Team:Lubbock TTU/Notebook



Start of iGEM!
The TTU Raider Research Team began member recruitment and team selection for the 2018 iGEM competition. Members began discussions on synthetic biology literature and identified a potential project to pursue. The first meeting with our advisers was held. Start of fundraising.
Adviser Meeting
In a meeting with our advisers- Dr. John D'Auria and Dr. Michael Latham - we adjusted our project details and planned a rough timeline of events. We also extended our proposed materials list to include basic lab supplies like plates and pipette tips.
Searching for Buried Scholarships
We began reaching out to on-campus sources of funding, including the Center for the Integration of STEM education and research (CISER) and the Center for Active Learning and Undergraduate Engagement (CALUE, now know as TrUE - Center for Transformative Undergraduate Experiences.)
Meme Team
At our weekly meeting, we revamped our social media profiles and gave access to Jeff Ptak. A new logo was created featuring the double T in tRNA form.

Tip: Click any image to enlarge.
W3110 Strain of E.Coli
The lab received the W3110 Strain of E. Coli.
Addgene Acount
The Addgene Acount was created to help design Plasmids
Lambda Red Recombinase System Plasmids
The Team design the plasmids for lambda Red Recombinase System Plasmid: pMDIS (Template plasmid for spectinomycin resistance), pMDIAI (Template plasmid for apramycin resistance), pREDKI (Recombinase and endonuclease plasmid), pKSI-1 (Donor Plasmid)
Refine the Design
Greg and Ben led the discussion about the project procedure, and identified what parts we would need to design and order.
Addgene Stab Culture arrived
The lab received the plasmid stab cultures from iGEM. Storage in 4C fridge.
Restreaked the Stab Cultures
The team re-streaked the plasmid stab cultures onto appropriate antibiotics .
E.Coli Genome
The team review E.Coli Genome editing papers
Lab Orientation
This week, Ben gave everyone a tour of the D'Auria lab. During this tour, we learned the locations of all of the safety materials. We also learned where different compounds and machines were stored. Lab coats were issued, and safety forms were completed.
Members of the team were assigned papers to read on genome editing.
Mini-Prep Plasmids
Mini-preps of the pREDKI, pKSI-1 and pMDISI plasmids.
Finals Week
Break for Finals Week! It's our policy that academics comes first, before anything. Team members took the week off to focus on their studies.
Homology Arms
Greg, Ben and Brandon wrote out the sequences for the LacI and ArgI homology arms with spec resistance. Material were order to start knock out.
Electrocompetent E.coli cells
The team made Electrocompetent E.coli cells following the Molecular Cloning (Sambrook) book Protocol.
The team performed a PFU PCR of LacI and ArgI anitbiotic template with homology arms. Unfortunately, The ArgI PCR was not successful.
The team decided to run another PFU PCR of ArgI antibiotic template with homology arms
Picture Day
The team took our group and individual photos. Too bad we can't edit our own genomes to be more photogenic!

Tip: Click any image to enlarge.
Meet the Team
The team planned Greg's presentation for the ME12 conference in Germany.
All of the iGEM members filled out a short survey made by Paula. This information is intended to be used on the team page.



Greg and Brandon secure funding for travel to the ME12 conference.
Darron ran a PCR of the LacI and ArgI G-Blocks. Ben Made Electro-competent E. coli Cells
Ben ran a PFU PCR of the ArgI antibiotic template with homology arms.
Sunday Fund Day
Preparation continued for the ME12 conference. Jeff Set up the Panda Express Fundraiser. Paula secured funding from Cedarlane!
Darron runs a second PCR of the ArgI gene.
Electrocomp 2
Ben and Jordan make electrocompetent W3110 Cells with the pREDKI plasmid. They also ran a PFU PCR of the ArgI antibiotic template with homology arms.
More PCR
Darron ran another PCR of the LacI gene with a new reverse primer.
Even More PCR
Darron ran yet another PCR of the LacI gene with new primers.
ME12 Prep
Jeff and Greg finalized Greg's presentation for the ME12 conference. The team discussed issues with the knockouts.
Jordan makes full and half strength Kanamycin plates.
Tech iGEM Goes International
Greg attends the ME12 conference in Germany. The rest of the team continues wet lab work in Lubbock. Jeff begins working on a potential fundraising opportunity with Eventide.
Jordan and Ben create the gene fragments for LacI, ArgI, SpeG, and SpeE with new restriction sites.
Induction Solutions
A PCR is run on the LacI and ArgI templates with homology arms. Arabinose and Rhamnose stock solutions are made.
The Power of German (Metabolic) Engineering
Greg returned from the ME12 conference. He relayed feedback he had been given by experts and other iGEM teams.
Most excitingly, Greg met Sang Yup Lee - one of the authors of the paper our project is based off of! Mr. Lee agreed to send us a sample of the strain we were trying to improve upon in order to compare the efficiency of our project.
Ben and Darron made liquid cultures of the pREDKI E. coli. Brandon and Paula made Arabinose, Kan and Spec plates.
Electroporation of W3110
Paula, Jordan, and Ben made electrocompetent W3110 cells with the pREDKI plasmid. They then transformed the LacI and ArgI PCR products and G blocks into the competent cells.
Clean-Up Prep and More Cells
Darron and Ben made electrocompetent W3110 cells with the pREDKI plasmid. They also prepared the buffer solutions for our PCR clean-up kit.
PCR Clean up
The Team ran a PCR clean-up of the ArgI and LacI PFU PCR products.
Inter-Lab Study
For this week, the group split up to tackle the inter-lab study experiments. Jeff and Paula started the plate reader protocol, which Ben finished later that day. However, due to scheduling issues, the flow cytometer was unavailable. We later repeated this experiment with the extra credit portion after asking for an extension from iGEM HQ.
We are having issues with our experiment. We decided to redesign primers for the lambda proteins on the pREDKI plasmid rather than on the I-SceI gene to insure that there were no issues with the recombination step.
Making Plates
We made Arabinose + Kan and Kan + Spec plates. Ben tried another electroporation of the pREDKI containing W3110 cells.
Electroporation day 2
After allowing the cells a longer incubation period, we came in to make a master plate and check on our colonies. They had grown very weakly, and we decided to run a colony PCR to ensure that the spec template had been inserted into the genome.
Colony PCR
We Ran a colony PCR, but the pREDKI plasmid didn't show up in our cells.
Colony PCR Take 2
After a meeting with our mentor, we redesigned our controls and re-ran the PCR. We still had issues.
We decided to redesign our primers and check their annealing temperatures. We found discrepancies with the temperatures we had been using and the actual annealing temperatures.
The (Lab) Notebook
We held our weekly lab meeting. We talked about our project progress, and keeping our individual lab notebooks up to date. We also re-did the InterLab study, with the optional flow-cytometry section.



We began from scratch with our electroporation. After allowing the plates to grow overnight, we noticed that our antibiotic concentrations were not high enough, as we had growth on our negative control plate..
Genomic PCR
Ben ran a PCR on the LacI and ArgI antibiotic template with homology arms using a gradient. We also ran a genomic PCR of the W3110 Lambda Red cells.
Ooh Wiki Wiki
Darron, Jeff and Brandon sat down in the library to work on the wiki page. Jeff and Darron began editing the main page. A document was made containing all of the information that would go on each page.
Paula made plates and LB agar.
Genomic PCR 2
Re-ran the Genomic PCR of W3110 with Lambda Red
Checking Boxes
Brandon led a discussion about all of the other requirements we needed to meet for this year's competition. Work began on fulfilling the human practices, collaboration and notebook requirements.
We had a meeting with Dr. Moreno about troubleshooting our protocol.
Re-Stocking Materials
Darron made more arabinose stock solution and electrocomp cells.
Team members met for our last weekly meeting before the fall semester started. We decided that after everyone settled into their classes, we would update our availability document.
Jeff and Paula completed a phone interview with Dr. Augustin Mohedas of Eventide. TTU iGEM was invited to present to Eventide after the iGEM competition.
New Arms
We performed a new PCR of the ArgI and LacI g-blocks with our newly designed homology arms.
PCR Clean UP
We performed a PCR clean up of our LacI and ArgI sequences with the new homology arms.
Human Practices Prep
This week we prepared our paintings for the art trail. Jeff and Paula painted artworks and gathered materials for our booth. Brandon, Darron and Ben transformed GFPs into DH5alpha cells and streaked them into stenciled-out shapes.
The team discussed adding new, young iGEM members.



New Member Recruitment
Jeff and Paula visited several colleges and gave talks to classes about the opportunities and benefits iGEM had to offer. Over 30 new undergraduates, from Freshmen to Seniors and consisting of dozens of majors showed interest in iGEM. Unfortunately for everyone, this made the application process very competitive!
In the lab, We worked on making chemical competent DH5alpha cells and performing a transformation of both pREDKI and the florescent plasmids.
First Friday Art Trail
The Lubbock_TTU team went to the monthly First Friday Art Trail in Lubbock to showcase our research and educate the public about synthetic biology.Thousands of people come to the downtown arts district for live music, food trucks, and of course - spectacular art. iGEM decided to take our own approach to art - streaking GFP transformed E.Coli onto plates in fantastic shapes! We also painted a few "traditional" paintings ourselves! We invited members of the community to view our artwork while we questioned them regarding basic knowledge of synthetic biology. Members of the team educated enthusiastic patrons about the field of synthetic biology, the iGEM competition, the protocols for the artwork, ethical dilemmas associated with biology, and the long term goals and effects of our project. Many people were surprised to find out that bacteria can be little factories for our medicines!

Tip: Click any image to enlarge.
New Member Interviews
The iGEM officers reviewed applications and potential new members were selected. In the lab, we ran a colony PCR of DH5alpha pREDKI and the LacI and ArgI truncated sequences.
Paula streaked BH10B/pSIM5 and BH10B/pSIM27 onto plates and made liquid cultures.
Ordered Primers
Brandon order VR/VF2 primers to confirm Bio-Brick/shipping vector assembly. Jeff and Darron ran a gel of the LacI and ArgI PCR products
PCR and Clean-Ups
Darron ran a Colony PCR screening for PKIS-1 plasmid and nothing showed up. He also cleaned-up our truncated LacI and ArgI sequences.
pSIM and pSB1C3
Paula made liquid cultures of pSIM 5 and pSIM 27. Jeff made liquid cultures of pSB1C3.
Coltyn Wagnon and Pranathi Bingi were interviewed and accepted as Junior iGEM members. Congrats!
Mini-Prep of Shipping Vector
Jeff Mini-prepped out the pSB1C3 shipping vectors. Paula re-stocked the lab's agar and tidied up the work space.
New Member Orientation
New members Coltyn and Pranathi completed their new member orientation, which included coming to their first weekly meeting and completing the TTU lab safety training.
The team assigned conversation topics and more importantly, food items for the potluck dinner at Julie's.
Austin iGEM Summit
Paula and Brandon drove down to Austin for a conference with UT iGEM as well as the high school iGEM team LASA.

Tip: Click any image to enlarge.
Potluck Dinner
The team all met on our adviser Julie Isom's farm for dinner. We enjoyed good food, and we also had a productive discussion about the future of iGEM. Paula and Brandon updated everyone on what happened at the UT Austin-LASA conference. Jeff updated everyone on his progress with the shipping vectors. Darron talked about his donor vector project. Coltyn got his first real taste of iGEM.
The team also worked on documents for finalizing the CALUE travel funding.



Ligation of Bio-Bricks in Shipping Vector
We attempted to ligate the Bio-Bricks into the standardized shipping vector, but were unsuccessful. Darron ran a PCR of our Bio-Bricks for another shot at it.
Liquid Cultures and PCR
The team made more pSB1C3 liquid cultures and cleaned up our Bio-Brick PCR.
The Final Push
We met casually to plan a timeline for completion of Bio-Bricks, Collaborations, and the Wiki Page.
the team digested our Bio-Bricks and shipping vectors.
TTU iGEM successfully ligated our Bio-Brick into our backbone. Later, we transformed the plasmid backbone into chemical competent Top10.
Liquid Cultures
The team made liquid cultures of our mini-prepped plasmid.
We mini-prepped out our Bio-Brick DNA.
Shipped our biobricks
We dried down our parts and sent them off to iGEM HQ.
Wiki Grind
We sat down and finished the wiki page as a team.
Though too late for the competition, we finally received the strain Sang Yup Lee agreed to send us at ME12.