iGEM 2017 — Team Toronto

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# Team:Toronto/Notebook

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* • [Human Practices](http://2017.igem.org/Team%3AToronto/Human_Practices)

# Notebook

## Preparing stocks, plates, cell cultures, etc.

### Week 1, day 1 (26/06)

* Prepared wash bottles of 70% Ethanol
* Made 4x500mL LB media
  + 2 with agar for plating
  + 2 without agar for preparing overnight cultures
* Made chloramphenicol (CAM) stocks
* Plated out LB + agar onto sterile plates with CAM

### Week 1, day 2 (27/06)

* Made 10mL 0.2M acetic acid/1M NaOH
* Prepared 50mL of RF1/RF2 buffer (to make RbCl competent cells)
* Streaked 4 plates (stored in 4°C):
  + MG1655ΔLacI onto 2 LB and 1 LB+CAM plates
  + DH10β onto 1 LB plate

### Week 1, day 3 (28/06)

Streaked 2 LB plates: MG1655ΔLacI & DH10β to replace Tuesday’s plates (because they over-grew)

* New plates put into 37°C incubator overnight, backup plates placed into 4°C fridge

Prepared 5mL LB cultures and placed in overnight shaker of:

* MG1655ΔLacI
* DH10β

### Week 1, day 4 (29/06)

* Prepared SOC medium
* Cultured DH10β cells in baffled flasks until OD reached 0.4-0.55

### Week 1, day 5 (30/06)

* Plated DH10β cells and incubated at 37°C overnight

### Week 1, day 6 (31/06)

* Checked on plated cells from 30/06:

### Week 3, day 1 (10/07)

* Made and autoclaved LB and LB+CAM liquid.
* Poured 13 LB plates.
  + LB+CAM plates were unsuccessful since incorrect amount of CAM was added (12.5g instead of 12.5mg).

### Week 3, day 2 (11/07)

* Made LB+CAM plates
* Plated and spread 50µL and 100µL DH5α cells onto LB agar plates using sterile loops.
  + Placed in overnight incubator

### Week 3, day 3 (12/07)

-Checked on DH5α plates from the incubator.

### Week 3, day 4 (13/07)

* KAN and CAM antibiotic stocks
* 500mL each of LB+CAM and LB+KAN
* 500mL 1X TAE buffer from 50X TAE buffer and MilliQ water
* Prepared overnight culture of one DH5α colony in 5mL LB at 37°C.

### Week 3, day 5 (14/07)

* Checked on DH5α culture
  + Initial OD600 was 0.011, so waited 110min for optimal OD600
  + Moved culture to ice once OD600 reached 0.466
  + Made DH5α stocks stored in -80°C, competence to be tested on Monday using iGEM competence cell test kit.

### Week 4, day 1 (17/07)

* Plated transformed cells.
  + Spread the DH5α cells onto 4 LB+CAM plates, each containing 100µL of the 100pg/µL, 50pg/µL, 10pg/µL, and 0pg/µL cells.
  + Spread the DH10β cells onto two LB+KAN plates, both containing 100µL of the control and either 100µL or 50µL of the pKDL071 cells
* Incubated the plates at 37°C overnight.

### Week 4, day 2 (18/07)

* Made LB media
* Made LB+CAM plates
* Checked on 17/07 transformed cells and moved them to the 4°C fridge.
  + Confirmed DH5α cells were competent
  + Note: LB+CAM plates made on 13/07 may have been made with too much CAM (1250µL CAM to 500mL LB). This may explain why there were so few colonies from 17/07 transformation of DH5α + RFP/pSB1C3.

* Obtained single colonies from transformed DH5α and DH10β plates from 17/07 and made overnight 37°C colonies.
  + The DH5α was placed in the 5mL of LB+CAM (5µL of stock CAM added to 5mL LB).
  + The DH10β was placed in 5mL of LB+KAN (25µL of stock KAN added to 5mL of LB).

### Week 4, day 3 (19/07)

* Examined plates from 18/07
  + Most plates had no cells likely due to the high CAM concentration (2.5x working concentration).

* Made glycerol stocks of DH10β with pKDL071

### Week 4, day 5 (21/07)

* Made LB+AMP plates

### Week 5, day 1 (24/07)

* Made LB+CAM plates

### Week 5, day 3 (26/07)

* Made LB+CAM and LB+KAN solutions for overnight cultures

### Week 5, day 4 (26/07)

* Made 17 LB+KAN plates

### Week 5, day 5 (28/07)

* Prepared 10X PBS buffer

### Week 6, day 1 (31/07)

* Made 50mL non-sterilized 1X PBS Buffer

### Week 6, day 3 (02/08)

* Made LB+CAM plates

### Week 6, day 5 (04/08)

* Made LB liquid media

### Week 7, day 4 (10/08)

* Made 17 LB+Kan plates

### Week 9, day 2 (22/08)

* Made LB+KAN plates

### Week 9, day 3 (23/08)

* Made LB+AMP plates

### Week 9, day 4 (24/08)

* Made 5mL LB+Kan in falcon tube

### Week 10, day 4 (01/09)

* Made LB+CAM plates

### Week 16, day 5 (06/10)

* Made LB+AMP plates

### Week 18, day 2 (17/10)

* Made LB+KAN and LB+CAM plates

### Week 19, day 7 (29/10)

* Made 20% stock D-glucose solution by adding 20g of glucose to 100ml MilliQ water then autoclaved
* Made Modified M9 Media (+0.2% glucose + 0.2% arabinose)

## Interlab

### Week 3, day 4 (13/07)

* Prepared overnight culture of one DH5α colony in 5mL LB at 37°C

### Week 3, day 5 (14/07)

* Checked on DH5α culture
  + Initial OD600 was 0.011, so waited 110min for optimal OD600
  + Moved culture to ice once OD600 reached 0.466
  + Made DH5α stocks stored in -80°C, competence to be tested on Monday using iGEM competence cell test kit.

### Week 4, day 1 (17/07)

* Transformed DH5α cells using iGEM competence cell test kit with RFP/pSB1C3 at concentrations 100pg/µL, 50pg/µL, 10pg/µL, and 0pg/µL (control)
* Plated the cells onto 4 LB+CAM plates, each containing 100µL of the 100pg/µL, 50pg/µL, 10pg/µL, and 0pg/µL cells.
* Incubated the plates at 37°C overnight.

### Week 4, day 2 (18/07)

* Checked on transformed cells from previous day and moved them to the 4°C fridge.
  + Confirmed DH5α cells were competent
  + Note: LB+CAM plates made on 13/07 may have been made with too much CAM (1250µL CAM to 500mL LB). This may explain why there were so few colonies from 17/07 transformation of DH5α + RFP/pSB1C3.

* Resuspended the iGEM kit parts containing the DNA required for the Interlab study (DNA was obtained from kit plate #6, which was then moved to the 4°C fridge for storage) and transformed the chemically competent DH5α cells (from 14/07) with resuspended DNA
  + Made 9 tubes: 8 GFP samples and one no plasmid control (labelled 20B, 20D, 20F, 20H, 20J, 20L, 20N, and 20P, respectively).
  + Originally, the goal was to make 17 tubes for two transformations (8 samples x 2 + 1 no plasmid control), however, there were not enough cells to to make a second set (missing second set of 20L and 20B).
  + Spread 100µL of transformed bacteria on LB+CAM plates to incubate and dry overnight at 37°C.

### Week 4, day 3 (19/07)

* Examined plates from yesterday
  + Most plates had no cells likely due to the high CAM concentration (2.5x working concentration).

07-19 plates

* Re-plated DH5α cells transformed with Interlab study plasmids from transformation tubes done on Tuesday and incubated overnight in 37°C shaker.

### Week 4, day 4 (20/07)

* Obtained 19/07 GFP plates from the 37°C shaker.
* Picked a single colony from each plate and incubated in 1.5mL LB media overnight in 37°C shaker and left to grow in the 37 degree C shaker.
* In total, there were 8 sample tubes containing the GFP interlab samples and one control tube of just LB.
* Made 5mL cultures of the DH5α cells transformed with GFP plasmids and placed them in the overnight shaker to grow.

### Week 4, day 5 (21/07)

* Plated transfromed DH5a

### Week 4, day 6 (22/07)

* Checked plates of DH5a cells transformed with Interlab plasmids (from 21/07).
* Colonies grew into a lawn of bacteria and no single colonies could be isolated.

### Week 5, day 2 (25/07)

* Obtained single colonies from the GFP sample plates (21/07) and plated them on LB+CAM plates. The plates were left at 37°C to grow overnight.

### Week 5, day 3 (26/07)

* Examined LB+CAM plates and saw single colonies

* Inoculated single colonies from plates into LB+KAN and LB+CAM and put in incubator

### Week 5, day 4 (26/07)

* Made 80x1mL glycerol stocks of 26/07 cultures and stored in -80°C freezer

### Week 5, day 5 (28/07)

* Ran LUDOX-S40 calibration

Table 1: LUDOX Calibration

| 100% LUDOX | H<sub>2</sub>O |
| --- | --- |
| 0.0508 | 0.0402 |
| 0.0561 | 0.0415 |
| 0.0525 | 0.0419 |
| 0.0527 | 0.0435 |

### Week 6, day 1 (31/07)

* Transferred samples 20J, 20L, 20N, and 20P from glycerol aliquots (from 26/07) to a 96-well plate and measured maximum emission and exciation wavelengths of GFP and YFP.
  + GFP: Literature said the maximum exictation wavelength is 395 nm, the maximum emission wavelength is 509 nm
    - We measured maximum exication: 390 nm, maximum emission: 400-750 nm.
  + YFP: Literature said the maximum excitation wavelength is 512 nm, the maximum emission wavelength is 529 nm.
  + *Note: No negative control was measured so data can’t be normalized.*

### Week 6, day 2 (01/08)

* Measured the emission spectra of GFP samples 20B, 20D, and 20F with the plate reader
* The interlab GFP samples use the mut3b GFP, which has a shifted excitation and emission spectra
  + Excitation range of 488 nm - 501 nm
  + Emission maximum at 511 nm
  + At a 395 nm excitation wavelength, this GFP mutant emits 0.2x the intensity of wild type GFP. However, at a excitation wavelength of 488 nm, the emission intensity is 21x that of wild type GFP.
* The emission spectra was tested using samples 20B (positive control), 20D (negative control), and 20F from glycerol aliquots.
  + In total, three different excitation spectra were tested: 488 nm, 508 nm, and 495 nm.

### Week 6, day 3 (02/08)

* Washed glycerol stocks and then suspended in LB+CAM to ensure consistent measurements from the plate reader
  + All subsequent interlab samples will be measured in LB+CAM
* Restreaked samples to obtain biological replicates
* Measured excitation spectra using plate reader in WB 316 of GFP samples 20B, 20D, and 20F.

### Week 6, day 5 (04/08)

* Restreaked 20D transfomed cell

* Took plate reader measurements according to Interlab protocol
  + Used LUDOX 100% and H2O to set the standard point
  + Found fluorescein fluorescence standard curve
  + Took measurements of all cell samples

## LacILOV

### Week 1, day 5 (30/06)

* Bacterial transformation of competent cells with RFP plasmid

### Week 2, day 5 (07/07)

* Electroporation of MG1655ΔLacI electrocompetent cells.
* Modified electroporation protocol to incubate transformed cells at 300rpm, 37°C after SOC recovery step.
* Bacterial transformation of electroporated cells with RFP plasmid (BBa\_j04450 in pSB1C3) to check transformation efficiency at 0, 10, 50 and 100pg/ul.
* MiniPrepped transformed DH10β (BBa\_j04450 in pSB1C3) to purify plasmid (sample 1/2 stored in -20°C)
* Performed RE digestion, nicked with EcoRI-HF (sample 1/2 stored in -20°C)

### Week 3, day 1 (10/07)

* Miniprepped RFP plasmids on the pSB1C3 backbone from chemically competent DH10β cells from 07/07.
* Conducted gel electrophoresis of RE digested and non RE digested dupicate samples from 07/07.
  + 10µL of each ladder was used, but in the future we should experiment with adding less ladder (conserve reagents!).
  + Each 10µg sample was mixed with 2µL loading dye and loaded on the gel along with two ladders. The sequences are as follows:
    1. EcoRI-HF Digested Sample 1 (D1)
    2. Undigested Sample 1 (UD1)
    3. 1kb DNA ladder for measurement
    4. EcoRI-HF Digested Sample 2 (D2)
    5. Undigested Sample 2 (UD2)
    6. 1kb DNA ladder for measurement
* Demonstration: Nanodropped each sample to show the protocol to general members.

### Week 3, day 2 (11/07)

Calculated optimization for Q5 (High Fidelity) PCR of UNS3/2 into ends of pSB1C3 backbone

* Optimized annealing step because our primer Tms are high (85°C), so using different additives/temperature gradients to optimize yield

“07-11 Tm Calculator.png”

Table 1: PCR Optimization

| Component | No treatment (µL) | 3% DMSO (µL) | 6% DMSO (µL) | 1X GC Buffer (µL) | No DNA control (µL) |
| --- | --- | --- | --- | --- | --- |
| 5X Q5 Reaction Buffer | 5 | 5 | 5 | 5 | 5 |
| 10mM dNTPs | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| 10µM Forward primers | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 |
| 10µM Reverse primers | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 |
| 1ng/µL Template DNA (prediluted) | 1 | 1 | 1 | 1 | 0 |
| Q5 HF DNA Polymerase | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
| 1X GC Buffer | 0 | 0 | 0 | 5 | 5 |
| DMSO | 0 | 0.75 | 1.5 | 0 | 0 |
| Nuclease-free water | 15.75 | 15 | 14.25 | 10.75 | 11.75 |
| Total volume | 25 | 25 | 25 | 25 | 25 |

\* Multiply five for 5 reactions (5 different annealing temperatures: 72, 71, 70, 69, and 68°C).

Table 2: Master Mixes

| Master Mix | No treatment (µL) | 3% DMSO (µL) | 6% DMSO (µL) | 1X GC Buffer (µL) | No DNA control (µL) |
| --- | --- | --- | --- | --- | --- |
| GC Buffer | 0 | 0 | 0 | 40 | 40 |
| DMSO | 0 | 6 | 12 | 0 | 0 |
| Nuclease-free water | 126 | 120 | 114 | 86 | 94 |
| Sum | 126 | 126 | 126 | 126 | 134 |

### Week 3, day 3 (12/07)

Performed Q5 HF PCR (discussed 11/07) with modifications:

* Revised PCR calculations based on Christian’s suggestions: 7 Q5 HF PCR 50µL reactions with 3% DMSO in each sample (one control tube with no DNA template).

Table 1: Revised PCR Calculations

| Component | Reaction (×7) | Control |
| --- | --- | --- |
| 5X Q5 Reaction Buffer | 10 | 10 |
| 10mM dNTPs | 1 | 1 |
| 10µM Forward primers | 2.5 | 2.5 |
| 10µM Reverse primers | 2.5 | 2.5 |
| 1ng/µL Template DNA (prediluted) | 1 | 0 |
| Q5 HF DNA Polymerase | 0.5 | 0.5 |
| 1X GC Buffer | 10 | 10 |
| 100% DMSO | 1.5 | 1.5 |
| Nuclease-free water | 31 | 32 |

* Prepared master mix for Q5 HF PCR reactions (with 50µL 3% DMSO). Template DNA is included in 6 tubes with 1 control without template DNA.
  + One template DNA tube only contains 25 uL, possibly due to pippetting error
    - Note: prepare a master mix of 8 reactions of 50 μL, instead of 7.1 reactions, to accommodate pippeting errors).
* Ran PCR. Tubes were meant to be optimized through a temperature gradient, but were placed incorrectly in the PCR machine, so they were run all at the same temperature.
* Ran a gel electrophoresis of psB1C3 backbone PCR. The gel was not successful. (Life: What a cruel mistress)
  + Note: Gel was made with 15 wells - too small for 10µl samples (15 wells should use ~6-8µl).

### Week 3, day 4 (13/07)

Performed Q5 HF PCR to amplify pSB1C3 backbone

* Prepared same master master mixes as calculated 12/07 for 6 samples with template DNA and one control without template DNA
* Ran the PCR of samples 1-6 at different temperatures, one each at 72.0, 71.4, 70.1, 68.3, 66, and 64.3°C respectively
  + Control was tested at 70.1°C
* Performed gel electrophoresis of Q5 PCR samples, along with EcoRI digested pSB1C3 with RFP control

* Advisor suggested results may be due to primer dimerization, possibly because of high primer Tm
* Planned to redesign primers to reduce this problem by removing base pairs from the 3’ binding up to ~15bp homology between primers and backbone
  + Aiming for Tm of ~68°C

### Week 3, day 5 (14/07)

Redesigned primers based on suggestions from 13/07 to have lower Tm

* New reverse primer for pKDL071 backbone
* New forward and reverse primers for pSB1C3 backbone

The Q5 polymerase used in previous PCRs is very bubbly which could have been a source of error, so instead we performed 2-step NEB Phusion HF PCR of pSB1C3 with RFP construct

* Used primers from 2016 and 2017 to compare results
* Tubes were labelled 16.1-16.5 (for 2016 primers) and 17.1-17.5 (for 2017 primers)
* Controls without DNA template were labelled NTC (no template control)
* Ran the samples on a temperature gradient to determine the optimal PCR settings

Table 1: Phusion PCR Reaction Component Table (for total volume of 50µL per tube)

| Component | 16.NTC | 16.1 | 16.2 | 16.3 | 16.4 | 16.5 | 17.NTC | 17.1 | 17.2 | 17.3 | 17.4 | 17.5 |
| — | — | — | — | — | — | — | — | — | — | — | — |
| 5X Phusion HF or GC Buffer | 10µL | 10µL | 10µL | 10µL | 10µL | 10µL | 10µL | 10µL | 10µL | 10µL | 10µL | 10µL |
| 10mM dNTPs | 1µL | 1µL | 1µL | 1µL | 1µL | 1µL | 1µL | 1µL | 1µL | 1µL | 1µL | 1µL |
| 10µM 2016 Forward Primer | 2.5µL | 2.5µL | 2.5µL | 2.5µL | 2.5µL | 2.5µL | 0µL | 0µL | 0µL | 0µL | 0µL | 0µL |
| 10µM 2016 Reverse Primer | 2.5µL | 2.5µL | 2.5µL | 2.5µL | 2.5µL | 2.5µL | 0µL | 0µL | 0µL | 0µL | 0µL | 0µL |
| 10µM 2017 Forward Primer | 0µL | 0µL | 0µL | 0µL | 0µL | 0µL | 2.5µL | 2.5µL | 2.5µL | 2.5µL | 2.5µL | 2.5µL |
| 10µM 2017 Reverse Primer | 0µL | 0µL | 0µL | 0µL | 0µL | 0µL | 2.5µL | 2.5µL | 2.5µL | 2.5µL | 2.5µL | 2.5µL |
| Template DNA | 0µL | 1µL | 1µL | 1µL | 1µL | 1µL | 0µL | 1µL | 1µL | 1µL | 1µL | 1µL |
| DMSO | 1.5µL | 1.5µL | 1.5µL | 1.5µL | 1.5µL | 1.5µL | 1.5µL | 1.5µL | 1.5µL | 1.5µL | 1.5µL | 1.5µL |
| Phusion DNA Polymerase | 0.5µL | 0.5µL | 0.5µL | 0.5µL | 0.5µL | 0.5µL | 0.5µL | 0.5µL | 0.5µL | 0.5µL | 0.5µL | 0.5µL |
| Nuclease-free water | 32µL | 31µL | 31µL | 31µL | 31µL | 31µL | 32µL | 31µL | 31µL | 31µL | 31µL | 31µL |

Table 2: Phusion PCR Temperature Gradient

| Temperature | Tubes |
| --- | --- |
| 62°C | 16.1, 17.1 |
| 63.7°C | 16.2, 17.2 |
| 68°C | 16.3, 16.NTC, 17.3, 17.NTC |
| 70.5°C | 16.4, 17.4 |
| 72°C | 16.5, 17.5 |

* Performed gel electrophoresis of Phusion PCR samples

“07-14 gel of Phusion PCR”

### Week 4, day 1 (17/07)

* Performed bacterial transformation
  + Transformed DH5α cells using iGEM competence cell test kit with pSB1C3+RFP at concentrations 100pg/µL, 50pg/µL, 10pg/µL, and 0pg/µL (control)
  + Transformed DH10β cells with pKDL071 or no plasmid control
  + Plated transformed cells.
    - Spread the DH5α cells onto 4 LB+CAM plates, each containing 100µL of the 100pg/µL, 50pg/µL, 10pg/µL, and 0pg/µL cells.
    - Spread the DH10β cells onto two LB+KAN plates, both containing 100µL of the control and either 100µL or 50µL of the pKDL071 cells
  + Incubated the plates at 37°C overnight.

### Week 4, day 2 (18/07)

* Checked on 17/07 transformed cells and moved them to the 4°C fridge.
  + Confirmed DH5α cells were competent
  + Note: LB+CAM plates made on 13/07 may have been made with too much CAM (1250µL CAM to 500mL LB). This may explain why there were so few colonies from 17/07 transformation of DH5α + RFP/pSB1C3.

* Obtained single colonies from DH5α and DH10β plates from 17/07 and made overnight 37°C colonies.
  + The DH5α was placed in the 5mL of LB+CAM (5µL of stock CAM added to 5mL LB).
  + The DH10β was placed in 5mL of LB+KAN (25µL of stock KAN added to 5mL of LB).

### Week 4, day 3 (19/07)

* Examined plates from 18/07
  + Most plates had no cells likely due to the high CAM concentration (2.5x working concentration).

* Miniprepped DH10β and DH5α to extract plasmides (pKDL071 and pSB1C3 with RFP respectively) and did Nanodrop on the results

Table 1: Nanodrop results from Miniprep

| Plasmid | 260/280 | 260/230 | ng/µL |
| --- | --- | --- | --- |
| pSB1C3 #1 | 1.82 | 1.42 | 86.6 |
| pSB1C3 #2 | 1.85 | 1.52 | 87.9 |
| psB1C3 #3 | 1.79 | 0.99 | 136.6 |
| pKDL071 #1 | 1.89 | 1.55 | 60.0 |
| pKDL071 #2 | 1.78 | 1.25 | 66.0 |
| pKDL071 #3 | 1.95 | 1.90 | 48.7 |

* Performed Phusion PCR of pSB1C3 with RFP construct using psB1C3 #2 and the pKDL071 FWD/RVS primers
  1. Made 1ng/uL of pDKL071 #3 for PCR via following steps:
  + 2μL of pDKL071 #3 Miniprep was mixed with 95.4 μL of nuclease free water
  + Made 100μL aliquots of 100μM from pKDL071 FWD UNS3 primer (shipped 05/07) resuspended in 785μL nuclease free water
  + Made 100μL aliquots of 100μM from pKDL071 REV UNS2 primer (shipped 05/07) resuspended in 761μL nuclease free water
  2. Made a Master mix without DNA template containing:
  + 17.5μL of 10 uM FWD\_UNS3 primer
  + 17.5μL of 10 uM REV\_UNS2 primer
  + 70uL of 5x phusion buffer
  + 7uL of 10mM dNTPS
  + 10.5uL 100% DMSO ( to reach a final concentration of 3%)
  + 3.5uL Phusion polymerase
  3. Took 18uL was taken from this master mix added 32 uL of Nuclease free water for a final volume of 50 uL as the No Template Control (NTC)
  4. Added DNA template to the Master Mix:
  + 6 uL 1ng/uL Miniprepped pKDL071 #3
  + 186 uL nuclease free water
  5. Added 50uL of the Master Mix to 5 PCR tubes
  6. Ran Phusion PCR on temperature gradient with samples 1-5 at 62, 63.7, 68, 71.3, and 72°C respectively
  + NTC was at 72°C
* Ran gel on 12μL samples of Phusion PCR results for 1 minute then left for 1 hour then run for 1 hour due to equipment issues.
  + Timing issues due might be why the ladder is not as intense.
  + Sample 3 appears to be our optimal temperature @ 68°C

### Week 4, day 4 (20/07)

Ran Phusion PCR of the pKDL071 plasmid with RFP using the pKDL071 plasmid from Miniprep #3 (19/07).

* Used the pKDL071 FWD/RVS primers shipped on 05/07 (the first set of primers designed in 2017)
* Made Master Mix without DNA template or primers (for 8 reactions)
  + 20 μL of 10 uM FWD\_UNS3 primer
  + 20 μL of 10 uM REV\_UNS2 primer
  + 80 uL of 5x phusion buffer
  + 8 uL of 10mM dNTPS
  + 12 uL 100% DMSO ( to reach a final concentration of 3%)
  + 4 uL Phusion polymerase
* 18 uL was taken from the master mix as the No Template Control (NTC) and mixed with 32 uL of Nuclease free water.
* Added DNA template to Master Mix (for 7 reactions)
  + 7 uL of 1ng/uL miniprep#3 pKDL071
  + 217 uL of nuclease free water
* 50 uL of the master mix were added to 5 PCR tubes
* 6 samples and one NTC run at 68°C, the optimal temperature determined on 17/07.

Optimization Phusion PCRs of pSB1C3 and pKDL071 plasmids with new UNS primers

* Made 5 samples and one NTC for PCR of the pSB1C3 plasmid using pSB1C3 miniprep#2 and the new forward and reverse UNS primers.
  + Annealing temperature was 64°C so samples 1-5 at 72, 70.1, 66, 64.3, and 62°C respectively with NTC at 64.3°C
* Made 5 samples and one NTC for PCR of the pKDL071 plasmid using pKDL071 miniprep#3, the old forward, and new reverse UNS primers
  + Annealing temperature was 62°C so samples 1-5 at 67, 65.1, 63.3, 61, and 57°C respectively with NTC at 63.3°C
* Made Master mixes with DNA
  + OLD\_FWD and NEW\_REV pDKL071 (7 reactions)
    - 17.5 μL of 10 uM FWD primer
    - 17.5 μL of 10 uM REV primer
  + NEW\_FWD\_USN3 and NEW\_REV\_UNS2 pSB1C3 (7 reactions)
    - 17.5 μL of 10 uM FWD\_UNS3 primer
    - 17.5 μL of 10 uM REV\_UNS2 primer
* Made Master mix without DNA template or primers (for 15 reactions)
  + 150 uL of 5x phusion buffer
  + 15 uL of 10mM dNTPS
  + 22.5 uL 100% DMSO ( to reach a final concentration of 3%)
  + 7.5 uL Phusion polymerase
* Added 91 uL of Master mix without DNA to the pSB1C3 & pDKL071 primer master mixes
* 18 uL was taken from each primer master mix as the No Template Control (NTC) and added to 32 uL of Nuclease free water to reach a final volume of 50 uL.
* Added DNA template to primer mixes:
  + pSB1C3 master with DNA template (for 6 reactions):
    - 5.5 uL of 1ng/uL miniprep pSB1C3
    - 170.5 uL of nuclease free water
  + pDKL071 master with DNA template (for 6 reactions)
    - 5.5 uL of 1ng/uL miniprep pDKL071
    - 170.5 uL of nuclease free water
* 50 uL of each master mix were added to 5 PCR tubes (10 tubes total + 2 NTCs)

Performed gel electrophoresis all PCR results

* 8 wells for Phusion PCR of the pKDL071 plasmid

* 15 wells for optimization PCR of pSB1C3 and pKDL071 plasmids
  + Wells 1-6 contained pSB1C3 samples 1-5 and NTC
  + Well 7 contained the 10kb ladder
  + Wells 8-13 contained the pKDL071 samples 1-5 and NTC

Made 5mL cultures of the DH5 alpha cells transformed with GFP plasmids and placed them in the overnight shaker to grow.

### Week 4, day 5 (21/07)

Nanodrop PCR products of pKDL071 from 20/07

* Note: NOT purified

Table 1: Nanodrop of pKDL071-UNS with old primers (at 68°C)

| Sample | ng/uL | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 1 | 200.7 | 1.34 | 0.47 |
| 2 | 379.0 | 1.32 | 0.62 |
| 3 | 357.5 | 1.37 | 0.62 |
| 4 | 346.1 | 1.33 | 0.64 |
| 5 | 363.9 | 1.33 | 0.59 |
| 6 | 353.6 | 1.37 | 0.61 |
| NTC | 339.3 | 1.34 | 0.57 |

Table 2: Nanodrop of pKDL071-UNS optimization with new REV primer (at 62-72°C)

| Sample | ng/uL | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 1 | 436.9 | 1.42 | 0.74 |
| 2 | 437.1 | 1.46 | 0.72 |
| 3 | 443.0 | 1.41 | 0.72 |
| 4 | 446.7 | 1.42 | 0.73 |
| 5 | 434.9 | 1.43 | 0.71 |
| NTC | 436.3 | 1.43 | 0.71 |

Dpn1 digest and purification of pKDL071 PCR product with new REV primer: 40uL digestion reaction each tube.

* 6 tubes in total, including 4 samples (D1-D4), 1 NTC and 1 positive control (miniprepped pKDL071)
* Master Mix contained:
  + 32uL of pKDL071
  + 4uL 10X CutSmart
  + 4uL Dpn1
* Nanodropped the results

Table 3: Nanodrop of the digested and purified pKDL071 PCR product with new REV primer

| Sample | ng/ul | 260/280 | 260/230 |
| --- | --- | --- | --- |
| D1 | 28.1 | 1.82 | 1.69 |
| D2 | 27.9 | 1.84 | 1.84 |
| D3 | 31.6 | 1.80 | 1.73 |
| D4 | 30.5 | 1.94 | 1.84 |
| NTC | 18.3 | 1.79 | 1.43 |
| Positive Control (minipreped pKDL071) | 22.3 | 1.81 | 1.70 |

Performed Phusion PCR of pSB1C3 with new FWD UNS & REV UNS with temperature gradient

* Made Primer Master mix (MM1) new FWD UNS3 & New REV UNS2 pSB1C3 (for 7 reactions) containing:
  + 17.5 μL of 10 uM NEW FWD\_UNS3 primer
  + 17.5 μL of 10 uM NEW REV\_UNS2 primer
* Made Master mix (MM2) without DNA template or primers:
  + 70 uL of 5x Phusion buffer
  + 7 uL of 10mM dNTPS
  + 10.5 uL 100% DMSO ( to reach a final concentration of 3%)
  + 3.5 uL Phusion polymerase
* Added MM2 to MM1 to make a Primer + Ploymerase mix
* Took 18uL from polymerase + primer mix as NTC and added 32 uL of nuclease free water for final volume of 50uL
* Added the following to make a Primer + ploymerase + DNA Mix (for 6 reactions)
  + 6 uL of 1 ng/uL miniprep pSB1C3 (RFP)
  + 186 uL of Nuclease free water
* Divided final mix into 6 tubes, 50 uL each
* Ran PCR with S1-6 in 62.3, 63.7, 66.3, 69.5, 71.3, and 62°C respectively, with NTC in 69.5°C
* Ran a gel of the results

### Week 5, day 1 (24/07)

* Dpn1 digest of pSB1C3+UNS PCR from 21/07
  + 4 sample tubes and 1 control without Dpn1
* Gel electrophoresis of the results

* Note: The absence of bands could be due to multiple reasons; the product was lost during digestion and purification or there was not enough product initially.
* Nanodrop of the purified samples was preformed to test concentration of DNA purified.

Table 1: Nanodrop results of purified Dpn1 digestion of pSB1C3+UNS

| Sample | 260/280 | 260/230 | ng/uL |
| --- | --- | --- | --- |
| D1 | 1.88 | 1.43 | 10.6 |
| D2 | 1.74 | 1.34 | 11.8 |
| D3 | 1.91 | 0.75 | 8.5 |
| D4 | 1.87 | 1.64 | 13.6 |
| Undigested pSB1C3 | 1.33 | 1.16 | 6.2 |

### Week 5, day 2 (25/07)

Assembled reporter and mCherry constructs

* Pooled the purified pKDL071+UNS product from 21/07 purification to obtain a greater amount of DNA necessary for assembly.
  + The resulting concentration of product, measured via Nanodrop, was 38.2 ng/uL (Contamination values: 260/280 = 1.93; 260/230 = 1.77).
* Created assembly according to the HiFi DNA assembly protocol. The reporter and mCherry master mix values are listed in the table below.

Table 1: HiFi DNA Assembly Calculations

| Sample | Reporter | mCherry |
| --- | --- | --- |
| pKDL071+UNS | 1.67uL | 1.67uL |
| Reporter mCherry | 10.81uL | N/A |
| Reporter YFP | 9.7uL | N/A |
| mCherry | N/A | 10uL |
| Total fragment volume | 22.18uL | 11.67uL |
| Buffer (HiFi MM) | 22.18uL | 11.67uL |
| Total Reaction Volume | 44.36uL | 23.34uL |

* Transformed the assembly parts into the NEB 5α competent cells (came with the HiFi DNA assembly kit).

Plated NEB 5α cells to grow overnight in the 37°C shaker.

* 12 LB+KAN plates(6 mCherry, 6 reporter)
* 4 LB+AMP plates (2 for the positive control transformation (pUC19), 2 for the positive assembly control)

### Week 5, day 3 (26/07)

* Examined NEB 5α plates from 25/07 and saw single colonies

* Inoculated single colonies from pKDL071 mCherry and Reporter plates into LB+KAN and LB+CAM and put in incubator

Ran Phusion PCR of pSB1C3 (minprep #2) with new primers

* Made 5 samples and 1 NTC
* Loaded samples qPCR machine at 68°C

### Week 5, day 4 (26/07)

* Gel Electrophoresis of pSB1C3 Phusion PCR from 26/07
  + Primer dimers & no amplified product
  + NEB’s 2-Log-DNA Ladders were used

* Miniprepped mCherry and reporter (YFP) constructs from our pKDL071 plasmid backbone from DH5a transformed bacteria (from 25/07) with subsequent nanodrop to measure concentration of DNA:
  + 9 minipreps in total for mCherry construct:
    - 6 from inoculation culture of colony A and B
    - 3 from inoculation culture of colony C
  + 6 minipreps in total for Reporter construct:
    - 3 minipreps from inoculation culture of colony A
    - 3 minipreps from inoculation culture of colony B
  + Nuclease free water was added to resuspend DNA after vacufuging the miniprepped product (to eliminate elution buffer)

Table 1: Nanodrop results of mCherry and Reporter pKDL071 plasmids

| Sample | DNA Concentration (ng/uL) | 260/280 | 260/230 | Nuclease-free water (uL) |
| --- | --- | --- | --- | --- |
| mCherry 1 (A) | 20.0 | 1.59 | 1.30 | 22.8571428571 |
| mCherry 2 (A) | 36.6 | 1.78 | 1.74 | 41.8285714286 |
| mCherry 3 (A) | 38.7 | 1.60 | 0.90 | 44.2285714286 |
| mCherry 1 (B) | 74.5 | 1.54 | 0.74 | 85.1428571429 |
| mCherry 2 (B) | 25.0 | 1.69 | 1.54 | 28.5714285714 |
| mCherry 3 (B) | 27.2 | 1.65 | 1.35 | 31.0857142857 |
| mCherry 1 © | 25.3 | 1.89 | 2.46 | 28.9142857143 |
| mCherry 2 © | 24.1 | 2.08 | 2.18 | 27.5428571429 |
| mCherry 3 © | 24.0 | 2.05 | 2.54 | 27.4285714286 |
| Reporter 1 (A) | 18.1 | 1.57 | 1.42 | 20.6857142857 |
| Reporter 2 (A) | 18.8 | 1.55 | 1.36 | 21.4857142857 |
| Reporter 3 (A) | 21.1 | 1.55 | 1.33 | 24.1142857143 |
| Reporter 1 (B) | 21.6 | 2.05 | 2.31 | 24.6857142857 |
| Reporter 2 (B) | 20.7 | 2.09 | 2.17 | 23.6571428571 |
| Reporter 3 (B) | 21.8 | 2.06 | 2.63 | 24.9142857143 |

* Set up and re-ran Phusion PCR of pSB1C3 with same settings as 26/07
* Streaked out 5 plates of pKDL071\_mCherry (from colonies a, b, c) and pKDL071\_Reporter (from colonies a and b) and put in 37°C incubator overnight

### Week 5, day 5 (28/07)

* Checked the plates with pKDL017\_mCherry and pKDL017\_Reporter from 27/07
* Ran the gel for 2nd PCR of PSB1C3 with new primers from 27/07

* Note: The exposure time might be too little (only 0.02), needs to be at least 0.35s.

### Week 6, day 1 (31/07)

* Ran PCR for pSB1C3 + new UNS primers (Tm Calculator indicated annealing temperature was 67°C)
  + 6 samples at 62°C, 62.9°C, 64.9°C, 68°C, 70.5°C, and 72°C
  + One NTC at 68°C
* Ran gel of the results

### Week 6, day 3 (02/08)

* Made electrocompetent MG 1655ΔLacI cells according to the electrocompetent cell protocol.
* Made 80 glycerol stocks of the electrocompetent MG1655ΔLacI cells. Stored cells in -80°C freezer.

### Week 6, day 4 (03/08)

* Vacufuged pKDL071 plasmids (w/ mCherry and Reporter) to concentrate DNA
* Transformed electrocompetent MG1655ΔLacI cells (from 02/08) with RFP competence test kit and placed in 37°C incubator overnight
* Resuspended vacufuged DNA samples in 7uL of nuclease free water to reach a concentration of 35ng/uL

### Week 6, day 5 (04/08)

* Checked competence of MG1655ΔLacI cells (from 03/08) - the results were good.
* Nanodropped mCherry and Reporter DNA samples from 03/08

Table 1: Nanodrop results of vacufuged mCherry and Reporter DNA

| Sample | ng/uL | 260/280 | 260/230 |
| --- | --- | --- | --- |
| mCherry A1 | 67.3 | 2.74 | 1.00 |
| mCherry A2 | 14.6 | 3.6 | 1.14 |
| mCherry A3 | 17.2 | 5.23 | 0.83 |
| mCherry B1 | 26.4 | 2.86 | 1.06 |
| mCherry B2 | 38.6 | 2.76 | 1.16 |
| mCherry B3 | 46.2 | 2.58 | 1.23 |
| mCherry C1 | 56.2 | 2.61 | 1.23 |
| mCherry C2 | 60.8 | 2.37 | 1.17 |
| mCherry C3 | 48.4 | 2.57 | 1.23 |
| Reporter A1 | 45 | 2.86 | 1.13 |
| Reporter A2 | 47.5 | 2.69 | 1.06 |
| Reporter A3 | 57.5 | 2.76 | 1.07 |
| Reporter B1 | 81.9 | 2.69 | 1.1 |
| Reporter B2 | 54.2 | 2.8 | 1.07 |
| Reporter B3 | 54.3 | 2.63 | 1.11 |

* Performed Colony PCR of mCherry + Reporter construct
* Ran gel of colony PCR results

### Week 7, day 2 (08/08)

* Ran 12-well gel of 04/08 PCR products: pKDL071 plasmid in wells 2-8; pSB1C3 plasmid in wells 9-12
  1. DNA ladder 10kb biobasic
  2. Reporter A2
  3. Reporter B3
  4. mCherry A1
  5. mCherry B2
  6. mCherry C2
  7. Positive control
  8. Negative control
  9. Sample 1
  10. Sample 2
  11. Sample 3
  12. Negative control.

* Repeated colony PCR of mCherry and reporter constructs in the pKDL071 plasmid
  + PCR was run with samples mCherry A1, B3, and C1, and Reporter A1 and B3.
  + Samples were first diluted to concentrations of 1 ng/uL based on nanodrop results from 04/08.
* Gel electrophoresis of colony PCR of mCherry and reporter constructs.

### Week 7, day 3 (09/08)

* Q5 PCR with Temperature Gradient (62-72°C) of PKDL071 with mCherry sample A1
  + Samples 1-5 at 72.0°C, 70.0°C, 68.3°C, 64.3°C, 62.0°C, and 66.0°C
  + NTC at 66.0°C
* NEB OneTaq Hot Start PCR with temperature gradient of pSB1C3 to add UNSs with new primers. Tm Calculator indicated annealing temperature of 54°C.
  + Samples 1-6 at 61.0°C, 59.1°C, 57.3°C, 53.5°C, 51.9°C, 51.0°C, 57.3°C
  + NTC at 57.3°C
* Transformation of MG1655ΔLacI with Reporter construct and mCherry construct respectively, both in pKDL071 bakcbone.
* Gel eletrophoresis of both PCRs

### Week 7, day 4 (10/08)

* Examined transformed MG1655ΔLacI with pKDL071 plasmid with insert from 09/08 (NC, Rep A, B, mCherry A, B, C)

* NEB OneTaq Hot Start PCR with temperature gradient of Christian’s inserts (using colony PCR primers to test if they are working)
  + Made Master Mix (8 reactions) (50uL)
    - 80uL GC Reaction Buffer
    - 8uL of 10uM dNTP
    - 8uL of 10uM FWD
    - 8uL of 10uM REV
    - 4uL of OneTaq Polymerase
    - 12uL of 100% DMSO (to make final concentration of 3%)
  + Made NTC with 15uL of MM and 35uL nuclease free water
  + Added DNA to MM:
    - 7uL of DNA Template
    - 238 uL nuclease free water
  + Added 50uL final mix to 6 tubes and ran the PCR
* Gel electrophoresis of PCR products showed colony PCR primers weren’t working

### Week 7, day 5 (11/08)

* Designed new pKDL071 colony PCR primers
  + BLASTED against pKDL071 w/ UNSs, also BLASTED against *E.coli* K-12
  + Highest off target E-value was 0.46 for both FWD and RVS.
  + When analyzed with IDT OligoAnalyzer Tool the highest Delta G: -6.75 kcal/mole (Base Pairs3) was found for hetero-dimerization between fwd and rvs primers.)

“08-11 primer design.png”

* Notes on how to interpret IDT OligoAnalyzer Output:
  + For self-dimer analysis, click on ‘Self-Dimer’ to bring up a new window with each possible self-dimer your oligo can form. For each diagram you will be able to see the calculated delta G value for this secondary structure. If you have a strong delta G (-9kcal/mol or more negative) this oligo could be problematic.
  + Heterodimer analysis and hairpin formation is same as above

“08-11 analysis.png”

### Week 8, day 4 (17/08)

* Received new pSB1C3 and pKDL071 w/ UNS2/UNS3 FWD/REV primers
* Phusion PCR with temperature gradient (62°C to 72°C) to put UNS primers onto pKDL071 and pSB1C3 backbone
  + 6 pKDL071 samples + NTC and 6 pSB1C3 samples + NTC
* Gel Electrophoresis of PCR products to determine size

* Redid Phusion PCR for pSB1C3 with new primers with increased template; also tested 2016 pSB1C3 primers with increased template
  + Made Master Mix (MM1): (these were done for BOTH old (2016) and new (2.0) primers) for 8 reactions (50 μL each)
    - 80uL of 5x Phusion HF Buffer
    - 8uL of 10mM dNTPs
    - 20uL FWD primer
    - 20uL REV primer
    - 12uL of 100% DMSO
    - 4uL Phusion polymerase
  + Made NTC with 18uL of MM1 and 32uL of nuclease free water
  + Added DNA to MM1 to make MM2
    - 50uL (1ng/uL) template
    - 174uL nuclease free H2O
  + Made 6 50uL PCR tubes from MM2
* Gel electrophoresis of PCR products

* Dpn1 digest of pKDL071 to get rid of template
* PCR purification and Nanodrop of digested pKDL071

Table 1: Nanodrop results of purified Dpn1 digested pKDL071

| Sample | ng/uL | 260/280 | 260/230 |
| — | — | — |
| 4 | 12.2 | 2.55 | 0.91 |
| 6 | 11.6 | 2.80 | 0.75 |
\*Note: samples 3/4 and 5/6 were combined into sample 4 and 6, receptively.

* Performed Gibson HiFi DNA assembly, adding 0.1 pmol of fragments and 0.05pmol of inserts

Table 2: Reaction Component Table for Gibson HiFi DNA Assembly

| Component | Reporter | mCherry |
| --- | --- | --- |
| pKDL071 + UNS BB | 5.23 uL | 5.23 uL |
| Reporter\_mCherry | 10.81 uL | - |
| Reporter\_YFP | 9.7 uL | - |
| LacILOV\_mCherry | - | 10 uL |

### Week 8, day 5 (18/08)

* Gibson plates from 17/08 (as well as the positive control supplied by Gibson kit) showed no colonies
* PCR of pSB1C3 UNS with 2.0 UNS primers using a linearized pSB1C3 backbone to control for supercoil interference. We also ran a temperature gradient: 50-71°C. Phusion HF polymerase was used as well as 3% DMSO.
* Gel electrophoresis of PCR products:

“08-18 pSB1C3 gel.png”

* Result: Primer Dimers and what appears to be a slightly larger product (we hypothesize that these could be the palindromic ends of our UNS primers (backbone complement) annealing and amplifying.
* Redid Gibson assembly based on new Nanodrop of samples

Table 1: Nanodrop results of purified Dpn1 digested pKDL071

| Sample | ng/uL | 260/280 | 260/230 |
| — | — | — |
| 4 | 16.8 | 1.63 | 1.23 |
| 6 | 16.1 | 1.63 | 0.72 |

Table 2: Reaction Component Table for Gibson HiFi DNA Assembly

| Component | Reporter | mCherry |
| --- | --- | --- |
| pKDL071 + UNS BB | 4 uL | 4 uL |
| Reporter\_mCherry | 10.87 uL | - |
| Reporter\_YFP | 9.7 uL | - |
| LacILOV\_mCherry | - | 10 uL |

### Week 9, day 1 (21/08)

* OneTaq PCR of Christian’s miniprepped pKDL071 plasmid with backbone specific PCR colony primers to test them out with temperature gradient from 45-55°C
* Gel electrophoresis of PCR product:

* Gel electrophoresis for assembled pKDL071 Reporter ® and mCherry (M) plasmids from 25/07 (1), 17/08 (2), and 18/08 (3)

* Made 5mL overnight LB+KAN cultures of pKDL071 mCherry colonies 1, 2, and 3 in preparation for miniprep the next day

### Week 9, day 2 (22/08)

* Miniprepped Gibson assembly colonies A through C
* Performed EZ-10 spin column PCR products purification kit of all PCR reactions using 2.0 primers on pSB1C3 backbone (samples 9-14 from 17/08 and samples 1-11 from 17/08)
  + Purpose: to find out if the backbone + UNS was actually amplified during PCR, and was not visible due to low yield.
  + The purified products would then be combined to obtain enough product to run a gel extraction.
* Nanodropped PCR purified pKDL071+UNS samples 4 and 6 from 17/08 and miniprepped plasmid with mCherry in pKDL071 (colonies A,B and C)

Table 1: Nanodrop results of PCR purified pKDL071+UNS and mCherry+pKDL071 colonies

| Sample | ng/uL | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 4 | 1.85 | 1.02 | 40.7 |
| 6 | 2.11 | 0.91 | 25.4 |
| A | 1.85 | 1.46 | 68.6 |
| B | 1.76 | 1.17 | 74.9 |
| C | 1.90 | 1.45 | 77.2 |

* OneTaq PCR of purified plasmid: 6 samples and 1 NTC
* Gel electrophoresis of PCR purified pSB1C3

“08-22 PCR purified pSC1C”

* Gel electrophoresis of LacILOV mCherry from colonies and colony PCR products

“08-22 OneTaq HotStart pKDL071 mCherry with 2.0 Colony PCR primers”

* Gibson assembly of the reporter constructs

### Week 9, day 3 (23/08)

* Vacufuged assembly from 22/08 and took Nanodrop each time
* Ran a gel of miniprepped reporter

### Week 9, day 4 (24/08)

* Gibson Assembly of mCherry\_LacILOV insert and YFP\_Reporter insert
  + Note: we put the tubes in the PCR at 50°C instead of using thermocycler
* Constructed the light array
* Transformed DH5α cells with Gibson assembled mCherry\_LacILOV\_YFP\_reporter insert

### Week 9, day 5 (25/08)

* Checked plates from yesterday and got 1 colony
* Colony PCR of reporter insert (resuspended colony in water)
* RE Digestion (pstI)
* Gel electrophoresis in order: 2-log NEB ladder, Christian’s insert, Reporter insert, NTC

### Week 10, day 1 (29/08)

* Phusion PCR with colony PCR primers on our Gibson Assembly product

* PCR of LacILOV\_mCherry insert WITH restriction enzyme primers

### Week 10, day 2 (30/08)

* Ran the gel for PCR purified Reporter and LacILOV.
* Gibson Assembly of mCherry and YFP in pKDL071
* RE Digest of reporter and pSB1C3
* NEB Transformation protocol (E2621) for HIFI Gibson
  + 3 transformations: reporter, transformation, and assembly
* T4 DNA ligation of pSB1C3+reporter
* Nanodropped LacILOV mCherry gel

### Week 10, day 3 (31/08)

* RE Digest (XbaI, PstI) of LacILOV & mCherry from gel
* Transformation of rep\_pSB1C3 into DH10B
* Gibson assembly of reporter + pJDK071 with 1:1 ratio insert:BB

### Week 10, day 4 (01/09)

* Colony PCR :
  + Transformed DH10B cells with ligated pSB1C3 & Reporter construct (3:1, reporter: Backbone) using OneTaq Hot Start polymerase.
  + Transformed reporter + pKDL071 HiFi DNA assembly with 1:1:1 of Reporter\_YFP: Reporter\_mCherry: pKDL071 w UNS, using OneTaq Hotstart
* Streaked reporter\_pSB1C3 and reporter\_pKDL071 colonies onto new LB+CAM plates
* Transformed ligated reporter + pSB1C3 (1:3, reporter: Backbone) and ligated LacILOV\_mCherry+ pSB1C3(3:1, insert:Backbone) into DH10B cells

### Week 11, day 2 (05/09)

* OneTaq Hot Start Colony PCR on colonies from 01/09 (pSB1C3\_reporter & pSB1C3\_mCherry\_LacILOV (transformed into DH10Beta))
* Phusion PCR: pSB1C3 backbone amplification using 2016 primers
* Overnight culture of LacILOV\_mCherry insert in pSB1C3 plasmid in DH5beta

### Week 11, day 3 (06/09)

* Redid Colony PCR w/ LacILOV\_mCherry & Reporter constructs and Phusion PCR for amplification of pSB1C3 backbone (with 2016 primers) from 05/09
* Miniprepped our overnight culture of DH10Beta with LacILOV construct in pSB1C3
* Phusion PCR of Gibson Mix with new UNS 2 REV and UNS 3 FWD primers

### Week 11, day 4 (07/09)

* Nanodrop results of pooled PCR purified samples from 06/09
* Redid Phusion PCR from 06/09
* RE Digest for PCR with RE primers of pSB1C3
* PCR purification of digested pSB1C3

### Week 11, day 5 (08/09)

* Phusion PCR of LacILOV\_mCherry insert from plasmid (inserted into both BBs) + directly from IDT stock to test UNS primers
* Ran gel for PCR purified reporter construct with RE sites from 29/08
  + Gel extract of single band on gel, following protocol from NEB Gel extraction kit
  + Nanodropped results
  + Phusion PCR of gel extracted reporter
* PCR purified PCR product from phusion PCR of reporter construct with RE sites using UNS primers (07/09)
  + Nanodropped product
  + Gel of purified product
* Gibson Assembly of pKDL071 + reporter with vector: insert (2:1 ratio) from 22/08
  + Transformed Gibson products following NEB Gel extraction protocol
* Ligated dephosphorylated + digested pSB1C3 BB from 07/09 and gel extraction product from today
* LacILOV\_mCherry colony and completely resuspended in microcentrifuge tube with LB+Kan

### Week 11, day 6 (09/09)

* We got colonies! From HiFi assembly of reporter+pKDL071 in 2:1 vector:insert ratio from 08/09
  + Colony PCR to test
* Phusion PCR with UNS primers for (A) Gibson mix HiFi DNA assembly from 08/09, and B) IDT LacILOV\_mCherry insert
* Phusion PCR with RE primers for Gibson mix (HiFi assembly) from 08/09 with temperature gradient

### Week 12, day 1 (11/09)

* Colony PCR of reporter construct assembled 08/09 using UNS primers to eliminate non-specific binding possibility of colony PCR primers
* Crossover PCR of reporter subpart YFP & reporter subpart mCherry to assemble reporter construct
* Inoculated 5ml overnight cultures with all reporter colonies
* Performed LacILOV\_mCherry end point measurement and start new assay

### Week 12, day 2 (12/09)

* Temperature gradient PCR with UNS 3 FWD and UNS 2 REV primers with LacILOV\_mCherry in pKDL071 and pSB1C3
* LacILOV\_mCherry pSB1C3 miniprep
* Overlap PCR of reporter inserts

### Week 12, day 3 (13/09)

* Miniprepping reporter construct in pKDL071
* New UNS 4 fwd and rev primers
* PCR purified reporter constructs from PCR amplification

### Week 12, day 4 (14/09)

* Phusion PCR of miniprep’d “reporter” plasmid in pKDL071 using colony PCR primers

### Week 12, day 5 (15/09)

* RE Digest of minipreped “reporter” plasmids R1, R2, and RA (used 10ul of plasmid in each rxn) from gibson assemblies with PKDL071 back bone.
* Ran gel of PCR purified reporter inserts
  + DNA Gel Extraction and Nanodropped results
* Phusion PCR temp gradient of reporter mcherry with UNS primers (65-55C)

### Week 13, day 1 (18/09)

* Redid reporter mCherry temperature gradient using PCR purified mCherry sample from 14/09

### Week 13, day 2 (19/09)

* Phusion PCR of LacILOV\_mCherry in pKDL071 using UNS primers to test if PCR reagents are good

### Week 13, day 3 (20/09)

* PCR amplification of YFP reporter & mCherry reporter from PCR purified product and gel extracted product.
  + Used 1:10 diluted PCR purified YFP and mCherry and Gel extracted YFP and mCherry will as template for PCR
* Nanodropped Reporter Parts
* Amplification of reporter insert from ALL previous Gibson Assemblies using UNS 3 FWD and UNS 2 REV
* Final PCR: PCR of LacILOV\_mCherry purified (1rxn + NTC + buffer)

### Week 13, day 4 (21/09)

* RE (BglII) digest of LacILOV\_mCherry in pKDL071

### Week 13, day 5 (22/09)

Note: From PCR results done on 21/09, PCRing after PCR purification is not usually successful due to incomplete amplicons from the first PCR

* RE Digest results:
  + Bgl II digest of LacILOV mcherry yielded unexpected results and digestion will be repeated today for a longer incubation period (~2.5h)
  + DpnI digest of PCR puified pKDL071; was not digested beforePCR purification, PCR purify after digest.
* Bgl II digest of LacILOV mcherry PKDL071, LacILOVmcherry pSB1C3, and Christan’s pKDL071 (digested for 2.5h)

### Week 14, day 1 (25/09)

* Ran temperature gradient PCR with colony PCR primers of LacILOV mCherry with Phusion HF to test if OneTaq gave artifacts
* Started overnight culture of LacILOV\_mCherry for assays
* Designed primers to add RE sites (xbai and psti) to LacILOV, reason: be able to clone LacILOV into pSB1C3 for parts submission

### Week 14, day 2 (26/09)

* PCR (25ul rxn, 6 rxns) using colony PCR primers to check if successfully cloned reporter insert into pKDL071
* Phusion PCR to amplify this to insert reporter\_mCherry
* DpnI digest of pKDL071 amplified backbone with UNS 2 at 5’ end and UNS 4 at 3’ end
  + combined PCR samples 1&2, 3&4
* PCR purification and Nanodrop of dpnI digested pKDL071

### Week 15, day 3 (27/09)

* Gibson Assembly of Reporter\_mCherry and pKDL071 BB with UNS 2 + 4
  + Reporter\_mCherry will be used from IDT stock
  + pKDL071 BB with UNS 2 and 4 will be used from sample 1 prepared 26/09
* Phusion PCR using UNS 4 FWD and UNS 3 REV to amplify pKDL071
* Transformed of DH5a cells with our mCherry reporter in pKDL071
  + Spreaded LB+Kan plates and placed in incubator overnight @ 37oC

### Week 15, day 4 (28/09)

* Got 28 colonies from 27/09!
* Colony PCR with OneTaq and colony PCR primers
* DpnI digest of pKDL071 BB using UNS4 and UNS3 primers
* PCR purification of DpnI digested pKDL071
* Nanodrop of dpnI digested + PCR purified pKDL071 BB

### Week 15, day 5 (29/09)

* Started an overnight culture of colonies 1,4,5,6,14,18,26,29 (streaked onto new plates) in LB+KAN
* Colony PCR of colony 29 from 28/09

### Week 15, day 6 (30/09)

* Miniprepped reporter\_mCherry\_pKDL071 colonies 1,4,5.6.14.18.26 &29
* PCR amplified those plasmids with UNS4 FWD & UNS2 REV primers

### Week 16, day 1 (02/10)

* Phusion PCR to add UNS 3 to reporter mCherry in pKDL071 - will make it easier to add YFP reporter (Gibson)

### Week 16, day 2 (03/10)

* Received LacILOV Fwd and rev primers adding cut sites XbaI and PstI and YFP reporter stock from IDT
* Phusion PCR for LacILOV PCR with RE sites
* PCR amplification of reporter\_mCherry colonies with UNS 4 FWD and pKDL071 UNS 3 rev to linearized plasmid and add UNS 3 for Gibson assembly of reporter\_YFP

### Week 16, day 3 (04/10)

* Prepared samples for sequencing:
  + Reporter\_mCherry in pKDL071
  + LacILOV\_mCherry in pSB1C3

### Week 16, day 4 (05/10)

* DpnI digest of LacILOV AND digestion of LacILOV using XbaI/PstI
* PCR purified digested LacILOV\_with\_RE sites

### Week 16, day 5 (06/10)

* Transformed ligated LacILOV in pSB1C3 to DH5B cells
* Gibson assembly of reporter YFP with reporter\_mCherry in pKDL071 from colony 1
* Transformed into NEB Competent cells DH5a, plated on LB+AMP and incubated overnight

### Week 16, day 6 (07/10)

* Colony PCR

### Week 17, day 2 (10/10)

* Received mCherry reporter construct from IDT, dCas9 on CamR/TetR controlled plasmid from AddGene
* DH5a-pKDL071-reporter colonies 13,20,21,22,23 were restreaked on LB+Kan and overnight cultures of these colonies were also made
* dCas9 containing cultures were streaked onto an LB+Cam plate and put in 37°C incubator

### Week 17, day 3 (11/10)

* Miniprepped pKDL071\_reporter plasmids from colonies 13, 20, 21
* Made overnight culture of pdCas9 cells to miniprep the next day
* Phusion PCR of reporter colonies with UNS 3 FWD and UNS 2 REV primers (7rxns, 25ul each)
* Prepared sequencing primers for sequencing reporter construct in pKDL071
  + seq 1,2,3,4,5,6
  + To amplify pSB1C3 out of LacILOV mCherry
    - primers for UNS3 REV and UNS 2 FWD
    - UNS 5 REV - sgRNA + antiCRISPR
    - add UNS 3 beginning and UNS 2 REV for antiCRISPR
* Transform MG1655ΔLacI cells with reporter pKDL071 colony 21 and 22
  + Reporter mCherry pKDL071 was also transformed into MG1655ΔLacI as a +ve control for mCherry expression in LacILOV assays
  + RFP\_pSB1C3 (10pg/ul) was used as +ve transformation control
* Miniprepped pdCas9 from overnight culture (1.5ml into 2 tubes)

### Week 17, day 5 (13/10)

* Phusion PCR for amplifying pSB1C3 backbone out of LacILOV with UNS 2 and UNS 3

### Week 17, day 6 (14/10)

* DpnI digest of pSB1C3 with UNS 2 and 3 PCR product
* PCR purification of DpnI digested pSB1C3 UNS2 and 3 PCR product

### Week 18, day 2 (17/10)

* Phusion PCR of reporter\_mCherry in pKDL071 and of AcrIIA4 and LacILOV with new UNS primers
* DpnI digest of LacILOV and pKDL071 PCR product
* PCR purification and Nanodrop of LacILOV, reporter\_mCherry\_pKDL071 with UNS 5, AcrIIA4, and reporter construct
* Calculated Gibson Assembly of AcrIIA4, LacILOV, and sgRNA

### Week 18, day 3 (18/10)

* Transformed into DH5a cells and incubated at 37oC overnight (3plates each):
  + AcrIIA4 in pSB1C3
  + sgRNA\_reporter mCherry in pKDL071
  + LacILOV in pSB1C3

### Week 18, day 4 (19/10)

* Colony PCR of 18/10 plates
  + AcrIIA4 using colony rev/fwd primers
  + LacILOV using colony rev/fwd primers
  + sgRNA\_reporter mCherry

### Week 18, day 5 (20/10)

* Measurement of LacILOV\_mCherry after diluting and growing overnight:
  + First measurement was 12hrs after dilution
  + Note: LacILOV\_pSB1C3 colony 9 was incubated from colony PCR sample after 5hrs from preparation as colony labeling on the plate was not clear. LacILOV\_pSB1C3 colony 9 overnight culture didn’t grow and was not miniprep’d
* Miniprepped overnight cultures from colonies obtained after assembly

### Week 18, day 7 (22/10)

* Phusion PCR of :
  + Reporter\_mCherry\_sgRNA\_pKDL071 with UNS 4 FWD and pKDL071 UNS 3 REV (3rxn + NTC + extra) to assemble full construct
  + Reporter\_mCherry\_sgRNA\_pKDL071 with UNS 5 REV and pKDL071 UNS 6 FWD (3rxn + ntc + extra) to assemble AcrIIA4 for antiCRISPR assays
  + Reporter\_pKDL071 colony 21 & 22 with UNS 3 FWD and UNS 4 REV (2rxn)
  + Reporter\_pKDL071\_colony 21 and 22 with UNS 4 FWD and UNS 2 REV (2rxn)
* DpnI digest of sgRNA\_reporter mCherry for full construct and sgRNA\_reporter mCherry\_pKDL071 for AcrIIA4 assembly
  + PCR purification of S1 and AS1
  + Nanodropped results
* Redid PCR of:
  + Reporter\_pKDL071 (colony 21 & 22) with UNS 3 FWD and UNS 4 REV
  + Reporter\_pKDL071 colony 21 and 22 with UNS 4 FWD and UNS 2 REV. (reporter\_mCherry\_pKDL071 will be used as +ve control) (3rxns)
* Gibson assembly:
  + AcrIIA4\_YFP subparts into sgRNA reporter\_mCherry\_pKDL071 (assembly of full construct)
  + AcrIIA4 alone into sgRNA\_reporter\_mCherry\_pKDL071 with UNS 5 and 6
* Transformed MG1655ΔLacI cells with:
  + sgRNA\_rep\_mCherry\_pKDL071 colony 1 and pdCas9 (1ul of each plasmid will be used)
  + BBa\_K523013 pSB1C3 to be used as +ve control for YFP found in plate 1 well 8H
* Colony PCR of:
  + Full construct (AcrIIA4\_YFP\_sgRNA\_repmCherry)
  + sgRNA rep\_mCherry in pKDL071 with UNS 5 and 6 and AcrIIA4
* Streaked MG1655ΔLacI pdCas9 + sgRNA\_pKDL071 on a new plate to get single colonies
  + Started overnight culture of MG1655ΔLacI pdCas9 + sgRNA\_pKDL071 for assays
  + Started overnight culture of MG1655ΔLacI YFP to trouble shoot YFP measurements

### Week 19, day 2 (24/10)

* Miniprep and Nanodrop colonies F3 and F7, S2 and S5
* Phusion PCR to take:
  + Reporter construct out of pKDL071 (reporter colony #21)
  + Full construct out of miniprep’d pKDL071 (to Gibson into pSB1C3 tomorrow)
* DpnI digest of reporter construct [50ul rxn]
* Gibson of reporter construct with pSB1C3…UNS 2 and 3

### Week 19, day 3 (25/10)

* Phusion PCR of pSB1C3 out of LacILOV pSB1C3 to add UNS 5 and extend UNS 3 for Gibson of full construct in pSB1C3
* PCR purification of full construct (F3 and F7)
* Colony PCR of reporter in pSB1C3 (twice)
* Redid Phusion PCR from 24/10
  + Full construct PCR out of pKDL071 (2rxns) using UNS primers
  + Full construct PCR out of pKDL071 (F3+F7) using phusion and pKDL071 colony PCR primers

### Week 19, day 4 (26/10)

* Sent samples for sequencing:
  + LacILOV\_pSB1C3 (L3 + L4)
  + AcrIIA4\_pSB1C3 (A1+A3+A7)
  + sgRNA\_repmCherry\_pKDL071 (S1)
  + AcrIIA4\_sgRNA\_repmCherry (S2+S5)
* Colony PCR of reporter\_pSB1C3 Colonies 7-20
  + Colonies 10,11,15 to be streaked onto new plates and miniprep’d

### Week 19, day 5 (27/10)

* Miniprep of reporter\_pSB1C3 colonies 10,11,15
* XbaI and PstI digest of :
  + LacILOV\_pSB1C3 (L3 and L4)
  + AcrIIA4, pSB1C3 (A1, A3 and A7)
  + Reporter\_pSB1C3
* Colony PCR (10ul/rxn, 14 rxns) + 1 buffer

### Week 19, day 7 (29/10)

* Minipreped full construct from overnight culture
* Transformed full construct plasmids + pdCas9 in MG1655 delta LacI
* Started overnight cultures:
  + reporter\_mCherry\_pKDL071 + pdCas9 MG1655ΔLacI in minimum media + 0.2% glucose
  + reporter\_mCherry\_sgRNA\_pKDL071 + pdCas9 MG1655ΔLacI in minimum media + 0.2% glucose
  + reporter\_mCherry\_sgRNA\_AcrIIA4\_pKDL071 + pdCas9 MG1655ΔLacI in minimum media + 0.2% glucose
* Streaked LacILOV\_mCherry\_pKDL071 MG1655ΔLacI on 4 LB+Kan plates to test LOKI
* Grew :
  + MG1655ΔLacI + YFP +ve to measure YFP
  + MG165ΔLacI repmCherry
  + pdCas9 cells

### Week 20, day 1 (30/10)

* Restreaked 9 colonies (labeled) of MG1655ΔLacI\_sgRNA\_Acr\_reportermCherry in pKDL071 backbone onto LB + CAM + KAN plates

#### Contents

#### Related Pages

* [Experiments](http://2017.igem.org/Team%3AToronto/Experiments)
* [Interlab](http://2017.igem.org/Team%3AToronto/InterLab)
* [Results](http://2017.igem.org/Team%3AToronto/Results)

### General Pages

* [Home](http://2017.igem.org/Team%3AToronto)
* [Description](http://2017.igem.org/Team%3AToronto/Description)
* [Team](http://2017.igem.org/Team%3AToronto/Team)
* [Attributions](http://2017.igem.org/Team%3AToronto/Attributions)
* [Collaborations](http://2017.igem.org/Team%3AToronto/Collaborations)
* [Safety](http://2017.igem.org/Team%3AToronto/Safety)

### Wet Lab

* [Design](http://2017.igem.org/Team%3AToronto/Design)
* [Parts](http://2017.igem.org/Team%3AToronto/Parts)
* [Hardware](http://2017.igem.org/Team%3AToronto/Hardware)
* [Experiments](http://2017.igem.org/Team%3AToronto/Experiments)
* [Notebook](http://2017.igem.org/Team%3AToronto/Notebook)
* [InterLab](http://2017.igem.org/Team%3AToronto/InterLab)
* [Results](http://2017.igem.org/Team%3AToronto/Results)
* [Demonstrate](http://2017.igem.org/Team%3AToronto/Demonstrate)

### Dry Lab

* [ODE](http://2017.igem.org/Team%3AToronto/ODE)
* [Analysis](http://2017.igem.org/Team%3AToronto/Analysis)
* [Protein Modelling](http://2017.igem.org/Team%3AToronto/Protein-Modelling)
* [Model](http://2017.igem.org/Team%3AToronto/Model)

### Human Practices

* [Interviews](http://2017.igem.org/Team%3AToronto/Interviews)
* [Genetic Code Workshop](http://2017.igem.org/Team%3AToronto/Genetic_Code_Workshop)
* [Podcast](http://2017.igem.org/Team%3AToronto/Podcast)
* [Icon-a-thon](http://2017.igem.org/Team%3AToronto/Icon-a-thon)
* [Guide](http://2017.igem.org/Team%3AToronto/Guide)
* [HP/Silver](http://2017.igem.org/Team%3AToronto/HP/Silver)
* [HP/Gold Integrated](http://2017.igem.org/Team%3AToronto/HP/Gold_Integrated)
* [Engagement](http://2017.igem.org/Team%3AToronto/Engagement)

---

iGEM Toronto

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