iGEM 2018 — Team Lubbock_TTU

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# Team:Lubbock TTU/Notebook

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### Notebook

[March-May](#janjun)
[June-July](#july)
[August](#august)
[September](#september)
[October](#october)

#### MARCH-MAY

##### Start of iGEM!

March 2018

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

April 2nd

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

April 8th

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

April 15th

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.
![](https://static.igem.org/mediawiki/2018/0/01/T--Lubbock_TTU--logo.jpg)
**Tip:** Click any image to enlarge.

##### W3110 Strain of E.Coli

April 17nd

The lab received the W3110 Strain of E. Coli.

##### Addgene Acount

April 18nd

The Addgene Acount was created to help design Plasmids

##### Lambda Red Recombinase System Plasmids

April 18nd

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

April 22nd

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

April 26nd

The lab received the plasmid stab cultures from iGEM. Storage in 4C fridge.

##### Restreaked the Stab Cultures

April 27nd

The team re-streaked the plasmid stab cultures onto appropriate antibiotics .

##### E.Coli Genome

April 29nd

The team review E.Coli Genome editing papers

##### Lab Orientation

April 29th

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

May 4th

Mini-preps of the pREDKI, pKSI-1 and pMDISI plasmids.

##### Finals Week

May 9th

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

May 16th

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

May 9th

The team made Electrocompetent E.coli cells following the Molecular Cloning (Sambrook) book Protocol.

##### PFU PCR

May 9th

The team performed a PFU PCR of LacI and ArgI anitbiotic template with homology arms. Unfortunately, The ArgI PCR was not successful.

##### PFU PCR

May 13th

The team decided to run another PFU PCR of ArgI antibiotic template with homology arms

##### Picture Day

May 22nd

The team took our group and individual photos. Too bad we can't edit our own genomes to be more photogenic!
![](https://static.igem.org/mediawiki/2018/2/22/T--Lubbock_TTU--TeamPhoto.jpg)
**Tip:** Click any image to enlarge.

##### Meet the Team

May 27th

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.

#### JUNE-JULY

##### Update

June 1st

Greg and Brandon secure funding for travel to the ME12 conference.

##### G-Blocks

June 8th

Darron ran a PCR of the LacI and ArgI G-Blocks. Ben Made Electro-competent E. coli Cells

##### PFU PCR

June 9th

Ben ran a PFU PCR of the ArgI antibiotic template with homology arms.

##### Sunday Fund Day

June 10th

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

June 13th

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

June 14th

Darron ran another PCR of the LacI gene with a new reverse primer.

##### Even More PCR

June 15th

Darron ran yet another PCR of the LacI gene with new primers.

##### ME12 Prep

June17th

Jeff and Greg finalized Greg's presentation for the ME12 conference. The team discussed issues with the knockouts.

##### Plates

June19th

Jordan makes full and half strength Kanamycin plates.

##### Tech iGEM Goes International

June 24th

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

June 30th

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

July 1st

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

July 3rd

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

July 9th

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

July 11th

The Team ran a PCR clean-up of the ArgI and LacI PFU PCR products.

##### Inter-Lab Study

July 15th

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

July 16th

We made Arabinose + Kan and Kan + Spec plates. Ben tried another electroporation of the pREDKI containing W3110 cells.

##### Electroporation day 2

July 17th

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

July 19th

We Ran a colony PCR, but the pREDKI plasmid didn't show up in our cells.

##### Colony PCR Take 2

July 20th

After a meeting with our mentor, we redesigned our controls and re-ran the PCR. We still had issues.

##### Re-Priming

July 23rd

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

July 27th

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.

#### AUGUST

##### Electroporation

August 1st

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

August 5th

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

August 6th

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

August 8th

Re-ran the Genomic PCR of W3110 with Lambda Red

##### Checking Boxes

August 13th

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

August 15th

Darron made more arabinose stock solution and electrocomp cells.

##### Back-to-School

August 19th

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

August 22nd

We performed a new PCR of the ArgI and LacI g-blocks with our newly designed homology arms.

##### PCR Clean UP

August 24th

We performed a PCR clean up of our LacI and ArgI sequences with the new homology arms.

##### Human Practices Prep

August 26th

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.

#### SEPTEMBER

##### New Member Recruitment

September 3rd-6th

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

September 7th

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!
![](https://static.igem.org/mediawiki/2018/thumb/c/c7/T--Lubbock_TTU--FFATteampic.jpg/1200px-T--Lubbock_TTU--FFATteampic.jpg)
![](https://static.igem.org/mediawiki/2018/thumb/3/31/T--Lubbock_TTU--BacterialPaintings.jpg/1200px-T--Lubbock_TTU--BacterialPaintings.jpg)
**Tip:** Click any image to enlarge.

##### New Member Interviews

September 10th

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.

##### pSIM

September 15th

Paula streaked BH10B/pSIM5 and BH10B/pSIM27 onto plates and made liquid cultures.

##### Ordered Primers

September 17th

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

September 19th

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

September 22nd

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

September 23rd

Jeff Mini-prepped out the pSB1C3 shipping vectors. Paula re-stocked the lab's agar and tidied up the work space.

##### New Member Orientation

September 24th

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

September 29th

Paula and Brandon drove down to Austin for a conference with UT iGEM as well as the high school iGEM team LASA.
![](https://static.igem.org/mediawiki/2018/7/76/T--Austin_UTexas--TTU_Collab1.jpeg)
**Tip:** Click any image to enlarge.

##### Potluck Dinner

September 30th

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.

#### OCTOBER

##### Ligation of Bio-Bricks in Shipping Vector

October 1st

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

October 3rd

The team made more pSB1C3 liquid cultures and cleaned up our Bio-Brick PCR.

##### The Final Push

October 4th

We met casually to plan a timeline for completion of Bio-Bricks, Collaborations, and the Wiki Page.

##### Digestion

October 6th

the team digested our Bio-Bricks and shipping vectors.

##### Ligation/transformation

October 7th

TTU iGEM successfully ligated our Bio-Brick into our backbone. Later, we transformed the plasmid backbone into chemical competent Top10.

##### Liquid Cultures

October 8th

The team made liquid cultures of our mini-prepped plasmid.

##### Mini-prep

October 9th

We mini-prepped out our Bio-Brick DNA.

##### Shipped our biobricks

October 10th

We dried down our parts and sent them off to iGEM HQ.

##### Wiki Grind

October 12th-17th

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.


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# Team:Lubbock TTU/Experiments

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### Protocols

**LB Media:**

250 ml d H20

5 g LB lenox media

1) Pour 250 ml of water into a plastic beaker with a magnetic spin vain.

2) Measure 5 g of LB lennox media on a weigh boat.

3) Add the LB lennox media slowly to the stirring water.

4) After the solution is homogeneous, pour 250 ml into a glass bottle.

5) Make sure the glass bottle is label and has autoclave tape.

6) Autoclave = liquid 30.
LB Agar:
250 ml d H20

5 g LB lenox media

3.75 g Agar

1) Pour 250 ml of water into a plastic beaker with a magnetic spin vain.

2) Measure 5 g of LB lennox media on a weigh boat.

3) Add the LB lennox media slowly to the stirring water.

4) Weight 3.75 g of Agar and pour it directly into a 500 ml glass bottle.

5) After the LB lennox solution is homogeneous, pour 250 ml into the glass bottle.

6) Make sure the glass bottle is label and has autoclave tape.

7) Autoclave = liquid 30.
**LB Liquid Cultures:**
1)Take 2 culture tubes with the lids on and place them in a glass beaker.

2)Microwave for 5 minutes at power level 4.

3)After the microwave is finished take the tubes into the clean bench.

4)Wipe the clean bench down with ethanol before and after use.

5)Label the tubes appropriately.

6)Add 5 ml of LB media to each tube and then add appropriate antibiotic.

7)Flame a loop and pick a colony from a petri dish.

8)Grow overnight at 28-30 °C.
**PLATES:**
1)250 ml LB Agar and microwave for 1 minutes, and then 30-second intervals until is completely melted. Do not let it boil.
let the Agar cold down to the point it only feels warm to the touch.

2)obtain two 50 mL conical vials.

3)after the agar is cold down enough pour it into one 50 ml into the conical vial.

4)dd appropriate concentration of antibiotic.

5)label plates.
**MINIPREP:**
This protocol was obtain from QIAprep Miniprep Handbook

QIAprep Spin Miniprep Kit using a Micro centrifuge

1) Make 1-5 ml overnight cultures of E.coli in LB media.

2) Pelleted Bacterial cell by centrifuging at 45000 g for 10 minutes.

3) Discard supernatant into a container.

4) Resuspend pelleted bacterial cell in 250 µl Buffer P1 and transfer to a microcentrifuge tube.

5) Add 250 µl Buffer P2 and invert the tube 4-6. The lysis reaction should not proceed for more than 5 minutes

6) Add 350 µl Buffer N3 and invert 4-6 times. (Solution should be cloudy)

7) Centrifuge for 10 minutes at 13,000 rmp.

8) Obtain the supernantants from step 4 and add 800 µl to a spin column by pipetting.

9) Centrifuge for 1 minutes and discard flow- through.

10) Wash spin column by adding 750 µl of Buffer PE and centrifuge for 1 minute. Discard Flow-through

11) Transfer spin column to a clean 1.5 ml Eppendorf tube and centrifuge for 1 minute.

12) Transfer spin column to a new clean 1.5 ml and elute DNA by adding 50 µl of water. (you can warm up the water to improve the efficient

13) Let it stand for 1 minutes and centrifuge for 1 minute,

14) Obtain the DNA concentration.

file:///C:/Users/pgaravit/Downloads/E2.QIAprep.Miniprep.Handbook.pdf
**Electrocompetent Protocol:**
This is according to the Molecular Cloning (Sambrook) book, page 178 book 1.

Day 1 - Make a 5mL starter culture:- 5mL LB media- Single colony from a plate- Appropriate antibiotics Incubate overnight at 37 °C with shaking (250rpm)

      Day 2 - Make a 50 mL cell culture:- Inoculate 50 mL of LB media with 2.5mL of the starter culture- Incubate at 30 °Cwith shaking (250rpm)- Take OD600 every 30-40mins- When OD reaches 0.35-0.4 Pour culture into a 50mL falcon tube (purple or blue lids) and place in an ice water bath for 15-30mins, swirl occasionally- Do NOT let cells get above 4 °C from here on!- Centrifuge at 1000g for 15mins at 4 °C- Decant and Resuspend pellet in 50mL COLD autoclaved water by pipetting- Centrifuge at 1000g for 20mins at 4 °C- Decant and Resuspend pellet in 25mL COLD sterile 10% glycerol- Centrifuge at 1000g for 20mins at 4 °C- Decant and Resuspend pellet in 1mL COLD sterile 10% glycerol by pipetting (be careful here)- Centrifuge at 1000g for 20mins at 4 °C- Immediately remove ALL of the supernatant!- Resuspend the pellet by gently swirling in 1mL of COLD GYT media
                                  **1% Agarose Gel protocol:**
                                  1)Measure 1 g of Agarose and 100 ml of 1x TAE buffer in specific flask designated for ethidium bromide

                                  2)Weigh the total of flask and mixture

                                  3)Microwave mixture for 1 minute and power level 10

                                  4)Stir and microwave once more for one minute at power level 10- The mixture should have gone from cloudy to clear

                                    1)Since water has evaporated, weigh the mixture and flask again and add enough water to compensate the amount that evaporated in the microwave

                                    2)Set up the casting apparatus- Put wells in place- Make surface level using the bubble level

                                        1)Add 4 µl ethidium bromide to the mixture and swirl bottle to homogenize

                                        2)Pour mixture into the apparatus. Pop the bubbles with a pipette tip. Keep the bubbles away from the wells

                                        3)Let the gel solidify for 15-20 minutes (should turn opaque when ready)

                                        4)Since you can only add 10 µl into the well:- Make enough small wells with parafilm to match the number of PCR samples- Add 1 µl of dye and 10 µl of PCR sample into the parafilm well and homogenize with pipette tip (dye damages the PCR sample so do not add directly to sample ever)

                                            1)Remove comb carefully from gel and apparatus

                                            2)Fill the apparatus with the 1x TAE buffer so that the gel is completely covered (If it's already full then don't fill it up with new buffer.)

                                            3)Grab the correct DNA ladder and pipette 10 µl into the far left lane

                                            4)Load 10 µl of each sample from the parafilm wells

                                            5)Turn on the machine at 110 V for 20-30 minutes and make sure bubbles form
                                            **PFU PCR:**
                                            Recipe:- 38 µL of Autoclaved DI water- 5 µL 10X PFU Buffer- 1 µL dNTP- 1 µL FWD primer- 1 µL REV primer- 1 µL template- 3 µL PFU (add PFU polymerase last and always keep the tubes on ice)

                                                          ![](https://static.igem.org/mediawiki/2018/b/b3/T--Lubbock_TTU--wiki5.jpg)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Lubbock TTU/Results

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### Using Lambda Red Recombinase System for Gene Knockout

**PFU PCR Linear DNA**

The LacI and ArgI with 280 bp homology arm gene were amplify using PFU PCR.- The size of the LacI template with spectinomycin resistance inserted between the homology arms is 1995 bp.- The size of the ArgI template with spectinomycin resistance inserted between the homology arms is 1990 bp

    ![](https://static.igem.org/mediawiki/2018/9/9b/T--Lubbock_TTU--results1.jpg)

### LacI and ArgI Transformation into E. Coli W3100

Amplified PCR LacI and ArgI products were electroporated into E. Coli W3110 harboring pREDKI plasmid. The transformed cells were plated into a gradient of different spectinomycin concertation; starting at 60 ug/ml, 70 ug/ml and 80 ug/ml and 100 ug/ml.- ArgI had grown in 80 µg/mL Spec Plates. There colonies were evaluated and determined to be false positives for recombination.- LacI had grown in 70 µg/mL Spec plates. There colonies were evaluated and determined to be false positives for recombination. However, the negative control had grown as well.

    ![](https://static.igem.org/mediawiki/2018/2/2d/T--Lubbock_TTU--results2.jpg)

### Genomic PCR of the W3110 Lambda Red Modified Cells

A genomic PCR was done to confirm the recombination.

* ArgI Genomic PCR did not work in terms of Lambda Red Recombination because the gel still shows the native gene size of 1549bp not the ArgI Homology arms with the spectinomycin template which would be closer to 1995bp, but the gel reflects the size of just above 1500bp.

  * LacI Genomic PCR did not work in terms of Lambda Red Recombination because the gel still shows the native gene size of 1616bp not the LacI Homology arms with the spectinomycin template which would be closer to 1990bp, but the gel reflects the size of just above 1500bp.

    If the recombination worked an expect band size near the 2000bp mark on the ladder. Since the band size are the native gene size it is safe to assume that the cells didn't accept the linear gBlock for the recombination event.

    The positive control was pREDKI which has a size of 1050 bp using the Exo Forward and Exo reverse primer sequences.

    ![](https://static.igem.org/mediawiki/2018/e/e8/T--Lubbock_TTU--results3.jpg)

    **Problems Faced*** Linear PCR fragments with the antibiotic template was frozen at -20C which could have caused issues because PCR products have nicks in the backbone.* The antibiotic concentration for selection of the genomic recombination was not clear and so gradient of antibiotic was used and this lead to some false positive colonies on these plates.* Concentration of arabinose for the pREDKI plasmid was not at the appropriate concentration for the most efficient expression of the lambda red recombination proteins.

          **Future Plans*** Optimize the pREDKI recombination protocol for higher efficiency.* Knock out nonessential genes to metabolically engineer E. coli cells.* Potentially knock in new genes to build a better metabolic pathway using other more efficient enzymes for putrescine production.* Using pSIM plasmid as the biofunctional helper plasmid for the recombination

**### Bio Bricks**

**Amplification of Truncated ArgI sequence using PFU PCR protocol**

The ArgI gene was amplify by PFU PCR using M13 primers.- The size of the Truncated ArgI template is 995 bp- Positive Control

    ![](https://static.igem.org/mediawiki/2018/e/ea/T--Lubbock_TTU--results4.jpg)

    The LacI gene was amplify by PFU PCR using T7 primers.- The size of the Truncated ArgI template is 835 bp- Positive Control

        **### Digestion/Ligation of LacI and ArgI into pSB1C3**

        The truncated LacI and ArgI PCR product were digested with EcoRI-PstI and ligated with T4 ligase into pSB1C3. The ligation product was plated on 3.7 ug/ml chloramphenicol plates. Colonies were confirmed by direct colony PCR.

        ![](https://static.igem.org/mediawiki/2018/0/0b/T--Lubbock_TTU--results5.jpg)

        ![](https://static.igem.org/mediawiki/2018/7/71/T--Lubbock_TTU--results6.jpg)

Lubbock\_TTU · Texas Tech University