Wetlab
EcnB part extracted from A(ABEF) synthesized plasmids and ligated with H backbone (pSB1C3). Parts were column purified, ligated, then transformed. Had some issues growing cultures with ligations. We are excited to start cloning!

Drylab
Dry lab discussed and finalized a design as well as the dimensions for the fish tag. Parts were designed through Solidworks in member’s own time.

Outreach
We began searching for fish farm contacts, and started our YOURS mentorship program. We introduced our mentors to the kids and taught them about what DNA is.

Wetlab
Week one steps were redone. Started gel purifications instead of column purifications, so plasmids were re-cultured (pre-ligation). We had some issues with low DNA concentrations after gel purifications, so we increased amount of DNA used in digestions.
Flavo Subteam
The flavo subteam made cultures of two strains of Flavobacterium, and measured growth of bacteria with spectrophotometer. In addition, daily OD readings were taken to determine growth rate.

Drylab
Dry lab continued with the CAD design of the fish tag. We also did research on a list of potentially compatible materials for the fish tag, including tubing, clamps, and syringes. .

Outreach
The YOURS kids got a tour of our lab and we taught them more about how DNA works in our body as the determining factor of our genetic characteristics. They were able to place their fingers on plates to “plate bacteria” and we showed them how to incubate the plates to help the bacteria grow. We helped them carry out an experiment that extracted DNA from strawberries and we showed them proper pipetting techniques. We also did a fun experiment with the kids with basic cooking ingredients to make a “volcano explode” to introduce chemical reactions.
We narrowed our search to a few fish hatcheries in the upstate New York region and began contacting them.

Wetlab
Internal (PstI) cut sites also found in AA & AE (switched to cutting the backbones with EcoRI and SpeI). AA+H (ZA) construct was sent for sequencing to verify the contents of the construct. The two primers added resulted in overlap, and sequencing was redone with one primer.
Flavo Subteam
Flavobacterium were re-plated, but they showed no growth after three days. Their growth is necessary to conduct ZOI (zone of inhibition) testing .

Drylab
We came up with and finalized the design of an applicator for the fish tag that improves upon the existing design. Parts were assembled through SolidWorks, and we planned to use the machine shop to build a metallic applicator. .

Outreach
We continued the topic of DNA in bacteria by talking about antibiotic resistance and how we only want certain genes in our own experiments. The kids learned how to pipet and load something into a gel and watched as we showed them how we run gel electrophoresis experiments everyday.

Wetlab
Had some issues growing cultures on plates. Most constructs were having issues in being cultured post-ligation and transformation.
Flavo Subteam
Flavobacterium was re-plated, but there was still no growth.

Drylab
Dry lab continued with the design of the applicator.

Outreach
We moved on to the topic of proteins to start to relate what we were introducing to YOURS kids with our own project so they would understand the relevance of our research. After discussing the basic properties of proteins, we helped them carry out an experiment that separated proteins from different dairy products. The visual results helped them better understand how chemical reactions can change molecular structures.
We contacted Bath hatchery and set up a meeting time in July so that we could drive there and talk to representatives in person and our project in perspective of current solutions.

Wetlab
We had to re-culture several plasmids (pre-ligation). We also received and made glycerol stocks of five new plasmids.
Flavo Subteam
Flavobacterium growth was tested on different types of plates to see if our plates were the issue causing lack of growth.

Drylab
To market the product, the tag needs an appealing name. We brainstormed many potential ideas that may be useful in constructing a name. In addition, we researched specific tubing, clamps and other materials that we intend to utilize for the fish tag

Outreach
Finally, we came a full circle and explained to the kids how bacteria can be potentially dangerous and helpful at the same time and how we can engineer their plasmids in ways to benefit other organisms. They learned about the ubiquitous nature of bacteria when they used cotton swabs to gather different bacterial samples from different areas in the building and let the samples grow overnight. Finally, we explained our own project’s goals and how we would be going to a hatchery the next week to see how farmers have to handle bacterial diseases that affect fish.

Wetlab
We cultured, ligated, and plated some of the new plasmids (A(LMNJKR) +H). We still had issues getting older plasmids past transformation stage.
Flavo Subteam
We determined that our plates (for the Flavobacterium) were the issue. We re-plated Flavobacterium on new plates(both strains).

Drylab
Dry lab came up with the design of a box that can be used to market our products. We listed materials, dimensions, and designs that would make up our fish tag kit. For each kit, we intended to include 20 tags, one applicator, one gel vial, syringe and needle, along with an information booklet. Using oak tag, we started a physical copy of the box.

Wetlab
ZE construct was sequenced and stored as glycerol stock along ZK, ZO, ZA, and ZB. The glycerol stocks are for later use in ZOI testing.
Flavo Subteam
Flavobacterium plates displayed lawn growth that will be used for ZOI tests.

Drylab
Dry lab continued with construction of the box. In addition, fish tags were being printed using high resolution 3D printers.

Outreach
We took the YOURS kids to the Bathe hatchery. We spoke to Bob Sweet who explained how they keep an eye on various conditions in the living conditions of the fish through the use of chemicals. He talked to us about the fish they raise, the types of diseases the fish face such as ferunculosis, and the overall day-to-day things the hatchery has to worry about like maintenance. The YOURS students also learned about how the fish are raised and transported in detail and were allowed to feed the fish. We learned that the hatchery has chosen not to use fish tags and instead clips fish fins to keep track. Sweet’s opinion of the future for the fish/agriculture industry was that the environment would put greater stresses on wild fish and therefore depending on farm-raised fish would be beneficial in supplying the demand for more food in a world with a rising population. We were also directed to speak to Andy Norse at Rome Hatchery who was more aware of chemicals used to treat water to prevent diseases, and we followed up with a call on Monday to plan a Skype meeting with Andy.
On Saturday, Grace and Saie went to the local Ithaca’s Farmer’s Market to find locals who had an opinion about GMOs and fish farming for our Humans and SynBio Facebook page.

Wetlab
ZG construct was sequenced and stored as glycerol stock. Started using Phusion PCR on all un-biobricked constructs (for increased yield).
Flavo Subteam
Attempted to sonicate ZA and ZB cultures, but they were only partially lysed.We also tried ZOI test on Flavobacterium plates (of both strains), with ZA, ZB, and Chloramphenicol and dH2O as controls.

Drylab
Dry lab outlined a comprehensive timeline for constructing business plan, fishtag testing, app design, and marketing. A feasibility analysis was conducted to assess the likelihood of fishPHARM in becoming a new startup. The app was intended to serve a preventative purpose.
In addition, we began researching sensors that could potentially complement the app.

Wetlab
We successfully sequenced and glycerol stocked ZR, ZL, and ZJ constructs. There were some weird results on gels of other constructs, possibly due to mutations in the backbone (H). We also started cloning MBP with Z constructs to stabilize the ecn-B peptides since they were likely being degraded in the cell.
Flavo Subteam
Chloramphenicol ZOI control didn’t work. We attempted ZOI test with ampicillin instead for positive control data.

Drylab
Meeting addressed fundamental parts of a business plan. Members conducted research into various aspects of the aquaculture industry, consumer behavior, and finances in order to construct a comprehensive business plan.

Wetlab
At this stage, Z(A,B,E,G,J,K,L,O,R) constructs have been bio-bricked. Continued using Phusion PCR for MBP + Z constructs. There were some smearing issues with MBP on gels (for ligation).
Flavo Subteam
We began to use terramycin for ZOI control testing since other antibiotics did not have visible results.

Drylab
Members designed potential business cards along with labels for the flavocide gel vials. The app development process is moving along smoothly.

Wetlab
We attempted Gibson Assembly with MBP-TEV insert and H (pSB1C3) backbone as an alternative cloning mechanism. This will increase ecnB peptide stability.
We also received synthesized plasmids from DNA 2.0 with all ecn-B inserts from biobricked Z constructs and MBP-TEV (not in pSB1C3).

Drylab
Dry lab continued with the design of labels and business cards. App development is proceeding without hurdles. The business plan was finalized.

Wetlab
We had issues with growing cultures for Gibson plates. This was likely due to poor selection on antibiotic plates, possibly due to antibiotic degradation. We removed inserts from synthesized plasmids and started cloning procedures into pSB1C3.

Drylab
A enlarged fishBIT tag model was 3D printed for display purposes. Sensor design for the app was started. We began looking into various tests that could be done to test the durability of our tag, including but not limited to tensile testing and wind tunnel testing.

Wetlab
We encountered issues with getting past ligation stage with synthesized plasmid inserts and H backbone.
To confirm ecnB expression, several SDS-PAGEs were done on constructs without stabilizing proteins to use for Western blots (to see their expression) and later ZOI assays .

Drylab
Business plan was reviewed by a professor with expertise in the area. Additional tags were printed, and samples were sent to prospective customers for feedback.

Wetlab
A Gibson construct (MJ) was successfully sequenced. Western blots showed possible expression of ZG and ZR, but not ZJ, though we didn’t have a control because it did not survive past the lag phase.
Switched focus to getting expression from all biobricked Z constructs and MJ to use in ZOI assays.
A second set of constructs (ZG, MJ, ZQ, ZT) were set up for western blots.

Drylab
Dry lab finalized plans for testing fishBIT. App design and sensor designs continue smoothly.

Wetlab
The second set of western blot revealed expression of ZG, MJ, and ZT. ZG was taken to be the positive control based on previous western blot.

Drylab
Dry lab tested the durability of fishBIT using agar gel samples and dead fish samples. In a hydraulics lab, fishBIT was tested for its resistance to current turbulence.