iGEM 2019 — Team CU-Boulder

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# Team:CU-Boulder/Notebook

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TEAM

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PROJECT

[Description](https://2019.igem.org/Team%3ACU-Boulder/Description)
[Design](https://2019.igem.org/Team%3ACU-Boulder/Design)
[Modeling](https://2019.igem.org/Team%3ACU-Boulder/Model)
[Experiments](https://2019.igem.org/Team%3ACU-Boulder/Experiments)

[Contribution](https://2019.igem.org/Team%3ACU-Boulder/Contribution)
[Results](https://2019.igem.org/Team%3ACU-Boulder/Results)

[Attributions](https://2019.igem.org/Team%3ACU-Boulder/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3ACU-Boulder/Parts)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ACU-Boulder/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ACU-Boulder/Public_Engagement)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=CU-Boulder)

# Notebook Timeline

---

###### Welcome to our notebook! As you scroll down the timeline you can see our actual notes for what we did each day.

2019

13
May

For the 2A9N - remove the H and the L chains from sequence (only want the M and I).

Need to do:

* Make the 9 Bsa1 sites
* Check NEB digest - get above 90% chance of working/not binding with the wrong overhang
* Check methylation sites

14
May

Bsa1 cutting sites--9 regions and 9 cutting sites

**Region 1 - linker region 1 (ScFv1 and AraC1)** - Bsa1 in ScFv1 is TTAC
- Bsa1 in AraC1 is AGTG
- 321 bases

**Region 2 - AraC1 and ribo linker and ScFv2**- Bsa1 in AraC1 is AGTG
- Bsa1 in ScFv2 is CAGA
- 580 bases

**Region 3 - Linker region 2 (ScFv2 and AraC2)**- Bsa1 in ScFv2 is CAGA
- Bsa1 in Arac2 is TGAA
- 361 bases

**Region 4 - AraC2 and part of plasmid (1)** - Bsa1 in AraC2 is TGAA
- Bsa1 in Plasmid (1) is TGCC
- 306 bases
There is extra stuff added on.
The Bsa1 overhang starts 14 bases in

**Region 5 - Plasmid (1) (Plasmid 1 w/ enzymes)**- Bsa1 1 is TGCC
- Bsa1 2 is AAAA
- 638 bases

**Region 6 - Plasmid (2) to Plasmid (3) (Plasmid 2 w/ enzymes)**- Bsa1 1 is AAAA
- Bsa1 2 is CCAG
- 1449 bases

**Region 7 - Plasmid (3) and Plasmid (4) (Plasmid 3 w/ enzymes)**- Bsa1 1 is CCAG
- Bsa1 2 is GCAA
- 1296 bases

**Region 8 - Plasmid (4) and Plasmid (4) with the ScFv1 (Plasmid 4 with enzymes)**- Bsa1 1 is GCAA
- Bsa1 2 is ATTC
- 1694 bases

**Region 9 - Plasmid (4) with the ScFv1**- Bsa1 in Plasmid (4) is ATTC
- Bsa1 in ScFv1 is TTAC
- 405 bases
48 bases from the plasmid are with the ScFv1 including the overhang

15
May

From Twist: 100 ng - 1 μg for each fragment- Concentration - in 2 ml tube with 10ng/μl

Fragments were ordered today.. expect to recieve them by June 12th.
Next order by date is June 12 - get by July 10th

23
May

Chain M (Scfv2) in the structure of Fluro antibody missing residue 149 (Gly)- ScFv1 = Chain I = Chain A (ScFv1 + AraC1)
- ScFv2 = Chain M = CHain B (ScFv2 + AraC2)

3
June

Continued to work on modeling and grants.

- Linker addition onto Arac1 (Chain C) = 10 amino acids -> 1 GSGSGSGRGQ 10
- Linker addition onto Arac2 (Chain D) = 15 amino acids -> 1 SGSGSGSGSGSGRGQ 15

A -> C = Scfv1 + Arac1.
B -> D = Scfv2 + Arac2

4
June

Continued modeling and fixing different linkers

5
June

\*\*\*Linker additions made to DNA fragments on 6/3
changed scfv1=A, scfv2=C, arac1=B, arac2=D on pymol files

- A -> B = Scfv1 + Arac1
- C -> D = Scfv2 + Arac2

6
June

For control, IgG1 ~ 1ngq antibody - humanized- M -> L = scfv2 + fab2
- I -> H = scfv1 + fab1

10
June

Received DNA from Twist, will bring up DNA to create stock solutions tomorrow.

11
June

TEST

12
June

**DNA IN EACH TUBE:**- 3.9 pmol = 3.9 e 10^-12 mol - plasmid4 + scfv1
- 4.9 pmol = linker region 1
- 2.75 pmol = arac1 + scfv2
- 4.3 pmol = linker region 2
- 4.9 pmol = acrc2 + plasmid 1
- 2.5 pmol = plasmid 1
- 1.11 pmol = plasmid 2
- 1.2 pmol = plasmid 3
- 0.92 pmol = plasmid 4

**AMOUNT OF Tris TO ADD TO BRING UP TWIST DNA:** - Add 97.5 μl to plasmid4 + scfv1
- Add 122.5 μl to linker region 1
- Add 68.75 μl to scfv1 + arac2
- Add 107.5 μl to linker region 2
- Add 122.5 μl to acrc2 + plasmid 1
- Add 62.5 μl to plasmid 1
- Add 27.75 μl to plasmid 2
- Add 30 μl to plasmid 3
- Add 23 μl to plasmid 4

Each piece of our DNA plasmid should now have a concentration 40 fmol/1μl

**ADDITION OF AMP TO AGAR MIX:**- 100 mg/ml - 2ml per 1 L

13
June

TEST

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# Team:CU-Boulder/Experiments

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PROJECT

[Description](https://2019.igem.org/Team%3ACU-Boulder/Description)
[Design](https://2019.igem.org/Team%3ACU-Boulder/Design)
[Modeling](https://2019.igem.org/Team%3ACU-Boulder/Model)
[Experiments](https://2019.igem.org/Team%3ACU-Boulder/Experiments)

[Contribution](https://2019.igem.org/Team%3ACU-Boulder/Contribution)
[Results](https://2019.igem.org/Team%3ACU-Boulder/Results)

[Attributions](https://2019.igem.org/Team%3ACU-Boulder/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3ACU-Boulder/Parts)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ACU-Boulder/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ACU-Boulder/Public_Engagement)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=CU-Boulder)

# Protocols

Golden Gate Set Up

#### Golden Gate Plasmid

1. Set up Amp. Plates (250 mL batch)

- 8.9g Easy Mix- per 250 ml of water- add 0.5 ml of AMP to autoclaved mix (cooled, now room temp)- pour mix into plates and let dry

      2. Make 10mM Tris-HCl

      3. Evaluate how many pmol/fmol are in each DNA tube

      - 3.9 pmol = plasmid4 + scfv1- 4.9 pmol = linker region 1- 2.75 pmol = scfv1 + arac2- 4.3 pmol = linker region 2- 4.9 pmol = acrc2 + plasmid 1- 2.5 pmol = plasmid 1- 1.11 pmol = plasmid 2- 1.2 pmol = plasmid 3- 0.92 pmol = plasmid 4

                      4. Bring up DNA to appropriate concentration (40fmol/1uL) using 10mM Tris-HCl

                      - Add 97.5 μl to plasmid4 + scfv1- Add 122.5 μl to linker region 1- Add 68.8 μl to arac1 + scfv2- Add 107.5 μl to linker region 2- Add 122.5 μl to acrc2 + plasmid 1- Add 62.5 μl to plasmid 1- Add 27.8 μl to plasmid 2- Add 30.0 μl to plasmid 3- Add 23.0 μl to plasmid 4

                                      5. Assemble

                                      - 11.5 μl Nuclease-free H2O- 2 μl of T4 DNA Ligase Buffer (10X)- Add 0.5μl of each fragment 1- Add 0.5μl of each fragment 2- Add 0.5μl of each fragment 3- Add 0.5μl of each fragment 4- Add 0.5μl of each fragment 5- Add 0.5μl of each fragment 6- Add 0.5μl of each fragment 7- Add 0.5μl of each fragment 8- Add 0.5μl of each fragment 9- 2 μl NEB Golden Gate Enzyme Mix- \*\*\* total volume should be 20 μl

                                                              6. Thermocycle/PCR

                                                              - For 5-10 inserts: (37°C, 1 min → 16°C, 1 min) x 30 → 60°C, 5 min- For 11+ inserts: (37°C, 5 min → 16°C, 5 min) x 120 → 60°C, 5 min- Going to 16°C is optional, and may create more mismatches

                                                                  7. PCR Purification

                                                                  1. Add 100μl of Buffer PB and all the PCR liquid into a QIAquick spin column- Centrifuge for 30-60sec- Discard flow through- Add 750μl buffer PE- Centrifuge for 30-60sec- Discard flow through- Centrifuge again for 30-60 secs- Discard flow- Get new 1.5ml microcentrifuge tube and label it GG and date- Add 50 μl water- Centrifuge for 1min and put in freezer

                                                                  8. Transform (into E.Coli cells)

                                                                  - Keep everything on ice constantly- Warm up plates and SOC to 37- Make sure cells are somewhat thawed
                                                                      1. Add 2μl of DNA to microcentrifuge tube- Add 25μl of E. coli cells to mc. tube- Stir the liquid in the mc. tube with the pipet- Pull all 27μl out of the tube- Drop in between the two slits at the bottom of the corvette- Perform electroporation ASAP, at 1.8 kV
                                                                                 - Record time constant (want to to be around 4.7)- Add 960μl of the SOC broth into the box- Pipet up and down a few times to mix- Pull all the broth back out and put back into the tube- Incubate at 37 for 1hr- Stir it a bit (invert it several times and flick it)- Plate 50μl per plate

PCR Protocol

1. From primer powder make 100uM/L solution of primer

2. Make 10uM/L stock solution of primer

- 45 uL water- 5 uL 100 uM/L primer solution

  3a. If using DreamTaq Green PCR Master Mix

  - 25 uL of Master Mix- 0.1-1 uM FWD Primer (.5 uL of stock)- 0.1-1 uM REV Primer (.5 uL of stock)- 10 pg-1 ug Template DNA (0.5 uL)- Up to 50 uL Nuclease free H2O

          3b. If no Master Mix

          - 5uL of 10x standard Taq buffer- 1.5ul of 50 uM MgCl2- 1uL of 10 uM dNTPs- 1 uL of FWD primer (from 10uM stock!)- 1 uL of REV primer (from 10uM stock!)- ~1.25uL of template DNA or from colonies- 0.25uL of Taq DNA polymerase- 39uL of dH2O (Up to 50 uL of water)

                        4a. If using DNA set Thermocycler to

                        - **Hold**- 94 C - 3 min- **25-40 cycles**- 94 C - 30 sec- Tm-5 C - 30 sec- 68 C - 1 min- **Hold**- 68 C - 5-15 min- 4 C - infinity /li>

                                          4b. If using colony as template DNA set Thermocycler to

                                          - **Hold**- 95 C - 5 min- **25-40 cycles** - 95 C - 30 sec- 58 C - 60 sec- 68 C - 1 min- **Hold**- 68 C - 5 min- 4 C - infinity

Restriction Digest Protocol

EcoRI Digest

- 8 uL DNA (150 ng/uL) = 0.15ug- 1 uL EcoRI- 1 uL CutSmart- 10 uL total volume- **Incubate 70 mins at 37 C**- **Heat inactivate for 20 mins at 65 C**

          Pst1 Digest

          - 8 uL DNA (150 ng/uL) = 0.15ug- 1 uL Pst1- 1 uL CutSmart- 10 uL volume total- **Incubate at 37 C for at least one hour**- **Heat inactivate at 80 C for 20 min**

Mini Prep/ Grow Up Protocol

Use the QIAprep Miniprep Kit

- Pellet 1-5ml of bacterial overnight culture by centrifugation at >8000rpm for 3min at room temperature- Resuspend the pelleted bacterial cells in 250μl Buffer P1 and transfer to a microcentrifuge tube- Add 250μl BufferP2 and mix throughly by inverting the tubes until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5min- Add 350μl Buffer N3 and mix immediatly and thoroughly by inverting the tube 4-6 times. If using the LyseBlue reagent the solution will turn colorless- Centrifuge for 10min at 13,000 RPM in a tabletop centrifuge- Apply 800μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting- Centrifuge for 30-60sec and discard flow through at 10,000 RPM- Wash the QIAprep 2.0 spin column by adding 0.5ml Buffer PB- Centrifuge for 30-60sec. Discard flow through- Wash the QIAprep 2.0 spin column by adding 0.75ml Buffer PE- Centrifuge for 30-60sec. Discard flow through- Centrifuge for 1 min to remove residual wash buffer- Place the QIAprep 2.0 column in a clean 1.5ml microcentrifuge tube.- Add 40μl of water and let stand for 1-2 min- Centrifuge for 1 min

LB Broth Recipe

Ingredients:

- 10g Tryptone- 5g Yeast Extract- 10g Sodium Chloride

    Steps:

    - Dissolve the ingredients in 1L water- Portion into flasks and cover with aluminum foil (fill flask to half its volume)- Autoclave for 20-30 minutes on LIQUID cycle

Electroporation Protocol

Steps:

- Prepare microcentrifuge at room temperature. Place NEB® 10-beta/Stable Outgrowth Medium in a 37°C water bath. Pre-warm plates at 37°C for 1 hour.- Place electroporation cuvettes and microcentrifuge tubes on ice- Positive control for transformation: dilute the control pUC19 by 1:5 to a final concentration of 10 pg/μl using ddH2O.- Thaw NEB 10-beta Electrocompetent cells on ice (about 10 min) and mix cells by flicking gently.- Transfer 25 μl of the cells to chilled microcentrifuge tubes and add 1 uL of DNA- Transfer the cell/DNA mix into chilled cuvette (without introducing bubbles)- Electroporate using the following conditions for BTX ECM 630 and Bio-Rad GenePulser electroporators: 1.8 kV. The typical time constant is 4.8 to 5.1 milliseconds.- Immediately add 975 uL of 37°C NEB 10-beta/Stable Outgrowth Medium to the cuvette- Gently mix up and down twice and then transfer to the microcentrifuge tube- Shake (250 rpm) or rotate at 37°C for 1 hour- Dilute the cells (as appropriate); spread 100-200 μl cells onto a pre-warmed plate.- Incubate plates overnight at 37°C.

Agar Recipe

Lorem Ipsum!

Gel Extraction Protocol

Steps:

- Excise the DNA band from the agarose el with a clean, sharp scalpel. Use a 1.5 ml microfuge tube for processing up to 250 mg agarose per tube.- Weigh the gel slice in a colorless tube. Add buffer QX1 according to DNA fragment size; 3 volumes with 2 volumes of water for >4kb.- Resuspend QIAEX II by vortexing for 30 sec. Add QIAEX II to the sample and mix: use 10 ul QIAEX II for ≤2 ug DNA; 30 ul for 2-10 ug DNA; andan additional 30 ul for each additional 10 ug DNA.- Incubate at 50 degrees for 10 min to solubilize the agarose and bind the DNA. Mix by vortexing every 2 min to keep QIAEX II in suspension. Check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 ul 3M sodium acetate, pH 5.0, and mix. The color should turn yellow. The incubation should then be continued for at least 5 min.- Centrifuge the sample for 30 sec and carefully remove supernatant with a pipet.- Wash the pellet with 500 ul buffer QX1. Resuspend the pellet by vortexing.- Centrifuge the sample for 30 sec and remove all traces of supernatant with a pipet. This step removes residual agarose contaminants.- Wash the pellet twice with 500 ul Buffer PE. Resuspend peet by vortexing.- Centrifuge the sample for 30 sec and carefully remove all traces of supernatant with a pipet. This step removes residual salt contaminants.- Air-dry the pellet for 10-15 min or until the pellet becomes white. If 30 ul QIAEX II suspension is used, air dry the pellet approx. 30 min.- To elute DNA, add 20 ul of water and resuspend the pellet by vortexing. Incubate according to the DNA fragment size: 5 min at 50 degrees for 4-10 kb.- Centrifuge for 30 sec, and carefully pipet the supernatant into a clean tube. The supernatant now contains the purified DNA.

BL21 Chemically Competent Cells Transformation Protocol

FOR C2527H CELLS

Perform these steps in the tube provided:

- Thaw a tube of BL21 cells on ice for 10 minutes until last bit of ice disappears- Add 1-5 uL containing 1pg - 100 ng of plasmid DNA to the cell mixture- Carefully flick the tube 4-5 times to mix the cells and DNA (do not vortex)- Place the mixture on ice for 30 minutes (do not mix)- Heat shock at exactly 42 C for exactly 10 seconds (do not mix)- Place on ice for 5 minutes (do not mix)- Pipette 950 uL of room temperature SOC into the mixture- Place at 37 C for 60 minutes - shake vigorously (250 rpm) or rotate

              These steps do not need to be in the tube:

              - Warm plates to 37 C- Mix the cells thoroughly by flicking the tube and inverting- Spread 20 uL, 50 uL, and 100 uL onto plates and incubate overnight at 37 C- Alternatively, incubate at 30 C for 24-36 hours or at 25 C for 48 hours

Growing Electrocompetent Cells Protocol

DAY ONE

- Streak out frozen glycerol stock of bacterial cells (Top 10, DHα, etc.) onto an LB plate (no antibiotics)- Grow plate overnight at 37°C

  #### DAY TWO

  1. Autoclave:- 2L ddH2O- 100 mL of 10% v/v glycerol- 1 L LB (or your preferred media)- 4 centrifuge bottles and caps- 2 50mL small centrifuge bottles- Lots of microcentrifuge tubes

            2. Chill overnight at 4°C:

            - ddH2O- 10% glycerol- Centrifuge rotor

                3. Prepare starter culture of cells:

                - Select a single colony of E. coli from fresh LB plate and inoculate a 10mL stater culture of LB (or your prepared media)- Grow culture at 37°C in shaker overnight- Notes: You can subsitute other media like SOB, 2xYT, etc for LB if preferred. All glassware should be detergent free. Trace detergent residue reduces competency.

                    #### DAY THREE

                    1.

                    - Inoculate 1 L of LB media at 37°C with 10mL starter culture and grow in 37°C shaker.- Measure the OD600 every hour, then every 15-20 mins when the OD gets above 0.2 AND STOP when OD reaches 0.35-0.40

                      2.

                      - When the OD600 reaches 0.35-0.4, immediately put the cells on ice.- Chill the culture for 20-30 mins, swirling occasionally to ensure even cooling- Place centrifuge bottles on ice

                          IMPORTANT NOTES:

                          - Carefully check and monitor often OD once the OD reaches 0.2- DO NOT let OD get above 0.4- It usually takes about 3 hours to reach an OD of 0.35 when using a 10mL starter culture.- Keep the cells at 4°C for the remainder of the procedure (any bottles or solutions that the cells come into contact with must be pre-chilled to 4°C

                                3.

                                - (SPIN 1) Split the 1 L culture into four parts by pouring about 250mL into ice cold centrifuge bottles.- Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-rotor) for 20 mins at 4°C

                                  4.

                                  - Decant the supernatant and resuspend each pellet in 200mL of ice cold ddH2O

                                  5.

                                  - (SPIN 2) harvest the cels by centrifugation at 1000g (~2400 rpm in the Beckman JA-rotor) for 20 mins at 4°C

                                  6.

                                  - Decant the supernatant and resuspend each pellet in 100mL of ice cold ddH2O

                                  7.

                                  - (SPIN 3) Combine resuspensions into 2 centrifuge bottles (so each contains about 200mL of cell suspension)- Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-rotor) for 20 mins at 4°C- Rinse two 50mL concical tubes with ddH2O and chill on ice

                                      8.

                                      - Decant the supernatant and resuspend each pellet in 40mL of ice cold 10% glycerol- Transfer each suspension to a 50mL concical tube

                                        9.

                                        - (SPIN 4)harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-rotor) for 20 mins at 4°C- Start putting 1.5mL microcentrifuge tubes on ice if not already chilled

                                          10.

                                          - Carefully aspirate the supernatant with a sterile pasteur pipette (pellets lose adherance in 10% gylcerol)- Resuspend each pellet in 1mL of ice cold 10% gylcerol by gently swirling- The final OD600 of the resuspended cells should be 200-250

                                              11.

                                              - Aliquot into sterile 1.5mL microcentrifuge tubes and snap freeze with liquid nitrogen- Store frozen cells in -80°C freezer

Protein Purification

#### THE PROTOCOL

1. PCR cleanup backbone

2. 30 uL of DNA (cleaned)

- 5 uL 10X Cutsmart buffer- 1 uL BsaI- 1 uL DpnI

    3. incubate for 4 hr at 37 C

    4. gel extract using 1.0% gel and ~90V (if needed)

    - 1 uL Quick CIP- 15 ul of DNA- 2 uL cutsmart buffer- 3 uL of dH2O ~ up to 20 ul

          5. incubate for 1 hr at 37 C

          6. heat inactivate at 80 C for 4 mins

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:CU-Boulder/Results

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TEAM

[Team Members](https://2019.igem.org/Team%3ACU-Boulder/Team)
[Collaborations](https://2019.igem.org/Team%3ACU-Boulder/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3ACU-Boulder/Description)
[Design](https://2019.igem.org/Team%3ACU-Boulder/Design)
[Modeling](https://2019.igem.org/Team%3ACU-Boulder/Model)
[Experiments](https://2019.igem.org/Team%3ACU-Boulder/Experiments)

[Contribution](https://2019.igem.org/Team%3ACU-Boulder/Contribution)
[Results](https://2019.igem.org/Team%3ACU-Boulder/Results)

[Attributions](https://2019.igem.org/Team%3ACU-Boulder/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3ACU-Boulder/Parts)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ACU-Boulder/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ACU-Boulder/Public_Engagement)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=CU-Boulder)

# Results

## Protein Design

---

With the use of protein modeling systems, a visual representation our antibody switch was made. We made two possible models, each one with a unique linker designs. The linkers were modeled and designed for flexibility and strength, in order to pull apart the antibody domains when the AraC domains bind to arabinose. In the absence of arabinose, the FITC-E2 domains will reinteract and be able to bind to fluorescein.

SHORT LINKER:

![Short Linker](https://static.igem.org/mediawiki/2019/e/ef/T--CU-Boulder--proto4.jpg)

LONG LINKER:

![long linker](https://static.igem.org/mediawiki/2019/a/ab/T--CU-Boulder--proto17.jpg)

## Plasmid Design

---

The created protein model was then designed for a vector system. Vectors were created using a pET52a vector system and specific double stranded DNA fragments (ordered from both IDT and Twist DNA). The completed vector, around roughly 7000 base pairs, was made up of nine fragments. The fragments all contained designed BsaI sites at each end, allowing for successful ligation of DNA fragments and incorporation into the pET52a plasmid via Golden Gate assembly.

![Complete Plasmid](https://static.igem.org/mediawiki/2019/7/70/T--CU-Boulder--CompletePlasmid.jpg)

Figure 1: Antibody Switch Plasmid. The places labeled as linker regions were designed to have BsaI sites between the end of the ScFv region and the AraC region so that we could insert various linker designs to determine which design worked the best to enable our switch. This plasmid construct was split into nine DNA fragments with BsaI sites unique BsaI sites between each fragment.

A control vector was also designed, which includes the fluorescein antibody ScFv domain attached to a FAB domain. The goal behind the control was to determine if attaching a second domain to the ScFv domain would inhibit the antibody from correctly binding to fluorescein, therefore the designed linkers were not used as a part of the control design.

![Control Plasmid](https://static.igem.org/mediawiki/2019/e/e9/T--CU-Boulder--ControlPlasmid.jpg)

Figure 2: Control Plasmid. The FITC-E2 + FAB domain region consists of the fluorescein binding antibody domains connected to a similar FAB domain. The designed linkers were not applied to the control because we wanted to determine if the general design of attaching a second domain to the ScFv domain would still allow the FITC-E2 domain to bind to fluorescein.

## Experimental Results

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Golden Gate assembly was used to assemble the DNA fragments for both the experimental and control plasmids.
Using PCR primers to check the overlapping regions between the DNA fragments, we were able to determine whether or not the fragments went correctly together in relation to one another. From multiple PCR checks, we were able to determine that the more fragments you had the more mismatching that could occur between fragments. So, once we correctly assembled the three pieces that made up the plasmid backbone, we performed a PCR that amplified the backbone region and then gel extracted to backbone fragment. From here, we performed all future Golden Gates with one backbone piece, which cut down our number of DNA fragments for each reaction.

![Gel 2](https://static.igem.org/mediawiki/2019/e/e1/T--CU-Boulder--Gel2.jpg)

Figure 3: PCR of the plasmid backbone. The backbone was just over 5000 base pairs so this gel determined that the backbone was successfully amplified. Two trials were done to determine if the addition of DMSO would help amplify the backbone. From the two trials, we found that the addition of DMSO did help the amplification.

All assembled vectors were transformed with electrical based techniques into Neb 10 Beta cells. Given that the control plasmid had fewer DNA fragments, we began with the assembly of that design first. Successful vector formation and transformation occurred with the pET52a plasmid and designed DNA fragments for the control, as seen in the figures below.

![Control Plate](https://static.igem.org/mediawiki/2019/f/f6/T--CU-Boulder--ControlPlate.jpg)

Figure 4: Successful colony growth after transformation of the control plasmid into Neb 10 Beta cells.

![Successful Results](https://static.igem.org/mediawiki/2019/f/fd/T--CU-Boulder--ResultsControl.jpg)

Figure 5: Colony PCR on Control - determined that the plasmid backbone went into the control DNA fragment (2 piece Golden Gate assembly). We designed a 300 base PCR product that spans one of overlaps between inserts. Therefore, by obtaining the expected 300 base pair results, we knew that the control fragment and the plasmid backbone were assembled correctly in relation to each other.

## Protein Identification

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Having successfully assembled and transformed our control plasmid, we then transformed the plasmid into chemically competent BL21 cells, as seen in the figure below.

![Colony Growth](https://static.igem.org/mediawiki/2019/e/e2/T--CU-Boulder--ColonyGrowth.jpg)

Figure 6: Successful colony growth after transformation of the control plasmid into BL21 cells.

Next we will be performing protein purification on these colonies and testing the control design.

# Analysis

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One of the unfortunate problems that we encountered with our experiment is that when we got to purification of our control protein, we discovered that our protein was incorrectly folding inside of the cytoplasm of the cell. We needed to add signal tags onto our protein so that it would sent into the periplasm of the cell, and therefore fold correctly. This mistake set our progress back because we had to order new DNA fragments with the included signal tags. Despite this mistake, we were still able to correctly assemble the control plasmid again with the signal tags included.

# Future Steps

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Our initial future steps would be to continue with Golden Gate experiments to successful assemble our full switch design with the two possible linker options. Once correctly assembled, we then would transform into BL21 cells before moving onto protein purification. Once purified, we will test our protein with both arabinose and fluorescein to determine if our switch model is feasible.

Our big picture future steps would potentially include humanizing the AraC domain and testing out more linker ideas.