically went through all of the different medal requirements regarding lab work and laid out plans of how we intended to fulfill each of them (i.e. Characterization, Part Improvement, and New Part Addition).

Results: Though our plans changed throughout the course of the summer, this meeting helped guide experimental planning and kept us on track throughout the entirety of the project.

Primer Design

Who? Jacob, Prajwal, Jason

Protocol: N/A

Notes: We continued research on our donor genome, and began to actually draft out primers (using Geneious software).

Results: N/A

Protocol Design/Evaluation

Who? Jacob, Prajwal, Jason, Chris

Protocol: N/A

Notes: We consulted our supervisors and did a literature search for all of the lab protocols that we would possibly need for the summer. We then put these in a common place (Benchling) so that we could easily reference them whenever needed.

Results: N/A

7/1-7/5

Primer Design

Who? Jacob, Jason, Prajwal

Protocol: N/A

Notes: We finally finished and got an approval on our primers, and how exactly we would piece together our desired part.

Results: Once approved by one of our supervisors, we sent out an order for our primers.

Literature and Lab Update

Who? Whole Team

Protocol: N/A

Notes: Now that we were about to start the “real” lab work, we all updated ourselves on necessary literature and reviewed our strategies for getting our main project to work and what the next steps were.

Results: N/A

7/8- 7/12

Genome Extraction

Who? Jacob, Prajwal

Protocol: Genomic DNA Extraction

Notes: We headed to Justin North’s lab (where our R. palustris was growing) and extracted gDNA from R. palustris for later use in PCR experiments.

Results: We brought a small microcentrifuge tube back to the lab, which served as our source of gDNA for the next few weeks.

Plate Mini-CTX1

Who? Prajwal

Protocol: N/A Three-phase streak??

Notes: Prajwal received a plate of Mini-CTX1 from Dr. Erin Gloag (who had a stock lying around) that we planned to use for transformation of our Gibson Assembly product.

Results: We had plentiful colonies that were all healthy and restreaked every few weeks to be kept fresh.

7/15-7/19

Miniprep of CTX-1

Who? Prajwal

Protocol: Liquid Culture Setup, Miniprep

Notes: Prajwal set up a liquid culture (5 mL) of Mini-CTX1, and then performed a miniprep the day after.

Results: This miniprep yielded a DNA concentration of 483.5 ng/uL.

Primer Resuspension

Who? Prajwal, Jason, Jacob

Protocol: IDT Primer Resuspension

Notes: Once we received our primers in the mail, we performed calculations and then resuspended our primers to usable concentrations for our PCR experiments.

Results: After completing the protocol, we got our primers to practical aliquots at a concentration of 10 uM.

Initial PCR

Who? Jacob, Jason, Prajwal

Protocol: 2-Step PCR

Notes: We performed an initial PCR on our gDNA (this included all 7 parts).

Results: These samples were taken out of the thermocycler after the end of the reaction, and were left to be analyzed on a gel on a different day.

Gel Electrophoresis

Who? Prajwal, Jason

Protocol: Gel Electrophoresis

Notes: We ran all 7 PCR products on the same gel, let it run, then looked at the gel under a UV lamp for analysis.

Results: Fragment 5 was the only positive result, everything else failed.

7/22-7/26

PCR of Fragment 6

Who? Jason, Prajwal

Protocol: 2-Step PCR

Notes: We set up another PCR reaction of just fragment 6, using the exact same protocol from the previous attempt.

Results: After gel analysis, fragment 6 appeared, and the PCR product was preserved.

Master Mix Design + PCR

Who? Jacob, Prajwal, Jason

Protocol: 2-Step PCR

Notes: We decided to reprogram our thermocycler to employ a linear gradient variation in the temperature that the reaction was run at (increasing by a decreased amount each “place” over in the thermocycler). We figured that since we were doing an unusual reaction, it’d be best to find optimal temperatures for the fragments we had. This included fragments 1, 2, 3, 4, and 7

Results: After gel analysis, we got fragments 1, 2, 3, and 7 to work. These products were preserved.

Fragment 4 Reattempt

Who? Jacob, Jason, Prajwal

Protocol: 2-Step PCR

Notes: We decided to make another master mix (for use in the temperature linear gradient scheme) of fragment 4, and then run a PCR of the fragment.

Results: Even with 12 samples at different temperatures, we were unable to get any successful products after running the samples on a gel.

7/29-8/2

Sequencing of Fragments 1, 2, 3, 5, 6 and 7

Who? Jacob, Prajwal

Protocol: N/A

Notes: These fragments were sequenced by an in-house department that just required products to be aliquoted and diluted to a certain concentration.

Results: Once we got sequencing data back, we opened it in Geneious software and learned that we did in fact have the products we wanted, and just needed fragment 4 to begin Gibson Assembly.

8/5-8/9

Fragment 4 Reattempt and Troubleshooting

Who? Jacob, Prajwal

Protocol: 2-Step PCR

Notes: We once again tried the same method of PCR for fragment 4. While it was running, we examined our primers for fragment 4 and realized that they were not optimized for our experiment and learned that they had considerable hairpin temperatures.

Results: After another unsuccessful gel, we decided to redesign our primers and immediately order them so that we can move on with our project (due to our design scheme, fragment 4 was a crucial element and the rest of the assembly could not move on without it).

8/12-8/16

Fragment 4 PCR with New Primers and Sequencing

Who? Jacob

Protocol: 2-Step PCR

Notes: After receiving our redesigned primers, we redid our PCR of fragment 4 with the linear gradient scheme.

Results: After running a gel, we finally got fragment 4 to work. We purified the PCR product and got it ready for Gibson Assembly.

Designing Microaerophilic Growth Experiments

Who? Jacob

Protocol: N/A

Notes: Now that we had our products ready for Gibson assembly, we began designing experiments that were supposed to be taking place in microaerophilic environments, and therefore required special precautions and considerations.

Results: N/A

8/19-8/23

Gibson Assembly

Who? Jacob

Protocol: Gibson Assembly

Notes: After re-verifying that we had the right reagents and a usable amount of PCR products we finally attempted our Gibson Assembly of our fragments. We then transformed the final Gibson product into NEB E. coli 10-Beta and left it to grow overnight on LB tetracycline plates.

Results: After overnight growth, we had colonies on the tetracycline plate, indicating that our transformation worked. We now just needed to send the product off for sequence verification.

8/24-8/30

Patching of colonies off Gibson plates to screen for insert

Who? Jacob

Protocol: n/a

Notes: Patching of colonies off of Gibson plates to screen for insert

Results:N/A

2-Step PCR

Who? Jacob

Protocol: 2-Step PCR

Notes: Colony PCR of patched colonies using insert verification primers

Results: PCR was not successful. Most wells evaporated and the ones that didn’t had reduced product

9/3-9/10

Gel Electrophoresis

Who? Jacob

Protocol: Gel Electrophoresis

Notes: Gel of remaining PCR wells (~11)

Results: All wells showed a long smear-not interpretable results

Miniprep

Who? Jacob

Protocol: Miniprep

Notes: The plasmids from each of the patched colonies corresponding to the wells run on the gel were miniprepped

Results: High DNA concentrations were obtained.

PCR

Who? Jacob

Protocol: PCR

Notes: Redid Colony PCR using Miniprepped plasmids

Results: N/A

Gel Electrophoresis

Who? Jacob

Protocol: Gel Electrophoresis

Notes: Gel of plasmid PCR

Results: All wells showed a long smear

Restriction Digest

Who? Jacob

Protocol: Restriction Digest

Notes: The plasmids miniprepped out of the patched colonies were digested in order to linearize them

Results: N/A

Gel Electrophoresis

Who? Jacob

Protocol: Gel Electrophoresis

Notes: Gel of Minipreps

Results: All colonies had a plasmid ~5.5kb, which is the expected size of our vector without the insert.

Nitrogen Limited Media

Who? Jacob, Prajwal

Protocol: Nitrogen Limited Media

Notes: Nitrogen Free, Nitrogen Limited, and Nitrogen Excess media were created for the culturing of diazotrophs (Jensen’s Broth)

Results: N/A

Replica Plating

Who? Jacob

Protocol: Replica Plating

Notes: The initial Gibson reaction platings were replica plated onto the nitrogen limited media and left to grow in the anaerobic chamber.

Results: After about 1 week a few colonies grew on each the nitrogen free and nitrogen deficient media. These were subcultured into LBTet and grown aerobically.

9/12-9/19

PCR

Who? Jacob

Protocol: PCR

Notes: Attempted to use our long range polymerase to amplify the Whole nif cluster in I shot

Results: N/A

Gel Electrophoresis

Who? Jacob, Prajwal

Protocol: Gel Electrophoresis

Notes: PCR of whole nif cluster

Results: No bands observed

Three-Phase Streak

Who? Jacob

Protocol: Three-Phase Streak

Notes: Pseudomonas Protegens was streaked out onto the nitrogen limited media and left to grow in the anaerobic chamber

Results: No growth on Nitrogen free, deficient media, small growth on Nitrogen enriched media after 1 week

9/20-9/23

PCR

Who? Jacob

Protocol: PCR

Notes: Colony PCR of colonies from anaerobic chamber

Results: N/A

Gel Electrophoresis

Who? Jacob

Protocol: Gel Electrophoresis

Notes: Gel of Colony PCR products

Results: No bands observed

10/7-10/13

Miniprep

Who? Jacob

Protocol: Miniprep

Notes: Miniprep of colonies isolated from anaerobic chamber

Results: Very low DNA concentrations coming out of these cultures; difficult to even visualize these preps on a gel, much less sequence or PCR.

Filter Mating

Who? Jacob

Protocol: Filter Mating

Notes: E.coli+pULB113 (RP4::mini-mu) and R.palustris were mated to try and get the plasmid into R.palustris and fragment its genome.

Results: Currently growing in anaerobic chamber to select for successful matings.

LasR-Dependent Promoter Characterization

6/24-6/28:

Parts Discussion

Who? Jacob, Prajwal, Jason

Protocol: N/A

Notes: We discussed how we were going to approach characterizing a part, which parts we should consider looking into, and how we would go about it.

Results: This discussion guided our experimental planning and activity for the coming months and helped to keep us on track, as well as keep us constantly evaluating whether or not we were doing our best to fulfill medal requirements.

Chemical Transformation of Biobricks

Who? Jason, Prajwal

Protocol: Mach1 Transformation

Notes: After selecting the biobricks that we wanted to use for experimentation (in this case, BBa_K575010 from the 2011 Northwestern team), we chemically transformed them to get them ready for further research.

Results: After growing overnight, we got successful colonies of our biobrick-containing plasmid.

7/8-7/12