iGEM 2018 — Team Pittsburgh

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# Team:Pittsburgh/Notebook

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* [Team](https://2018.igem.org/Team%3APittsburgh)
  + [Team Members](https://2018.igem.org/Team%3APittsburgh/Team)
  + [Collaborations](https://2018.igem.org/Team%3APittsburgh/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3APittsburgh/Attributions)
* [Project](https://2018.igem.org/Team%3APittsburgh)
  + [Description](https://2018.igem.org/Team%3APittsburgh/Description)
  + [Design](https://2018.igem.org/Team%3APittsburgh/Design)
  + [Experiments](https://2018.igem.org/Team%3APittsburgh/Experiments)
  + [Results](https://2018.igem.org/Team%3APittsburgh/Results)
  + [Notebook](https://2018.igem.org/Team%3APittsburgh/Notebook)
  + [InterLab](https://2018.igem.org/Team%3APittsburgh/InterLab)
* [Modeling](https://2018.igem.org/Team%3APittsburgh)
  + [Model](https://2018.igem.org/Team%3APittsburgh/Model)
  + [Software Tool](https://2018.igem.org/Team%3APittsburgh/Software)
* [Parts](https://2018.igem.org/Team%3APittsburgh/Parts)
* [Human Practices](https://2018.igem.org/Team%3APittsburgh)
  + [Human Practices](https://2018.igem.org/Team%3APittsburgh/Human_Practices)
  + [Education & Engagement](https://2018.igem.org/Team%3APittsburgh/Public_Engagement)
* [Safety](https://2018.igem.org/Team%3APittsburgh/Safety)


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# Team:Pittsburgh/Experiments

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* [Team](https://2018.igem.org/Team%3APittsburgh)
  + [Team Members](https://2018.igem.org/Team%3APittsburgh/Team)
  + [Collaborations](https://2018.igem.org/Team%3APittsburgh/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3APittsburgh/Attributions)
* [Project](https://2018.igem.org/Team%3APittsburgh)
  + [Description](https://2018.igem.org/Team%3APittsburgh/Description)
  + [Design](https://2018.igem.org/Team%3APittsburgh/Design)
  + [Experiments](https://2018.igem.org/Team%3APittsburgh/Experiments)
  + [Results](https://2018.igem.org/Team%3APittsburgh/Results)
  + [Notebook](https://2018.igem.org/Team%3APittsburgh/Notebook)
  + [InterLab](https://2018.igem.org/Team%3APittsburgh/InterLab)
* [Modeling](https://2018.igem.org/Team%3APittsburgh)
  + [Model](https://2018.igem.org/Team%3APittsburgh/Model)
  + [Software Tool](https://2018.igem.org/Team%3APittsburgh/Software)
* [Parts](https://2018.igem.org/Team%3APittsburgh/Parts)
* [Human Practices](https://2018.igem.org/Team%3APittsburgh)
  + [Human Practices](https://2018.igem.org/Team%3APittsburgh/Human_Practices)
  + [Education & Engagement](https://2018.igem.org/Team%3APittsburgh/Public_Engagement)
* [Safety](https://2018.igem.org/Team%3APittsburgh/Safety)

[Antibiotics and Inducers](#section1)
[TE Buffer](#section2)
[Liquid LB Media](#section3)
[LB Agar Plates](#section4)
[Liquid Cultures](#section5)
[Transformation: DH5a](#section6)
[Making Competent Cells](#section7)
[Transformation S1030](#section8)
[TBE Buffer](#section9)
[Gel Electrophoresis](#section10)
[Gel Extraction](#section11)
[Miniprep](#section12)
[PCR](#section13)
[DNA Clean](#section14)
[Annealing DNA Oligos](#section15)
[Digetion](#section16)
[Ligation](#section17)
[Gibson](#section18)
[TOPO Cloning](#section19)
[Annealing RNA oligos](#section23)
[Cas9 digestion](#section24)
[CAMERA](#section20)
[Base Editor Improvement](#section21)
[CrisPy](#section22)

Antibiotics and Inducers
:   | Antibiotic/Inducer | Stock Concentration | Working Concentration | Solvent | Storage |
    | --- | --- | --- | --- | --- |
    | Ampicillin/Carbenicillin (Amp/Carb) | 100 mg/mL | 100 μg/mL | ddH2O | -20℃ |
    | Chloramphenicol (CAM) | 35 mg/mL | 35 μg/mL | 100% ethanol | -20℃ |
    | Spectinomycin (Spec) | 100 mg/mL | 100 μg/mL | ddH2O | -20℃ |
    | Streptomycin (Strep) | 100 mg/mL | 50 μg/mL | ddH2O | -20℃ |
    | kanamycin (Kan) | 50 mg/mL | 50 μg/mL | ddH2O | -20℃ |
    | anhydrous tetracycline (aTc) | 250 μg/mL | 250 ng/mL | 50% ethanol | -20℃, in foil wrap (avoid light exposure) |
    | Isopropyl β-D-1-thiogalactopyranoside (IPTG) | 0.1 M (24 mg/mL) | 0.1 mM | ddH2O | -20℃ |
    | Arabinose | 20% (0.2 g/mL) | 0.2% | ddH2O | Room temperature |
    | Xylose | 20% (0.2g/mL) | 2% | ddH2O | Room temperature |

TE Buffer[1]
:   | Component | Volume (Total 500 mL) |
    | --- | --- |
    | 1M Tris (pH 8) | 5 mL |
    | 0.5 M EDTA (pH 8) | 1 mL |
    | ddH2O | 496 mL |

Liquid LB Media
:   1. To make 1L liquid LB media, weigh 25g of anhydrous LB Broth and add it to 1000 mL of Millipore water.
    2. Screw the cap partially and place the media bottle in the autoclave tray along with a water bath about 3-5 cm high. Apply Autoclave tape to the top of the bottle.
    3. Autoclave the bottle under the “liquid” setting.
    4. Allow to cool on bench top before use or adding antibiotics.

LB Agar plates
:   1. Turn on the water bath to 60℃.
    2. To make 500 mL media, add 12.5g of anhydrous LB Broth and 7.5g of BD BactoTM Agar to 500 mL of the Millipore water.
    3. Screw the cap partially and place the media bottle in the autoclave tray along with a water bath about 3-5 cm high. Apply Autoclave tape to the top of the bottle.
    4. Autoclave the bottle under the “liquid” setting.
    5. Allow to cool in water bath before use or adding antibiotics.
    6. Add antibiotic for selectivity as desired and distribute LB broth onto the plates to cover about halfway.
    7. Allow the plates to cool on the counter and store inverted in the 4℃ refrigerator.

Liquid Culture
:   1. Aliquot 5ml of LB into each 15 mL conical tube. Add antibiotic as desired.
    2. Remove plates from the fridge and use a marker to label the desired single origin colonies that are easy to pick.
    3. Using the p10 pipette tip, gently pick the colony and place it in the tube with liquid media.
    4. Screw the cap partially and apply lab tape to the top of the tube.
    5. Place the liquid culture tubes on the shaker at 37℃ and 220 rpm overnight (16-18 hours).

Transformation of E.coli DH5a Cells
:   1. Thaw chemically-competent DH5a cells on ice.
    2. Add 1-5 μL containing 1 pg-100 ng of plasmid DNA to 50 μL of competent cells.
    3. Place the mixture on ice for 30 minutes.
    4. Heat shock at 42℃ for 30 seconds.
    5. Transfer tubes to ice for 5 minutes.
    6. Add 950 μl of room temperature LB or SOC media to each tube.
    7. Incubate the tube at 37℃ for 60 minutes. Shake vigorously (250 rpm).
    8. Centrifuge the cells at 6800 rpm for 1 minute.
    9. Remove 900μL of the supernatant. Resuspend the cells and pipette 100 μL onto the selection plates.
    10. Place the plates upside down and incubate overnight at 37℃ (18-20 hours).

Preparation of chemically competent E. coli cells
:   Using Zymo Mix & Go E. coli Transformation Kit with a few differences:
    1. Use 0.5 mL of fresh, overnight E.coli culture to inoculate 50 mL LB in a flask. Shake culture vigorously (150-250 rpm) at 37℃ until the OD600 is 0.4-0.6.
    2. Prepare fresh buffer on ice: Add 2.5 mL Dilution Buffer and 2.5 mL 2X stock Wash Buffer to make 5 mL 1X Wash Buffer. Add 2.5 mL Dilution Buffer and 2.5 mL 2X stock Competent Buffer to make 5 mL 1X Competent Buffer.
    3. Transfer the culture from step 1 to ice. After 10 minutes, pellet the cell by centrifuge at 3300 rpm for 10 minutes at 4℃.
    4. Remove the supernatant and resuspended the cell in 5 mL 1X ice-cold Wash Buffer. Repellet the cells as step 3.
    5. Completely remove the supernatant and resuspended the cell in 5 mL 1X ice-cold Competent Buffer.
    6. Aliquot 0.1 mL of cell suspension into sterile eppendorfs. Cells are now ready for transformation or can be stored in -80℃.

Transformation of E. coli S1030 cells
:   1. Add 1-5 μL plasmid DNA to a tube of thawed Mix & Go cells on ice.
    2. When selecting with Ampicillin, spread 100 μl of the mixture onto a pre-warmed (37°C) plate.
    3. When selecting with other antibiotics, incubate the cells on ice for 5-10 minutes.
    3.Add 400 μL of SOC and incubate for 1 hour at 37°C with gentle shaking at 250 rpm.
    4.Then, spread 100 μL mixture directly onto pre-warmed plates.

TBE Buffer
:   1. To make 1L 5x TBE stock solution, add 54g of Tris Base, 27.5g of boric acid and 20 mL of 0.5M EDTA.
    2. Dissolve in 800 mL ddH2O.
    3. Using stir bar and heat plate set to low.
    4. Bring final volume to 1L.
    5. Dilute to 1x TBE buffer before using for gel electrophresis.

Gel Electrophresis
:   1. Put 0.5g of agar powder and 50 mL of New 1x TBE. Swirl gently and microwave for 45 seconds.
    2. Add 1uL of ethidium bromide to the hot gel and pour the gel with the desired comb.
    3. After the gel is solidify, add running buffer (Used 1x TBE) to completely submerge the gel.
    4. Add loading dye in a 5:1 DNA/loading dye ratio. (Ex. 25μL of DNA and 5μL of dye)
    5. Add 5-10 μL of ladder to the first well. Add controls and digested DNA to the other wells as desired.
    6. Run gel at 120 V for desired time.

Gel Extraction - Zymoclean Gel DNA Recovery Kit
:   1. Excise the DNA fragment from the agarose gel and transfer it into an eppendorf.
    2. Add 3 volumes of ADB to each volume of agarose excised from the gel.
    3. Incubate at 55℃ for 5-10 minutes until the gel slice is completely dissolved.
    4. Transfer the melted agarose solution to a Zymo-Spin™ Column in a Collection Tube.
    5. Centrifuge at maximum speed for 45 seconds. Discard the flow-through.
    6. Add 200 μL of DNA Wash Buffer to the column and centrifuge 30 seconds. Discard the flow-through. Repeat the wash step.
    7. Add 10-20 μL DNA Elution Buffer or nuclease free water directly to the column matrix. Place column into an eppendorf and centrifuge for 30 seconds to elute DNA.
    8. Nanodrop and record the concentration.

Miniprep - Zyppy™ Plasmid Miniprep Kit
:   1. Add 1.5 mL of bacterial culture grown in LB medium to a eppendorf and centrifuge for 30 seconds at maximum speed. Discard the supernatant. Repeat this step as needed.
    2. Add 600 μL of TE buffer to resuspend the pellet.
    3. Add 100 μL of 7X Lysis Buffer (Blue) and mix by inverting the tube 4-6 times.
    4. Add 350 μL of cold Neutralization Buffer (Yellow) and mix thoroughly. A yellowish precipitate will form.
    5. Centrifuge at maximum speed for 4 minutes.
    6. Transfer the supernatant (at most 800 μL) into the provided Zymo-Spin™ IIN column. Place the column into a Collection Tube and centrifuge for 15 seconds.
    7. Discard the flow-through. Reat Step 6 if necessary.
    8. Add 200 μL of Endo-Wash Buffer to the column. Centrifuge for 30 seconds.
    9. Add 400 μL of Zyppy™ Wash Buffer to the column. Centrifuge for 1 minute.
    10. Transfer the column into a labeled eppendorf then add 30 μL of Zyppy™ Elution Buffer or nuclease free water directly to the column matrix and let stand for one minute at room temperature.
    11. Centrifuge for 1 minute to elute the plasmid DNA.
    12. Nanodrop and record the conentration.

PCR - NEB Q5® High-Fidelity 2X Master Mix
:   Reaction Setup:

    | Component | 25 μL Reaction | 50 μL Reaction |
    | --- | --- | --- |
    | Q5 High-Fidelity 2X Master Mix | 12.5 µL | 25 µL |
    | 10 µM Forward Primer | 1.25 µL | 2.5 µL |
    | 10 µM Reverse Primer | 1.25 µL | 2.5 µL |
    | Template DNA (~25/50 ng) | variable | variable |
    | Nuclease-Free Water | to 25 µL | to 50 µL |

    Thermocycling Conditions for a Routine PCR:

    | Step | Temperature | Time |
    | --- | --- | --- |
    | Initial Denaturation | 98°C | 30 seconds |
    | 30 Cycles | 98°C | 10 seconds |
    | Ta for specific primers | 20 seconds |
    | 72°C | Elongation time 1 min/kb |
    | Final Extension | 72°C | 2 minutes |
    | Hold | 4°C | Infinity |

Column Purification - Zymo DNA Clean & Concentrator (DCC) Kit
:   1. For PCR product, add 5 volumes of DNA Binding Buffer to each volume of DNA sample. Mix briefly by vortexing.
    2. Transfer mixture to a provided Zymo-Spin™ Column in a Collection Tube.
    3. Centrifuge for 30 seconds. Discard the flow-through.
    4. Add 200 µL DNA Wash Buffer to the column. Centrifuge for 30 seconds. Repeat
    the wash step.
    5. Add ≥ 6 µL DNA Elution Buffer or nuclease-free water directly to the column matrix and incubate at room temperature for one minute. Transfer the column to an eppendorf and centrifuge for 30 seconds to elute the DNA.

Annealing Oligos
:   | Component | Amount |
    | --- | --- |
    | Phosphorylated oligos | 500 ng each (1 μg total) |
    | NEB 10X T4 Ligase Buffer | 15 μL |
    | Nuclease-free water | To 50 μL |

    Incubate the mixed oligonucleotides to 85°C for 10 minutes and cool down slowly (do 30 min ramp from 85 down to 20).

Restriction Enzyme Digestion
:   1. Reaction Setup:

    | Component | 50 μL Reaction |
    | --- | --- |
    | Purified DNA | 250-1000 ng |
    | NEB CutSmart Buffer | 5 μL |
    | Restriction Enzyme | 1 μL of each |
    | Nuclease-free water | to 50 μL |

    2. Incubate at 37°C for 30 minutes if doing diagnostic digestion or 2 hours if getting the DNA.

Ligation
:   1. Reaction Setup:

    | Component | 15 μL Reaction |
    | --- | --- |
    | NEB T4 DNA Ligase Buffer (10X) | 1.5 μL |
    | Vector DNA | 50 ng |
    | Insert DNA | X ng |
    | Nuclease-free water | to 15 μL |
    | NEB T4 DNA Ligase\* | 1 μL |

    \*T4 DNA Ligase should be added last.
    2. Incubate at 16°C overnight or room temperature for 30 minutes.
    3. Chill on ice and transform 5 μL of the reaction into 50 μL competent cells.

Gibson Assembly - NEBuilder HiFi DNA Assembly
:   1. Reaction Setup on Ice:

    |  | 2-3 Fragment Assembly | 4-6 Fragment Assembly | Positive Control |
    | --- | --- | --- | --- |
    | Recommended DNA Molar Ratio | vector:insert = 1:2 | vector:insert = 1:1 | - |
    | Total Amount of Fragments | 0.015 - 0.1 pmols\* | 0.1 - 0.25 pmols | 5 μL |
    | NEBuilder HiFi DNA Assembly Master Mix | 5 μL | 5 μL | 5 μL |
    | ddH2O | Volume to 10 μL | Volume to 10 μL | 0 |
    | Total Volume | 10 μL\*\* | 10 μL\*\* | 10 μL |

    \*pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)
    \*\*Can be cut down to half reaction.
    2. Incubate samples in a thermocycler at 50℃: 15 minutes - when 2 or 3 fragments are being assembled; 60 minutes - when 4–6 fragments are being assembled.
    3. Transformation:
    Follow the Transformation of E.coli DH5a Cells protocol with a few difference:
    Add 2 μL of product to 50 μL of competent cells.
    After heat shock, incubate on ice for 2 minutes instead of 5 minutes.

TOPO cloning - Zero Blunt™ TOPO™ PCR Cloning Kit
:   1. Reaction Setup\*:

    | Component | 6 μL Reaction |
    | --- | --- |
    | Fresh Blunt-end PCR Product | 0.5-4 μL |
    | Salt Solution | 1 μL |
    | Sterile Water | to 6 μL |
    | pCR™-Blunt II-TOPO® | 1 μL |

    \*For transformation of chemically competent E. coli only.
    2. Mix gently and incubate for 5 minutes at room temperature.
    3. Place on ice. Proceed to transformation.

Annealing RNA oligos - GenScript CRISPR Ribonucleoprotein User Manual
:   1. Centrifuge RNA oligos at 12000 rpm for 2 minutes at 4℃.
    2. Resuspend oligos in nuclease free water to a final volume of 100 μM.
    3. Vortex for 15 seconds and Centrifuge for 1 minute at 12000 rpm.
    4. Reaction Setup: To anneal components of 25 μM final duplex concentration

    | Component | 40 μL Reaction |
    | --- | --- |
    | Nuclease-free water | 12 μL |
    | Annealing Buffer (5X)\* | 8 μL |
    | crRNA Oligo (100 μM) | 10 μL |
    | tracrRNA Oligo (100 μM) | 10 μL |

    5. Heat at 95℃ for 5 minutes.
    6. Remove from heat and put in 60℃ water, and let it to cool to room temperature.
    7. Store at -20℃.
    \*Annealing Buffer (5X): 300mM KCl, 30mM HEPES, 1mM MgCl2, pH7.5

NEB In vitro digestion of DNA with Cas9 Nuclease
:   1. Reaction Setup at room temperature:

    | Component | 30 μL Reaction |
    | --- | --- |
    | Nuclease-free water | 20 μL |
    | 10X Cas9 Nuclease Reaction Buffer | 3 μL |
    | 300nM sgRNA | 3 μL |
    | 1 µM Cas9 Nuclease, S. pyogenes (M0386S) | 1 μL |
    | Pre-incubate for 10 minutes at 25⁰C | |
    | 30 nM substrate DNA | 3 μL |

    2. Mix thoroughly and pulse-spin in a microfuge.
    3. Incubate at 37°C for 15 minutes.
    4. Add 1 ul of Proteinase K to each sample, Mix thoroughly and pulse-spin in a microfuge.
    5. Incubate at room temperature for 10 minutes.
    6. Proceed with fragment analysis.

Test CAMERA Base Editing System
:   Goal: See that base editor (CAMERA W2.1) works with CAMERA R1. Confirm that mutation rate is linear and test the base editing efficiency.
    96 deep well plate:
    ![](https://static.igem.org/mediawiki/2018/1/1b/T--Pittsburgh--CAMERA.jpg)
    Steps:
    1. To start the layout, fill every well expect C5-C8 with 800 μL of sterile water. Fill C5-C8 with 800 μL of fresh LB and appropriate amount of antibiotics (1000X Carb and Spec). Add inducers (200 ng/mL aTc and 0.1 mM IPTG) into C5 and C7. Add overnight cultures into C5-C8 and make sure they are 1:500 dilutions. Secure with 96 well plate film. Allow the plate to shake at 37℃ for 24 hours.
    2. After 24 hours. take out the water in D5-D8 and refill these wells with 800 μL of fresh LB. Dilute the cultures in previous row (C5-C8) 500-fold into fresh LB media containing the corresponding inducers and antibiotics. Miniprep the rest of cultures in the previous row. Secure the plate with film and shake at 37℃ for another 24 hours.
    3. Repeat Step 2 in the following two days.
    4. Sanger sequencing results will then be analyzed by our software tool CrisPy.

Base Editing Improvement
:   Goals: Test if shortening the time between the dilutions would improve the editing rate.
    96 deep well plate:
    ![](https://static.igem.org/mediawiki/2018/4/46/T--Pittsburgh--CAMERA_dilution.jpg)
    Steps: Follow CAMERA protocol. Dilute the cultures every 12 hours. Send for sequencing every 24 hours.

CrisPy
:   Goals: Test the ability of our software tool, CrisPy, for characterizing CRISPR on-target and off-target edits through Sanger sequencing.
    96 deep well plate:
    ![](https://static.igem.org/mediawiki/2018/0/01/T--Pittsburgh--CrisPy.jpg)
    Steps: Follow CAMERA protocol, however change the antibiotics to CAM and Spec.

### References

[1] Aitken, A. (2012, November 10). TE buffer (Tris-EDTA buffer). Retrieved from http://www.nhm.ac.uk/content/dam/nhmwww/our-science/dpts-facilities-staff/Coreresearchlabs/te-buffer.pdf

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# Team:Pittsburgh/Results

Results

* [Overview](#section1)
* [CAMERA](#section2)
* [Improve](#section3)
* [Base Editor](#section4)
* [Individual gRNA](#section5)
* [Oscillatory gRNA](#section6)
* [CrisPy](#section7)

# Overview

* Characterize a previous base editor system (CAMERA)
* Improve editing rate of the base editor
* Assemble our own base editor
* Assemble individual gRNA design system
* Model our system to visualize readout

+ Utilize that model to redesign our project in order to get clearer readouts

* Assemble a new oscillatory gRNA design system
* Build and characterize a software tool to analyze Sanger sequencing readouts and analyze off-target sequences
* Build and characterize nCas9 part for the registry

# CAMERA

Firstly, we tested a previously characterized CRISPR/Cas9 base editing system obtained from the Addgene repository of Dr. Liu’s lab at Harvard University. The system consisted of a high copy number recording plasmid (R1) and a low copy number writing plasmid with a base editor and one gRNA (W2.1). To assemble this system we transformed the two plasmids into Escherichia coli S1030 obtained from Addgene. This strain of E. coli is a version of the K12 strain with regulatory elements for the 2 inducible promoters PtetO and PLacO encoded into the genome.

The results of the single nucleotide mutations were analyzed through sequencing. The original paper utilized High Throughput Sequencing (HTS) to analyze single nucleotide mutations produced by the base editor. However, due to budget restrictions, we utilized Sanger Sequencing combined with analysis from our software tool CrisPy. For analysis of this software please see our software tool page.

Figure 3 below displays the mutation frequency (the percent of total plasmids edited) over time. This matches the expected results, in that we see a linear relationship of editing over time. However, we did not reach the same amount of overall editing that we demonstrated in the paper. Our data shows an overall editing rate of about 12% editing efficiency after 96 hours, which is similar to the paper who got about 55% editing after four days.

![](https://static.igem.org/mediawiki/2018/8/8a/T--Pittsburgh--CAMERA_v1_induced.png)
![](https://static.igem.org/mediawiki/2018/a/ae/T--Pittsburgh--CAMERA_v1_control.png)

In order to optimize our protocol for testing the CAMERA base editing system, we made two modifications to the previous protocol. We used the corresponding antibiotics for each plasmid to mainly ensure that the low copy number plasmid would not be kicked out by the bacterium. Additionally, we used fresh medium and inducers each time we diluted instead of filling all the wells with medium at the beginning and then diluting into them.

From this new data, Figure 5 and 6 below, we can see that our optimizations from the previous protocol increased our editings efficiency. Once again this data matches the expected results, in that we see a linear relationship of editing over time. The data shows an overall editing rate of about 40% editing efficiency after 96 hours. Figure 6 demonstrates the control data, which is uninduced editing, there is about 20% baseline editing because of the leaky transcription of PtetO and PLacO which is backed by promoter characterization in the literature.

![](https://static.igem.org/mediawiki/2018/6/6e/T--Pittsburgh--CAMERA_v2_ind.png)
![](https://static.igem.org/mediawiki/2018/1/18/T--Pittsburgh--CAMERA_v2_control.png)

We performed 2-way ANOVA tests to demonstrate that this data represents a significant editing difference between each time point.

The purpose of these experiments were 2-fold. First, we needed to demonstrate the functionality of the base editor. Secondly, a fundamental part of our project utilizes the linear editing property of the base editor in order to establish a time scale. The ability of our project to establish a time point readout of a stimulus is what separates our project from previous work that utilizes a base editor as a recording mechanism for cellular stimuli.

# Improve Base Editing Efficiency

Based on feedback we got from Dr. Seth Childers during our Human Practices we set out to test a theory that increasing the replication rate of our plasmids would increase our base editing rate. From the literature, we discovered that the limiting factor to editing was not the matter of the Cytidine Deaminase modifying the base but rather the release of the complex from the DNA so that it could edit another plasmid. This base editor utilized dCas9 to localize the complex to the correct location for a base change. dCas9 is the catalytically inactivated version of Cas9; meaning that once it binds to the DNA it does not have any function. dCas9 is commonly used by scientists for the specific purpose that once bound it will stay bound. This is called CRISPR interfering or CRISPRi and is used to disable genes or block binding domains such as those for an RNA polymerase. Moreover, due to the fact that it is staying bound, we are not getting proficient use of the enzyme to edit multiple plasmids. Finally, in our research, we discovered that during replication of a plasmid all bound proteins are released as the polymerase replicates the plasmid.

To take advantage of this discovery, we wanted to test if shortening the time between the dilutions would improve the editing rate. By diluting every 12 hours instead of every 24 hours we not allowing the cells to reach stationary phase and therefore are constantly replicating in the exponential phase. We used our CAMERA protocol and adapted it to dilute into fresh medium with antibiotics and inducers every 12 instead of 24 hours.

![](https://static.igem.org/mediawiki/2018/2/2d/T--Pittsburgh--dilution_actual_control.png)
![](https://static.igem.org/mediawiki/2018/6/66/T--Pittsburgh--Dilution_control.png)

The difference in mutation frequencies from the two experiments (24 hr dilutions and 12 hr dilutions) was found at each time point. The standard error of the mean was also calculated. Because each difference lies more than two deviations from zero, we are at least 95% confident that the more frequent dilutions increase the mutation rate.

![](https://static.igem.org/mediawiki/2018/4/42/T--Pittsburgh--CAMERA_dil_diff.png)

Based on these results we are confident that our hypothesis, keeping the cells in exponential phase would increase the rate of base editing, is true. With this information, all of our future experiments with base editing will involve diluting every 12 hours instead of 24 hours. Alternatively, we can utilize a bioreactor, which will constantly supply the bacteria with new media and therefore keep the cells in exponential phase and improve base editing efficiency.

# Base Editor

Due to the fact that the base editor we used did not conform to iGEM’s BioBrick (RFC 10) compatibility, we synthesized DNA from IDT for the CDA and ugi proteins, which were codon optimized to remove BioBrick sites, and utilized a dCas9 from the registry BBa\_K1323002 in an effort to assemble our own base editor.

Our first attempt at assembling this plasmid involved using Gibson assembly with 30 bp overhangs between each part and the pSB4A5 low copy number backbone. We attempted varying concentrations, amounts, and ratios of pieces, but none of our experiments produced colonies.

After multiple attempts at assembly using Gibson, we transitioned into using overlap extension PCR. Then we attempted golden gate assembly. Using PCR we added BsaI or BbsI type II restriction enzymes sites to the ends of each of our parts each with a unique 4 bp overhang. These parts would then be ligated together and transformed. Once again we attempted using various concentrations and ratios of pieces; however, we never produced colonies with the correct assembly. We additionally attempted assembling the base editor stepwise, 1 piece at a time, but to no avail.

![](https://static.igem.org/mediawiki/2018/9/9c/T--Pittsburgh--GG_BE.png)

We learned from our advisors that sometimes plasmids will not successfully transform or be grown up when in a low copy backbone. So we transitioned into assembling it into the pSB1C3 backbone before we transitioned it into its pSB4A5 backbone for final use. Once again we continued to have no success. Finally, we assembled each of the individual parts into a TOPO vector along with the restriction enzymes sites necessary for golden gate assembly to ensure that our sequences from IDT were correct and that our enzymes were successfully cutting the parts before ligation. Assembling in TOPO confirmed that our sequences were correct and our restriction enzymes were cutting correctly. However, our ligations sill never produced any colonies.

After going through numerous attempts of assembly and seeking the advice from various experts about our issues we were unsuccessful in assembling our Base Editor. However, we still had the opportunity the use the base editor plasmid obtained from Addgene for our experiments, but it would not be able to be submitted to iGEM due to violations of the BioBrick RFC10 compatibility.

# Individual gRNA System

All recording plasmids for our MC gRNA system were ordered from IDT as blocks and assembled into pSB1C3 using Gibson assembly. Due to the fact that DNA synthesis is problematic in the production of repeating sequences of DNA, we resorted to designing our gBlocks with only 2 repeats and inserting more repeat sequences using type II restriction enzyme assembly (BsaI) and DNA oligos. The following plasmids were all fully assembled and sequence confirmed.

* BBa\_K2785000 - Multiple Cell Recording (0 Repeat Control)
* BBa\_K2785001 - Multiple Cell Recording (0 Repeat)
* BBa\_K2785002 - Multiple Cell Recording (1 Repeat)
* BBa\_K2785003 - Multiple Cell Recording (2 Repeat)
* BBa\_K2785004 - Multiple Cell Recording (3 Repeat)
* BBa\_K2785005 - Multiple Cell Recording (4 Repeat)
* BBa\_K2785006 - Multiple Cell Recording (5 Repeat)

Our two gRNAs for this system were ordered from IDT as separate gBlocks to be annealed using Gibson assembly. Due to the fact that repeating sequences are difficult to synthesize, IDT was not able to create a sample of gRNA 1 which met their quality control standards. They sent us their best effort attempt of producing it which contained an estimated 80% sample of correct sequence. In order to isolate the correct sequence, we cloned the gRNA into pSB1C3. All samples that we sent for sequencing did not contain the correct sequence of gRNA 1. We repeated this process multiple times, but could not find a correct sequence for gRNA 1. Furthermore, in an attempt to isolate a correct sequence of gRNA 1 we used TOPO cloning to insert our gRNA 1 into the TOPO vector in order to isolate a sequence-confirmed sample of gRNA 1 but had no success finding a correct sequence.

Due to the fact that our base editor could not be assembled, we attempted to incorporate our gRNA into the CAMERA W2.1 base editor. Using PCR we added restriction enzyme sites to W2.1 and our gRNA and attempted assembly using standard restriction enzyme cloning. Unfortunately, our ligations never produced colonies that had an insertion of our gRNAs.

Due to our struggles in cloning the gRNA and the fact that our model demonstrated that this system would not produce the outcomes desired for this system to work we decided to redirect our focus to the oscillatory gRNA system.

# Oscillatory gRNA System

Our single cell oscillatory system recording and writing plasmids were ordered from Twist Bioscience as complete plasmids. However once again we had to insert more repeat sequences into the recording plasmid using type II restriction enzyme assembly (BsaI) and DNA oligos as we did with our individual gRNA system. Additionally, we had to remove a spacer sequence from our writing plasmid using type II restriction enzyme assembly (BsaI). This was successfully assembled and sequence-confirmed. Additionally, we successfully modified the CAMERA W2.1 base to remove the gRNA in order to work with this system because our own base editor could not be assembled.

Once all plasmids were assembled and sequence confirmed, we attempted to co-transform all three plasmids into our chassis organism E. coli S1030. However, after multiple attempts, we were unsuccessful in isolated colonies that had successfully up took all three plasmids.

We are continuing to work on co-transforming these plasmids so that we can start this experiment.

# CrisPy

Our plasmid containing three off-target gRNA recognition sequences along with 1 target sequence was fully assembled using standard restriction enzyme protocol into the pSB1C3 backbone and sequence confirmed. This plasmid was co-transformed with CAMERA W2.1 base editor plasmid in S1030. We are currently awaiting results and analysis of our on and off-target editing efficiencies, which will be analyzed with our CrisPy software tool. This data will be used to characterize the tools accuracy in identifying and interpreting gRNA binding sequences.

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