iGEM 2018 — Team UConn

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

# Team:UConn/Notebook

[![](https://static.igem.org/mediawiki/2018/b/bf/T--UConn--Logo.svg)](https://2018.igem.org/Team%3AUConn)

* [![](https://static.igem.org/mediawiki/2018/b/bf/T--UConn--Logo.svg)](https://2018.igem.org/Team%3AUConn)[Home](https://2018.igem.org/Team%3AUConn)
* [![](https://static.igem.org/mediawiki/2018/9/95/T--UConn--ExperimentIcon.svg)](https://2018.igem.org/Team%3AUConn/Project)[Project](https://2018.igem.org/Team%3AUConn/Project)
  + [Background](https://2018.igem.org/Team%3AUConn/Project#background)
  + [Description](https://2018.igem.org/Team%3AUConn/Project#description)
  + [Background](https://2018.igem.org/Team%3AUConn/Project#background)
  + [Description](https://2018.igem.org/Team%3AUConn/Project#description)
* [![](https://static.igem.org/mediawiki/2018/f/f1/T--UConn--NotebookIcon.svg)](https://2018.igem.org/Team%3AUConn/Notebook)[Notebook](https://2018.igem.org/Team%3AUConn/Notebook)
  + [Interlab](https://2018.igem.org/Team%3AUConn/Notebook#interlab)
  + [Lab Notebook](https://2018.igem.org/Team%3AUConn/Notebook#labnotebook)
  + [Protocols](https://2018.igem.org/Team%3AUConn/Notebook#protocols)
  + [Interlab](https://2018.igem.org/Team%3AUConn/Notebook#interlab)
  + [Lab Notebook](https://2018.igem.org/Team%3AUConn/Notebook#labnotebook)
  + [Protocols](https://2018.igem.org/Team%3AUConn/Notebook#protocols)
* [![](https://static.igem.org/mediawiki/2018/2/2f/T--UConn--PartsIcon.svg)](https://2018.igem.org/Team%3AUConn/Parts)[Parts](https://2018.igem.org/Team%3AUConn/Parts)
  + [List of Parts](https://2018.igem.org/Team%3AUConn/Parts#listofparts)
  + [List of Parts](https://2018.igem.org/Team%3AUConn/Parts#listofparts)
* [![](https://static.igem.org/mediawiki/2018/3/3e/T--UConn--HumanIcon.svg)](https://2018.igem.org/Team%3AUConn/HumanPractices)[Human Practices](https://2018.igem.org/Team%3AUConn/HumanPractices)
  + [Community Outreach](https://2018.igem.org/Team%3AUConn/HumanPractices#communityoutreach)
  + [Collaborations](https://2018.igem.org/Team%3AUConn/HumanPractices#collaborations)
  + [Community Outreach](https://2018.igem.org/Team%3AUConn/HumanPractices#communityoutreach)
  + [Collaborations](https://2018.igem.org/Team%3AUConn/HumanPractices#collaborations)
* [![](https://static.igem.org/mediawiki/2018/0/02/T--UConn--SafetyIcon.svg)](https://2018.igem.org/Team%3AUConn/Safety)[Safety](https://2018.igem.org/Team%3AUConn/Safety)
* [![](https://static.igem.org/mediawiki/2018/1/1f/T--UConn--TeamIcon.svg)](https://2018.igem.org/Team%3AUConn/Team)[Team](https://2018.igem.org/Team%3AUConn/Team)
  + [Attributions](https://2018.igem.org/Team%3AUConn/Team#attributions)
  + [Attributions](https://2018.igem.org/Team%3AUConn/Team#attributions)

# Notebook

![](https://static.igem.org/mediawiki/2018/f/f1/T--UConn--NotebookIcon.svg)

## Interlab

The purpose of this year’s interlab is to address the high level of variability in fluorescence measurements. Typically, fluorescence is measured using the optical density (OD), or the absorbance of light by a sample when measured at 600nm using a plate reader, nanodrop, or spectrophotometer. However, since this method can be inaccurate, Interlab seeks to improve the standard method of measuring fluorescence in populations of cells. Since the number of cells in a sample can vary, if the fluorescence is normalized to the cell count of the sample, then the average amount of fluorescence can be calculated, which is likely more accurate than that of a population of unknown size. Therefore, Interlab will investigate if normalizing fluorescence measurements to colony-forming units (CFUs) will yield more precise results than OD.

Each iGEM team was asked to normalize the absorbance to the number of cells in two ways: by comparing sample absorbance to absorbance of a sample of known concentration of beads similar in size to E. coli cells and by counting CFUs from each sample.

### OD

For the first method, absorbance was measured for a sample and serial dilution of silicon beads. These beads, similar in size and shape to E. coli cells, are expected to scatter light in a similar manner, providing an analogous sample of known concentration. The first part of this process was to get a reference point for OD600 to easily interchange between Abs600 and OD600, accounting for differences in volume between wells. In this measurement, four equal samples LUDOX CL-X (a 45% colloidal silica suspension) were compared to samples of ddH2O, and the OD was measured using a plate reader. By finding the mean of the replicates, correcting by subtracting the mean of the water to get just the absorbance of the silica, and dividing by the OD600 from the provided Excel sheet, the conversion factor (OD600/Abs600) was calculated to be 3.500.

A particle standard curve was also developed using a serial dilution of monodisperse silica microspheres to plot the concentration versus absorbance, thus allowing the estimation of the number of cells based off the absorbance. The serial dilution was performed four times, and the Abs600 was measured to create the following standard curves:

![](https://static.igem.org/mediawiki/2018/e/ee/T--UConn--ParticleStandardCurve.svg)

![](https://static.igem.org/mediawiki/2018/9/9a/T--UConn--ParticleStandardCurveLog.svg)

Finally, a fluorescence standard curve was created to have a point of comparison between outputs of different plate readers, getting rid of the arbitrary units assigned by different instruments. Fluorescein is used as an easier, lower-cost alternative to GFP as it has similar excitation and emission properties. Four identical serial dilutions were completed for fluorescein, and the Abs600 was measured to create the following standard curves:

![](https://static.igem.org/mediawiki/2018/4/43/T--UConn--FluoresceinStandardCurve.svg)

![](https://static.igem.org/mediawiki/2018/a/ab/T--UConn--FluoresceinStandardCurveLog.svg)

### CFU

This part of the Interlab found the volume of a 0.1 OD600 culture and the number of cells in a known volume by growing a sample and counting the number of colonies that grew. For the “Starting Sample Preparation,” the OD600 of diluted overnight cultures of positive and negative controls were taken along with that of blank media. Based on the starting sample, overnights were diluted in triplicate, and the OD600 was taken and adjusted using the blank, yielding the absorbance. To find the number of cells in a given volume, a serial dilution was performed for each sample, and the last three dilutions were plated and grown overnight. Colonies were counted, and, with the understanding that one cell grows into one colony, the number of cells per unit of volume was found. To standardize across dilutions, we used the equation:

number of colonies x final dilution factor = CFU/mL

#### Dilution 3

| Plate | Number of Colonies | CFU/mL 0.1 OD600 solution |
| --- | --- | --- |
| 1.1 | 185 | 14800000 |
| 1.2 | 28 | 2240000 |
| 1.3 | 79 | 6320000 |
| 2.1 | Over 300 | N/A |
| 2.2 | Over 300 | N/A |
| 2.3 | Over 300 | N/A |
| 3.1 | 121 | 9680000 |
| 3.2 | 261 | 20880000 |
| 3.3 | 170 | 13600000 |
| 4.1 | 94 | 7520000 |
| 4.2 | 270 | 21600000 |
| 4.3 | Over 300 | N/A |

#### Dilution 4

| Plate | Number of Colonies | CFU/mL 0.1 OD600 solution |
| --- | --- | --- |
| 1.1 | 18 | 14400000 |
| 1.2 | 7 | 5600000 |
| 1.3 | 6 | 4800000 |
| 2.1 | 65 | 52000000 |
| 2.2 | 105 | 84000000 |
| 2.3 | 76 | 60800000 |
| 3.1 | 42 | 33600000 |
| 3.2 | 26 | 20800000 |
| 3.3 | 12 | 9600000 |
| 4.1 | 31 | 24800000 |
| 4.2 | 24 | 19200000 |
| 4.3 | 33 | 26400000 |

#### Dilution 5

| Plate | Number of Colonies | CFU/mL 0.1 OD600 solution |
| --- | --- | --- |
| 1.1 | 1 | 8000000 |
| 1.2 | 0 | 0 |
| 1.3 | 1 | 8000000 |
| 2.1 | 1 | 8000000 |
| 2.2 | 2 | 16000000 |
| 2.3 | 8 | 64000000 |
| 3.1 | 1 | 8000000 |
| 3.2 | 3 | 24000000 |
| 3.3 | 1 | 8000000 |
| 4.1 | 0 | 0 |
| 4.2 | 5 | 40000000 |
| 4.3 | 3 | 24000000 |

## Lab Notebook

Click on each section to view the week's notebook.

### May

Week 2: 5/21/2018

#### May 22, 2018

#### Competent Cell Prep

#### Liz, Peter, Ryan, Shannon, Sricharan

We’re preparing competent cells (E. coli, XL1 Blue) for the summer.

Craig and Sricharan tried to do this in December, but it didn’t work since the shaker was at 200rpm instead of at 250rpm.

Today: prepping stock solutions, streaking plate

#### TfbII (50 mL)

| Reagent | Concentration | Amount needed | Amount measured |
| --- | --- | --- | --- |
| MOPS | 10mM | 0.104g | 0.1040g |
| CaCl2 | 75mM | 0.417g | 0.4160g |
| RbCl2 | 10mM | 0.061g | 0.0616g |
| Glycerol | 15% (v/v) | 7.5mL | 7.5mL |

* Measured amounts of reagents in flask (next time, use beaker)
* Added most of water (don’t top off, so you can adjust pH)
* pH (w/stirring if possible) to 6.5, using 1M KOH dropwise - got to 6.52… (make sure to standardize with pH 7 and 4 buffers)
* Topped off in graduated cylinder
* 0.45µm filter sterilized
* No need to autoclave this solution
* Stored in Tigger (4°C)

#### TfbI (200 mL)

| Reagent | Concentration | Amount needed | Amount measured |
| --- | --- | --- | --- |
| Potassium Acetate | 30mM | 0.588g | 0.589g |
| MnCl2 | 50mM | 1.98g | 1.99g |
| CaCl2 | 10mM | 0.222g | 0.224g |
| RbCl2 | 10mM | 0.242g | 0.242g |
| Glycerol | 15% (v/v) | 30mL | 30mL |

* Measured amounts of reagents in flask (next time, use beaker)
* Added most of water (don’t top off, so you can adjust pH)
* pH (w/stirring if possible) to 5.8, using .2M acetic acid dropwise - got to 5.82… (make sure to standardize with pH 7 and 4 buffers)
* Topped off in graduated cylinder
* 0.45µm filter sterilized
* No need to autoclave this solution
* Stored in Tigger (4°C)

#### ᴪa (500 mL)

| Reagent | Concentration | Amount needed |
| --- | --- | --- |
| Bacto yeast extract | 5g/L | 2.5g |
| Bacco tryptone | 20g/L | 10g |
| MgSO4 | 5g/L | 2.5g |
| KOH | 1M | Added dropwise until solution reached pH 7.6 |

* Dry substances mixed in bottle (would be easier to use a beaker)
* Water topped off to almost 500mL
* Pour into flask to adjust pH using spinner

+ Standardized pH meter using 7 and 10 buffers, pain to do

* Pour back into graduated cylinder to top off to 500 mL
* Use sterile filter
* Aliquot 100mL into two 300mL flasks with caps, remaining 300mL in bottle
* Autoclave at liquid 30, 45 minutes dry
* Removed from autoclave, allowed to cool, and transferred to 4℃ fridge (Tigger)

Also streaked Ψa plate

* Sterile environment (with portable burner nearby)
* Stabbed competent XL1 Blue cells (frozen stock) with a pipet tip
* Gently spread frozen piece of cells onto plate with tip (be careful not to puncture agar)
* Spread cells on plate with Pasteur spinner (not the plating beads, @Craig)
* Put plate into incubator at 39°C (not 37°C, ugh)

#### May 22, 2018

#### Interlab Calibration

#### Liz, Peter, Ryan, Shannon, Sricharan

#### Calibration 1, OD600 Reference Point - LUDOX protocol:

* 100µL LUDOX CL-X in wells A1, B1, C1, D1
* 100µL ddH2O in wells A2, B2, C2, D2

#### Absorbances

|  | 1 | 2 |
| --- | --- | --- |
| A | 0.045 | 0.024 |
| B | 0.041 | 0.025 |
| C | 0.042 | 0.025 |
| D | 0.042 | 0.025 |

* Wavelength: 600nm
* Optics: top
* Temperature: 25.9°C

#### Calibration 2

Stock Solution (Vortex silica beads for 30 sec. before adding):

| Reagent | Amount |
| --- | --- |
| Silica beads | 96 µL |
| ddH2O | 904 µL |

* Add 100 µL ddH2O to wells A1, B1, C1, D1, …, A12, B12, C12, D12
* Add 200 µL stock solution to A1, B1, C1, and D1 (vortex 10s before adding)
* Transfer 100 µL from A1 to A2, pipet to mix, transfer 100 µL from A2 to A3, pipet to mix, etc. until A11. Repeat for B, C, and D.

+ Cells A1, B1, C1, and D1 should be pure stock
+ Cells A12, B12, C12, and D12 should be pure ddH2O

#### Absorbance

|  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A | 1.075 | 0.545 | 0.343 | 0.152 | 0.087 | 0.055 | 0.051 | 0.032 | 0.028 | 0.031 | 0.028 | 0.025 |
| B | 1.199 | 0.666 | 0.306 | 0.161 | 0.086 | 0.057 | 0.04 | 0.033 | 0.028 | 0.026 | 0.026 | 0.025 |
| C | 1.191 | 0.651 | 0.346 | 0.176 | 0.091 | 0.03 | 0.051 | 0.043 | 0.031 | 0.028 | 0.027 | 0.025 |
| D | 1.325 | 0.587 | 0.299 | 0.168 | 0.088 | 0.057 | 0.039 | 0.032 | 0.027 | 0.026 | 0.025 | 0.025 |

* Wavelength: 600nm
* Optics: top
* Temperature: 26°C

#### Calibration 3: Fluorescence Standard Curve

#### Fluorescein stock solution:

| Reagent | Amount |
| --- | --- |
| Fluorescein | Provided in Kit (5 tubes) |
| 1x PBS pH 7.4-7.6 | 10 mL |

* Spun down fluorescein in tube so the pellet was at the bottom
* Made ~ 40 mL 1x PBS in falcon tube (originally used 10 x accidentally for first 3 attempts/ tubes)
* Used 1 mL of 1x PBS to resuspend fluorescein in kit tube > makes 10x fluoresceiµLtocol calls for pH of 7.4-7.5. Problem? > email iGEM
* Diluted 10 fluorescein stock with 900 µL of 1x PBS and 100 µL of 10x fluorescein stock to make 1x fluorescein solution (10 M)

Serial Dilutions:

* Used clear flat-bottom 96 well plates
* Put 200 µL of 1x fluorescein into column 1 rows A, B, C, D
* Put 100 µL of 1x PBS in columns 2-12 for rows A, B, C, D

+ Column 12 for all rows (A, B, C, D) is control and only contains 100 µL 1x PBS

* Transferred 100 µL of fluorescein stock from A1 to A2
* Mixed A2 > pipetting up and down 3 times
* Transferred 100 µL from A2 to A3 and mixed A3
* Repeated until after mixing A11 > then 100 µL removed from A11 and put into liquid waste
* Process described for row A repeated for all other rows (B, C, D)

#### Fluorescence

|  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A | 497 | 266 | 144 | 75 | 41 | 23 | 14 | 9 | 8 | 6 | 6 | 5 |
| B | 532 | 302 | 153 | 81 | 44 | 24 | 25 | 10 | 7 | 7 | 5 | 5 |
| C | 532 | 295 | 154 | 81 | 45 | 25 | 15 | 10 | 7 | 7 | 6 | 5 |
| D | 547 | 297 | 159 | 83 | 45 | 25 | 16 | 11 | 8 | 7 | 6 | 5 |

* Excitation: 485 nm /20 nm
* Emission: 528 nm /20 nm
* Optics: top
* Temperature: 25.8°C (room temp)

#### May 23, 2018

#### Competent Cell Prep

#### Sricharan

* Took the Ψa plate out around 9am

+ It was densely packed with colonies (tan color), but some isolated ones appeared towards one edge of the plate

* Kept plate at 4°C cold room during day
* Overnight cultures prepared near portable burner (to the delight of Toni watching me try and refill the burner)

+ Picked 3 colonies with P200 tip
+ Each colony into a separate round bottomed tube with loose caps, filled with Ψb media
+ Tubes placed in shaker at 37°C and 250rpm around 4:30pm

#### May 24, 2018

#### Competent Cell Prep

#### Liz, Peter, Shannon, Sricharan

* Took the overnight cultures out around 9:15am

+ They all are cloudy compared to the original media, so decent growth it seems
+ Will start subcultures around noon
+ Cultured in shaker for an hour and 22 minutes and we over grew both cultures by a little
+ Prepared another overnight culture

#### May 24, 2018

#### Competent Cell Prep

#### Liz, Peter, Shannon, Sricharan

* Took the overnight cultures out around 9:00am

+ Growth in both cultures
+ Pre-warmed Ψb media before subculturing
+ Subcultured in Ψb media for 45 minutes (Flask A around 0.36 OD600; Flask B around 0.38 OD600)
+ Checked again at 55, then 58 minutes and each was around 0.48 OD600
+ Spun cells in pots in MSE 18 to isolate pellet and removed supernatant
+ Resuspended in TfbI and spun again and removed supernatant
+ Resuspended in TfbII, pooled A and B, and chilled on ice for 15 minutes
+ Aliquoted 200 µL of cell solution into autoclaved microcentrifuge tubes and snap-froze using liquid nitrogen
+ Put competent cells into -80 fridge on second floor

Week 3: 5/28/2018

#### May 29, 2018

#### Interlab Transformation

#### Shannon, Liz

#### Plasmid Re-suspension

Add 10 µL dH2O to wells 2D, 2B, 2F, 2H, 2J, 2L, 2N, and 2P on plate 7 from the interlab kit and pipette to resuspend the DNA in each well. Let sit for a few minutes. Transfer to 1.5 mL tubes.

* Note: 2D and 2B were accidently added to the same 1.5 mL tube, so these two were re-done using 21B and 21D from kit plate 7 from the 2017 interlab kit, which have the same corresponding DNA.

#### May 29, 2018

#### Chloramphenicol Plate Preparation

#### Ryan, Peter, Sricharan

#### Preparing Chloramphenicol Stock

* Made 10 mL stock of 35 mg/mL chloramphenicol in EtOH (this is 1000x as specified by iGEM)
* Took from Rachel’s floor (we bought it though), 0.3496g made up to 10mL volume

#### Preparing Chloramphenicol LB Plates (24)

* Collected ingredients for LB media and added to beaker
* Q/s’d with dH20 to 400
* Mixed in beaker for 20 min and Q/s’d to about 450 mL with spray bottle to get solid particles off of the sides of the beaker and into the solution
* Tried pouring into grad cylinder to finish Q/s but still goupy
* Poured ~ 50 mL of dH20 into grad. Cylinder to capture the goop stuck to the bottom after pouring as much as we could back into the beaker. Poured this 50 mL back into the beaker as well
* Turned up temp and stir speed on hot plate and stirred for another 25 minute in beaker
* Poured into bottle and stirred (stopping frequently to shake bottle) for ~ 30 minutes
* Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
* Added 0.5 mL of chloramphenicol (1000x, 35 mg/mL) to solution
* Plated LB + chloramphenicol onto 24 plates and put in cold room (-4 degrees C)

+ Rushed putting the plates away, so there might be some condensation on plates

#### May 30, 2018

#### Interlab Transformation

#### Shannon, Liz, Ryan

* Thawed DH5-alpha competent cells on ice
* Pipet 50 μL competent cells into empty 1.5 mL tubes
* Pipet 1 μL resuspended DNA (from yesterday, see page above) into individual tubes (according to well number) with competent cells

+ DNA from tubes: 21B, 21D, 2F, 2H, 2J, 2L, 2N, and 2P in box 1 from freezer

* Pipet 1 μL control DNA (10pg/μL) into control tube with competent cells
* Incubate tubes on ice for 30 min (0:00-30:23)
* Heat shock at 42℃ for 45s (30:23-31:08)
* Pipet 950 μL SOC media at room temperature into each tube
* Incubate at 37℃ for 1 hr at 300 rpm
* Plate 100 μL transformation (lower concentration)

+ Condensation got on agar, tried tapping plates to remove water but some remained
+ Spread water with spinners before putting transformation on plates
+ Unsure if plates N and P got switched

* Spin down cells at 6800g for 3 min and discard 800 μL supernatant
* Resuspend pellet in the remaining 100 μL and plate (higher concentration)

+ Amount of liquid remaining for resuspension varied from tube to tube

* Incubate at 37℃ for 14-18 hours

+ Put into incubator ~6pm
+ Incubator is kept at 39℃
+ Checked after 15.5 hours, barely any growth

#### May 30, 2018

#### Competent Cell Transformation (Scott-Simanis, Test Kit)

#### Peter, Sricharan

* 2 vials of Scott-Simanis competent cells from 5/25/18 thawed at RT (200 μL in each tube)
* After thawed, placed on ice for 10 minutes

+ At 8 minutes, aliquoted 50 μL (150 μL from each tube) into 6 separate tubes
+ At 10 minutes, added 1 μL of DNA from Competent Cell Test Kit

- Duplicates of 10, 50, 100 pg/μL BBa\_J04450, RFP construct

* Chilled aliquots on ice 30 min
* Heat shocked aliquots at 42 °C for 90 s
* Incubated on ice for 90 s
* Added 200μL Ψb media to each aliquot
* Incubated at 37 °C, 300rpm for 1 hour
* Plated onto LB plates with chloramphenicol (35 μg/mL) with spinners, under portable burner
* Incubated at 39 °C incubator downstairs starting at 6pm

#### May 31, 2018

#### LB + Chloramphenicol Media Preparation (w/o agar)

#### Peter, Sricharan, Liz, Shannon, Ryan

* Used for inoculation for O.C. & dilutions + control for transformation fluorescence readings

#### Preparing Chloramphenicol LB media (500 mL)

* Collected and measured out ingredients for LB media and added to bottle
* Q/s’d with dH20 to 400 mL and shook bottle to dissolve ingredients
* Stirred in beaker for 15 min
* Poured into grad cylinder to finish Q/s-ing to 500 mL
* Poured back into bottle and stirred for ~ a minute
* Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
* Added 0.5 mL of chloramphenicol (1000x, 35 mg/mL) to solution

#### May 31, 2018

#### Interlab Overnight Cultures

#### Peter, Sricharan, Liz, Shannon, Ryan

* Condensation on plates = osmotic stress on cells…. Not a lot of growth

+ Checked plates at 9am - not a lot of growth
+ Took plates out at 12pm, at least a few colonies on most plates with concentrated transformations, except Wells 21D (2017), 21B (2017)

* Made 2 OCs for each well with most well defined/isolated colonies from each well

+ Put into “new” old shaker in classroom (cleaned in morning with 70% ethanol)
+ 220rpm, 37 °C

#### June 1, 2018

#### Interlab Fluorescence Measurements

#### Peter, Sricharan, Liz, Shannon, Ryan, Toni

* Grabbed OCs @ 9:15am from shaker
* Diluted 0.5mL of OCs 10x, put foil on diluted tubes
* Took Abs600 readings on plate reader (100μL samples)

| Samples on Plate | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| G | 21D (‘17) | 2F-1 | 2H-1 | 2J-1 | 2L-1 | 2N-1 | 2P-1 |
| H |  | 2F-2 | 2H-2 | 2J-2 | 2L-2 | 2N-2 | 2P-2 |

Since Abs=ebc, and we need to dilute to a target Abs600 of 0.02, and final volume of 12mL, Valiquot=0.24/Abs600mL. Also, Vmedia = 12mL-Valiquot

Need to put in values from data on computer

| Abs600 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| G |  |  |  |  |  |  |  |
| H |  |  |  |  |  |  |  |

| Valiquot(mL) | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| G | 7.7 | 4.6 | 2.3 | 2.3 | 1.7 | 2.8 | 2.1 |
| H |  | 1.8 | 2.2 | 2.3 | 2.0 | 3.1 | 2.4 |

| Valiquot(mL) | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| G | 4.3 | 7.4 | 9.7 | 9.7 | 10.3 | 9.2 | 9.9 |
| H |  | 10.2 | 9.8 | 9.7 | 10.0 | 8.9 | 9.6 |

* @12pm Put diluted & absorbance-corrected cultures back in shaker

+ 220rpm, 37 °C for 6 hours
+ Used 0.5mL aliquot of these cultures for t = 0h readings

- Placed on 96-well plate as in protocol
- Note we don’t have negative or positive controls… Need to redo next week

#### June 1, 2018

#### LB + Chloramphenicol Plates Preparation

#### Peter, Ryan

* To be used for competent SS cell test & positive and negative controls for Interlab

#### Preparing Chloramphenicol LB plates (24)

* Collected and measured out ingredients for LB media and added to bottle
* Q/s’d with dH20 to 400 and shook bottle to dissolve ingredients
* Stirred in beaker for 30 min
* Poured into grad cylinder to finish Q/s to 500 mL
* Poured back into bottle and stirred for ~ a minute
* Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
* Added 0.5 mL of chloramphenicol (1000x, 35 mg/mL) to solution
* Let plates cool for an hour on lab bench
* Put them in the hood with the plates half open w/ agar side on the bottom to get rid of condensation (should have done this immediately to dry them)

#### June 1, 2018

#### Title

#### Names

#### June 1, 2018

#### Title

#### Sricharan, Liz

|  | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| A (absorbance) |  |  |  |  |  |  |  |
| A (fluorescence) |  |  |  |  |  |  |  |
| B (absorbance) |  |  |  |  |  |  |  |
| B (fluorescence) |  |  |  |  |  |  |  |
| C (absorbance) |  |  |  |  |  |  |  |
| C (fluorescence) |  |  |  |  |  |  |  |
| D (absorbance) |  |  |  |  |  |  |  |
| D (fluorescence) |  |  |  |  |  |  |  |
| E (absorbance) |  |  |  |  |  |  |  |
| E |  |  |  |  |  |  |  |
| F(absorbance) |  |  |  |  |  |  |  |
| F (fluorescence) |  |  |  |  |  |  |  |
| G |  |  |  |  |  |  |  |
| G (fluorescence) |  |  |  |  |  |  |  |
| H |  |  |  |  |  |  |  |
| H (fluorescence) |  |  |  |  |  |  |  |

### June

Week 4: 6/4/2018

#### June 4, 2018

#### LB + Chloramphenicol Plate Prep

#### Ryan

See protocol from 6/1/18 for LB + Chloramphenicol Plate Prep

#### Notes:

* Made 23 plates (whoops)
* Burned serological pipette...
* … and part of a plate

#### June 4, 2018

#### Interlab Transformation of 21B and 21D

#### Liz

See protocol from 5/29/18 transformation

#### Deviations/Notes

* Plates were taken out of fridge and warmed to room temperature

+ No condensation on plates because they were allowed to mostly cool on the bench and then transferred to the hood with the lids partially off in the process of making the plates

* Only doing DNA from wells 21B and 21D (from 2017 kit plate 7)

+ Colonies didn’t grow on plates last time (5/29)
+ For each sample, made 1 dilute plate and 2 concentrated plates with the hope of actually getting colonies this time

* Forgot to prechill tubes before putting in competent cells and resuspended DNA
* Centrifuged at 12:45 during 30 minute incubation on ice
* Only ended up making 1 concentrated plate each for 21B and 21D
* Put in incubator at 4:20pm

#### Times

0:00-30:00 - incubate on ice for 30 minutes
30:00-30:45 - heat shock at 42℃ for 45 s
30:45-35:45 - incubate for ice for 5 min

#### June 4, 2018

#### SS Competent Cell transformation Test (Day 1)

#### Peter

See protocol “Competent Cell Transformation (Scott-Simanis, Test Kit)” from 5/29/18 transformation test

#### Deviations/Notes

* Plates were taken out of fridge and warmed to room temperature

+ No condensation on plates because they were allowed to mostly cool on the bench and then transferred to the hood with the lids partially off in the process of making the plates

* Ψb taken out of fridge to warm to room temperature
* Only made samples from tube 1 for 10 pg/ μL
* Took two tubes of competent cells from -80 degrees celsius
* Took ~ 7 minutes to add DNA (too long for cells to be warm during this step)
* Centrifuged at 12:45 during 30 minute incubation on ice (should have done this before)
* Re-plated 100 pg/ μL plate 1 (labeled new plate “1b”) because burned cells with pasteur pipette for plate 1a
* Potentially killed cells for 10 pg/ μL on plate 1b
* Put in incubator at ~ 4:20pm

#### June 5, 2018

#### SS Competent Cell transformation Test (Day 2)

#### Peter

* Checked cells @ 9:15 am. Some growth on 100 pg/ μL plate 1a and no visible growth on others
* Checked again @ 10:15 am and same growth situation

#### June 5, 2018

#### Checking Distribution Kit Plasmids

#### Sricharan, Liz, Peter, Shannon

Needed to run a gel for 2017 kit 7 wells 21B and 21D to check to see if the plasmid was there because the transformation yielded no colonies again

* Used 2017 kit 7 wells 21B and 21D, 2017 kit 3 well 8P, and 2017 kit 2 well 6F
* Originally thought we had to do a digest of plasmids before running gel so measured concentration of DNA in samples using nanodrop (measure 1μL)

+ Blank with water first

* Results

+ Kit 7 well 21B: 134.4 ng/μL
+ Kit 7 well 21D: 109.0 ng/μL
+ Kit 3 well 8P: 114.7 ng/μL
+ Kit 2 well 6F: 131.7 ng/μL

* Made 2 small agarose gels (1%)
* 25mL 1x TAE, 0.25g agarose warmed in microwave
* 2.5 µL SYBR Safe added

All wells in gel from 2017 Kits

| Well | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Item | 2-Log Ladder | Kit 7, Well 21D | Kit 7, Well 21B | Kit 2, Well 6F | Kit 3, Well 8D | 2-Log Ladder | Test Kit 100pg/µL | Test Kit 50pg/µL | Test Kit 10pg/µL | Linearized pSB1C3 |

* Ran 52 minutes @ 50V on mini gel rig

+ No condensation (new TAE)

* Image on transilluminator hard to see
* Similar gel image - only clear lanes were ladder and linearized backbone

+ Diffuse spots for plasmids in wells 2-5, we should be fine

![](https://static.igem.org/mediawiki/2018/4/49/T--UConn--Gels-June-5.jpg)

#### June 6, 2018

#### Redoing Interlab and Scott-Siamanis Transformations

#### Sricharan, Liz, Peter, Shannon

* Redoing transformations from earlier this week (since they didn’t work)

+ Using water bath at 42°C instead of heat block for shocking cells
+ Keeping all tubes prechilled/on ice
+ Prewarming SOC media
+ Using incubator to shake cells <300rpm instead of thermomixer

- This incubator thing is probably overkill, we’ll go back to thermomixer later

* Got cells (2 tubes SS, 1 tube DH5-alpha) from -80°C

+ SS thawed off ice ~3min
+ DH5-alpha thawed on ice ~5min

* Immediately portioned 50µL aliquots of cells into several 1.5mL tubes

+ Scott-Simanis cells - samples

- Competent Cell Test Kit

* 100pg/µL
* 50pg/µL
* 10pg/µL

- Well 2F, Plate 7, 2018 Kit

+ DH5-alpha cells - samples

- Competent Cell Test Kit, 10pg/µL
- pUC19 Control
- Plate 7, 2018 Kit

* Well 4B (0.2 µL)
* Well 4B (1 µL)
* Well 4D (0.2 µL)
* Well 4D (1 µL)
* Well 2F (1 µL)

* For SS

+ Incubate 10 minute on ice, add DNA (1 µL), incubate on ice 30 min

* For DH5-alpha

+ Add DNA immediately (1 µL or 0.2 µL if indicated on sample name), incubate on ice 30 min

* Heat shock @ 42°C in water bath, 45 seconds (DH5-alpha) or 90s (SS)

+ Accidentally dropped DH5-alpha samples into water, held in water after

* Incubate on ice 5 min (DH5-alpha) or 90s (SS)
* Add (prewarmed) 950µL SOC media to each DH5-alpha tube, and 200µL Ψb into each SS tube
* Incubate @ 37°C in shaker in classroom ~250rpm
* Plated 100µL on LB+Cam plates

+ For DH5-alpha, followed iGEM protocol to concentrate solutions
+ Only plated for DH5-alpha controls after concentrated

* Incubated at 39°C downstairs ~4:30pm

#### June 7, 2018

#### Interlab Positive and Negative Controls

#### Sricharan, Liz, Peter, Shannon

* Took plates from yesterday out ~9:30am

+ Good growth on DH5-alpha plates (Interlab)

- Except pUC19 control, which was aliquoted after comp cell tube was dropped into ethanol

+ No growth on most Scott-Simanis plates (not even Well 2F)

- We know cells are not competent now - plates and protocol are fine

* Will do overnight cultures for Interlab cells and for new batch of competent cells this afternoon

#### June 7, 2018

#### Psi Solutions

#### Liz, Peter

#### Psi A

| Substance | Concentration | Protocol Amount | Actual Amount |
| --- | --- | --- | --- |
| Bacto yeast extract | 5 g/L | 0.625 g | 0.6357 g |
| Bacco tryptone | 20 g/L | 2.5 g | 2.5090 g |
| MgSO4 | 5 g/L | 0.625 g | 0.6261 g |
| Bacto agar | 14 g/L | 1.75 g | 1.7512 g |

* Fill with dH2O to just under 250 mL

+ This was our mistake!! We used enough chemicals to make 125 mL but put in too much water, making the solution useless

* Add 1M KOH dropwise until pH reached 7.63
* Mixed in beaker using stir plate

+ Didn’t mix fast enough so agar clumped on sides and at bottom
+ Shook a lot in a bottle and poured back and forth to get out as much agar as possible

* Autoclaved on liquid setting
* Was about to pour plates but realized that we can’t use because too diluted

| Substance | Concentration | Protocol Amount | Actual Amount |
| --- | --- | --- | --- |
| Bacto yeast extract | 5 g/L | 2.5 g | 2.50 g |
| Bacco tryptone | 20 g/L | 10 g | 10.0002 g |
| MgSO4 | 5 g/L | 2.5 g | 2.5026 g |

* Fill with dH2O to just under 500 mL
* Mixed in beaker using stir plate
* Adjusted pH to 7.60 by adding 1M KOH dropwise
* Topped off to 500 mL
* Put through a sterile filter and aliquoted 100mL each into 3 flasks and the rest into a bottle
* Autoclaved on liquid setting
* Store in fridge (4°C)

#### June 7, 2018

#### Chloramphenicol plate prep

#### Shannon and Toni

* See protocol from 6/1 - made 1L
* Mixture was goopy when transfering from bottle to graduated cylinder to Q/s to 500mL, so had to put back in bottle and shake/continue mixing on hot plate. Rinsed graduated cylinder with dH2O to get goop back into bottle (not enough to go past 500mL).

Week 5: 6/11/2018

#### June 11, 2018

#### Interlab CFU Day 1

#### Sricharan, Liz, Shannon

* Circled three colonies (labeled a, b, c) from plates 4B 1𝜇L and 4D 1𝜇L (made 6/6/18)
* Pipetted 5 mL LB broth into OC tubes

+ Six tubes: 4Ba, 4Bb, 4Bc, 4Da, 4Db, 4Dc
+ 5mL in each tube

* Used 200 1𝜇L pipet tip to take colony a from plate 4B and drop into tube 4Ba
* Repeat with other circeled colonies into corresponding tubes
* Put into shaker at about 37℃ and 220 rpm overnight

+ In: 3:30 pm

#### June 11, 2018

#### SS Competent Cells Trasformation Test Day 1

#### Peter, Deivydas, Ryan

See protocol “Competent Cell Transformation (Scott-Simanis, Test Kit)” from 5/29/18 transformation test

#### Deviations/Notes

* Plates were taken out of fridge and warmed to room temperature

+ No condensation on plates because they were allowed to mostly cool on the bench and then transferred to the hood with the lids partially off in the process of making the plates

* Ψb taken out of fridge to warm to room temperature
* Made one sample/ plate for each concentration (10 pg/ μL, 50 pg/ μL, 100 pg/ μL) adn well 2F from interlan study as a control
* Took one tube of competent cells from -80 degrees celsius
* Let ice thaw for ~ one minute
* Centrifuged during 30 min. To make sure is mixed (should not have done this- cells are fragile)

#### June 12, 2018

#### SS Competent Cells Trasformation Test Day 2

#### Peter, Deivydas, Ryan

* Little to no growth - ask Rachel what we are doing wrong
* Three colonies on 100 pg/ μL

#### June 12, 2018

#### Interlab CFU Day 2

#### Sricharan, Liz

* Took overnight cultures of positive (4B) and negative (4D) controls
* Following CFU protocol

+ C1V1 = C2V2→ V1 = C2V2/C1 = 0.1(1000mL)/8(1:8OD600-blank OD600)

#### Wells

|  | 1 | 2 |
| --- | --- | --- |
| F | 1 (positive control) | 2 (positive control) |
| G | 3 (negative control) | 4 (negative control) |
| H | Blank media | Blank media |

#### OD600

|  | 1 | 2 |
| --- | --- | --- |
| F | 0.237 | 0.122 |
| G | 0.250 | 0.208 |
| H | 0.033 | 0.034 |

#### Culture Volume (checked OD600 with plate reader)

V1: 61μL culture, 939μL media
V2: 141μL culture, 859μL media
V3: 58μL culture, 942μL media
V4: 72μL culture, 928μL media

Shannon is making more LB+Cam media for our dilutions

#### Plate notation: A.B.C

A=culture number (1,2=positive control, 3,4=negative control)
B=sample number
C=dilution number (3, 4, and 5 are plated)

Diluted according to protocol, plated cells in dilutions 3-5

* Reused 15mL Falcon tubes for dilutions 1-3

Incubate at 39℃ downstairs overnight

* In at ~4:15

#### June 12, 2018

#### SS Competent Cells Trasformation Test Day 1

#### Peter

#### \*\*\*New Protocol\*\*\* - we were doing stuff wrong

* Retrieved 3 vials of Scott-Simanis competent cells (XL1 Blue from 5/25/18, 6/8/18, and Rachel’s cells) from -80 and brought upstairs on ice (200 μL in each tube)
* Thawed at RT for ~ one minute
* NO ALIQUOTING AS PIPETTING SHREDS FRAGILE CELLS
* Added 1μL of DNA immediately after thawed (BBa\_J04450 100 pg/μL from 2018 CC test kit)
* NO CENTRIFUGING TO MIX DNA IN
* Chilled samples on ice for 30 minutes
* Heat shocked samples WITH WATER BATH at 42 °C for 45 S
* Incubated on ice for ~ 90 s
* Added 800μL (4 times volume of samples) 2YT media to each sample
* Incubated at 37 °C ON HEAT BLOCK (NO SHAKING) for 60 min
* Plated out w/ flame two plates for each sample: 100 μL on plate 1 and 200 μL on plate 2
* Incubated overnight @ 37 °C

#### June 13, 2018

#### SS Competent Cells Trasformation Test Day 2

#### Peter

* Retrieved from the incubator @ 9:15 (~ 17 hours in the incubator)
* FINALLY IT WORKED MOSTLY!!!

#### Colony Growth:

|  | Rachel (Date?) | iGEM 5/25/18 | iGEM 6/8/18 |
| --- | --- | --- | --- |
| Plate 1 (100 μL) | ~ 15 colonies | 1 colony?? | 13 colonies |
| Plate 2 (200 μL) | 69 colonies | 3 colonies | ~42 colonies |

#### June 13, 2018

#### Counting Interlab Colonies

#### Liz, Sricharan, Shannon, Peter

#### Dilution 3

| Plate | Number of Colonies |
| --- | --- |
| 1.1 | 185 |
| 1.2 | 28 |
| 1.3 | 79 |
| 2.1 | Over 300 |
| 2.2 | Over 300 |
| 2.3 | Over 300 |
| 3.1 | 121 |
| 3.2 | 261 |
| 3.3 | 170 |
| 4.1 | 94 |
| 4.2 | 270 |
| 4.3 | Over 300 |

#### Dilution 4

| Plate | Number of Colonies |
| --- | --- |
| 1.1 | 18 |
| 1.2 | 7 |
| 1.3 | 6 |
| 2.1 | 65 |
| 2.2 | 105 |
| 2.3 | Over 76 |
| 3.1 | 42 |
| 3.2 | 26 |
| 3.3 | 12 |
| 4.1 | 31 |
| 4.2 | 24 |
| 4.3 | 33 |

#### Dilution 5

| Plate | Number of Colonies |
| --- | --- |
| 1.1 | 1 |
| 1.2 | 0 |
| 1.3 | 1 |
| 2.1 | 1 |
| 2.2 | 2 |
| 2.3 | 8 |
| 3.1 | 1 |
| 3.2 | 3 |
| 3.3 | 1 |
| 4.1 | 0 |
| 4.2 | 5 |
| 4.3 | 3 |

Week 6: 6/18/2018

#### June 18, 2018

#### Transforming Parts from Registry

#### Sricharan

Resuspended some parts from 2018 Distribution Kit to transform tomorrow

| Plate | Well | Part | Name (BBa\_) |
| --- | --- | --- | --- |
| 4 | 17D | Anderson Promoter (Strong) | J23100 |
| 4 | 19D | Anderson Promoter (Medium) | J23110 |
| 4 | 19J | Anderson Promoter (Weak) | J23114 |
| 1 | 17H | Ptac | K864400 |
| 5 | 17J | ‘tesA | K1472601 |
| 5 | 7K | TUDelft mtrCAB | K1316012 |
| 6 | 21K | ADC (Petrobrick) | K590031 |
| 6 | 7K | AAR (Petrobrick) | K590032 |

Resuspending each well w/ 10μL of dH2O, let sit a few minutes, aliquoted into 1.5mL tubes.

#### June 19, 2018

#### 2xYT Medium

#### Liz

Wanted to make 500mL for use for transformations/potentially overnight cultures (point is we don’t need a ton)

In a bottle measured:

* 450mL dH2O
* 8g tryptone (measured 8.00g)
* 5g yeast extract (measured 5.00)
* 2.5g NaCl (measured 2.50)

Shook bottle between adding each substance to dissolve

When dissolve adjusted pH to 7.0 with 5M NaOH

* Added dH2O up to ~480mL before adjusting pH
* Only had 1M NaOH
* Had to use pH 4 calibration solution because start out acidic

Top off to 500mL

Autoclave for 20 min at 15 psi (1.05kg/cm3) on liquid cycle

* Used 30 minute liquid setting

Put in cold room to cool before using for transformations

#### June 19, 2018

#### Making CAM and Amp plates

#### Peter

#### \*\*\*See protocol from 6/1/18\*\*\*

#### Notes/Deviations

* After autoclaving, split 500 mL into two flasks of 250 mL each
* Added 250 μL of chloramphenicol (35 mg/ mL stock) to one flask
* Added 250 μL of Ampicillin (35 mg/ mL stock) to other flask

#### June 19, 2018

#### SS Competent Cells Trasformation Test Day 1

#### Peter, Shannon, Charan

Measured 0.5043g ampicillin (Na salt) from Rachel’s lab to make 100mg/mL stock solution (1000x) in 50% EtOH

#### \*\*\*See protocol from 6/12 (week 5)\*\*\*

#### Notes/Deviations

* Retrieved 8 vials of second set of Scott-Simanis competent cells (XL1 Blue from 6/8/18) from -80 and brought upstairs on ice (200 μL in each tube)
* DNA added: BBa\_J23100, BBa\_J23110, BBa\_J23114, BBa\_K864400, BBa\_K1472601, BBa\_K1316012, BBa\_590031, BBa\_590032
* Heat shocked four at a time- tried to fit all 8 in at once but four hands wouldn’t fit in the water bath to hold them all so 4 were in briefly then taken out and put back on ice until the the first four were finished and this could have caused problems
* Used 2YT media (richer than Ψb)
* Did not originally add 800μL media to tube with BBa\_590031 tube and did not realize until it was on heat block for ~10 minutes and then added so this could have caused problems
* Plated out 200 μL of cells + media except for BBa\_K1472601 and BBa\_J23100 which we did two plates, one 200 μL and one 300 μL
* Trouble keeping flame going (low on butane)
* Put in 37 °C incubator at 4:20 p.m.

#### June 20, 2018

#### SS Competent Cells Trasformation Test Day 2

#### Peter

#### Notes

* Observed growth on both the 200 μL and 300 μL plate for BBa\_K1472601 and BBa\_J23100, the BBa\_J23110 plate, and the BBa\_J23114 plate
* ~ 15 colonies (give or take 5) on average per plate
* Will redo transformations of parts that were not successful (BBa\_K864400, BBa\_K1316012, BBa\_590031, BBa\_590032)

#### June 20, 2018

#### SS Competent Cells Trasformation Test Day 1 Repeat

#### Liz, Toni

Also resuspended BBa\_B0015 from 2018, Plate 3, Well 3F (Cam resistance)

Retrieved 5 vials of SS competent cells from -80°C downstairs on ice

* competent cells from 6/8/18

Let cells thaw at room temp for ~1 min

Added 1 μL DNA (K1316012, K590032, K864400, K590031, BBa\_B0015) to each tube

* Competent cell tube for K590031 was empty, had to go downstairs and get another tube so everything was done 6.5 minutes late

Chilled on ice for 30 minutes

* K590031 started ice at 6:36 on stopwatch and went till 36:40

Heat shock in 42°C water bath for 45s (tubes held in water bath)

Added 800μL 2xYT media to each tube

* Media prewarmed in warm room

Incubated at 37°C on heat block for 60 min

* Heat block temporarily dipped to 35°C then went back up to 37°C
* Flick tubes every 10 min
* K1316012, K590032, K864488, BBa\_B0015 in at 2:32, K590031 in at 2:35

Tubes removed from heat block and spun down at 6800g for 3 minutes
~800μL supernatant removed and discarded and cells resuspended in remaining media
Cells plated and put in 39°C overnight (in at ~4ish)

* Flame ran out of butane

#### June 21, 2018

#### SS Competent Cells Trasformation Test Day 1

#### Peter, Ryan

#### \*\*\*See protocol from 6/12 (week 5)\*\*\*

#### Notes/Deviations:

* Retrieved 4 vials of second set of Scott-Simanis competent cells (XL1 Blue from 6/8/18) from -80 and brought upstairs on ice (200 μL in each tube)
* DNA added: BBa\_K864400, BBa\_K1316012, BBa\_590031, BBa\_590032
* Used 2YT media (richer than Ψb)
* Plated out 200 μL of cells per plate
* Used beads in hood instead of flame w/ pasteur pipette

#### June 22, 2018

#### Making Media for iGEM Competent Cells

#### Shannon and Ryan

Made 1L SOB and 1L CCMB80 according to the iGEM protocol.

#### SOB

| Reagent | Amount (g) | Measured Amount (g) |
| --- | --- | --- |
| Yeast extract | 5 | 5.0006 |
| Tryptone | 20 | 20.0009 |
| NaCl | 0.584 | 0.5846 |
| KCl | 0.186 | 0.1886 |
| MgSO4 \* 6H2O | 4.93 | 4.9333 |

#### CCMB80

| Reagent | Amount (g) | Measured Amount (g) |
| --- | --- | --- |
| KOAc | 5mL of 1M stock (0.49g) | 0.4938 |
| CaCl2 anhyd. | 4.44 | 4.4473 |
| MnCl2 \* 4H2O | 2.0 | 2.0062 |
| MgCl2 \* 6H2O | 1.0 | 1.0081 |
| Glycerol (100%) | 5mL | 5mL |

* SOB was pH adjusted to pH 7.54
* 100mL SOB was aliquoted and 1.6078g agar was added and autoclaved, and then used to make 5 plates. The agar did not dissolve when stirred, but did dissolve in the autoclave. These plates were left in the hood for the weekend.
* At first, we tried to make only 500mL of CCMB80, but we used 1M HCl, which decreased the pH much too rapidly, bringing it to around pH 5.8 after 1 drop. So, we doubled the amount and used 0.1M HCl. The final pH was 6.35.

#### June 22, 2018

#### Glycerol Stocks for Overnight Cultures

#### Liz

Made 40% glycerol stock (25mL) in blue falcon tube

* 10mL 100% glycerol
* 15mL DI water

Store extra in freezer (-20)

Put 0.75mL 40% glycerol into 10 microcentrifuge tubes (2 tubes per overnight culture, labeled as such)

Put 750 microliters of one culture into two different microcentrifuge tubes

* Repeat for each culture, should have 2 microcentrifuge tubes per culture
* Pipet up and down in culture to mix cells before transferring

Vortexed tubes to mix

Stored in -80 where DH5alpha cells used to be in a blue box

Week 7: 6/25/2018

#### June 25, 2018

#### Zyppy miniprep

#### Liz and Peter

Centrifuge tubes labeled:

\*need to look this up\*

Added 600 microliters culture to labeled 1.5 mL microcentrifuge tubes

Added 100 microliters 7x lysis buffer (blue) and mixed by inverting 4-6x

* Turns opaque to clear blue when lysis is done

\*time sensitive!!!\* add 350 microliters neutralization buffer (yellow) to each tube

* Neutralization buffer should be kept cold
* Mixture turns yellow and forms yellow precipitate when neutralized
* Invert 2-3x after color change

Centrifuged at 16,000rpm for 3 min then again at 11,000rpm for 1 minute

* Pellets got disturbed and had to be repelleted)

Supernatant (~900 microliters) transferred into zymo-spin IIN column (don’t disturb cell pellet)

Put column into collection tube and centrifuge 15 s

Discarded flow-through and put column back into collection tube

Added 200 microliters Endo-wash buffer and centrifuged for 30s

Added 400 microliters zyppy wash buffer and centrifuged 1 min

Put column into clean 1.5mL tube and added 30mL zyppy elution buffer

* Let sit 1 minute at room temp

Centrifuged 30s to elute plasmid DNA

#### June 25, 2018

#### Primer Rehydration

#### Liz and Peter

Spun down dry tubes

Added 1xTE buffer to make 100 micromolar solutions

| primer | Amount TE added (microliters) |
| --- | --- |
| SB prep-3P-1 | 201 |
| SB prep-2Ea | 221 |
| fadD\_P1F | 201 |
| fadD\_P1R | 231 |

Vortexed

Stored in -20 degree freezer in primer box

#### June 25, 2018

#### SOB plates

#### Ryan

* SOB plates failed/ dried up because they were left in the hood over the weekend (agar was thin and had a wavy texture, we felt it was better to make new plates).
* 100mL SOB was aliquoted and 1.6 g agar was added
* The solution was heated and mixed, and then autoclaved for an hour (set at liquids 30, P03)
* The agar did not completely dissolve when stirred, but did dissolve in the autoclave.
* This was used to make 5 plates, which were of proper thickness

#### June 26, 2018

#### Making new competent JM109 cells

#### Liz and Peter

* Streaking a SOB plate w/ Rachel’s JM109 cells (SOB plates specific to Top10 cells…)
* Put in incubator at 1:45 p.m.
* Results (6/27): good growth on plate

#### June 26, 2018

#### PCR of fadD

#### Liz, Peter, and Shannon

#### Thermocycler Settings

| Cycle | Time | Temp. (C) |
| --- | --- | --- |
| Initial denaturation | 30s | 98 |
| 35 Cycles | 10s | 98 |
|  | 30s | 61 |
|  | 20s for fadD (should have been 40 s because 2kb) 50s for backbone | 72 |
| Final extension | 10 min | 72 |
| Hold | indefinitely | 4 |

#### Notes

* For 61C during the 35 cycles, the time was found using NEBTm calculator
* fadD was accidently put at 20s instead of 40s at 72C during the 35 cycles
* We aren’t sure if fadD was held at 4C because it didn’t feel cold when opened

#### Add to PCR tube (for fadD):

| Component | Amount (µL) |
| --- | --- |
| Nuclease-free water | 12.2 (20 - sum of other components) |
| Phusion buffer (HF) | 4 |
| 10mM dNTPs | 0.4 |
| 10mM forward primer | 1 |
| 10mM reverse primer | 1 |
| Template (E. coli) DNA | 0.56 |
| Phusion polymerase (add last) | 0.2 |
| DMSO | 0.6 |

#### Notes

* Made a 10mM stock solution for dNTPs (1µL of dATP, dTTP, dCTP, dGTP)
* Diluted the forward and reverse primer to 10µM 100µM(V) = 10µM(1µL)

  2x[0.1µL stock, 0.9µL H2O]
* For template DNA, needed 40ng, stock was 40ng

  40ng x 1µL/71ng = 0.56µL

Procedure repeated for backbone

* 4 backbones (PSB1A3, PSB1K3, PSB1T3, PSB1C3), put into 4 labeled PCR tubes
* For primers use SB Prep-3P-1 and SBPrep - 2Ea
* Annealing temp is 69C
* Template DNA: 40ng x 1µL/25ng = 1.6 µL = 1.6 µL backbone DNA

#### Add to each tube:

| Component | Amount (µL) |
| --- | --- |
| Nuclease free water | 11.2 |
| Backbone DNA | 1.6 |
| Phusion buffer (HF) | 4 |
| dNTPs | 0.4 |
| 10mM forward primer | 1 |
| 10mM reverse primer | 1 |
| DMSO | 0.6 |
| Phusion polymerase (add last) | 0.2 |

Note: thermocycler did not feel cold when taking out, don’t know if this actually worked

#### June 27, 2018

#### PCR Purification

#### Liz and Shannon

Used protocol from PureLink PCR Purification Kit

* Pipet 20µL PCR sample (PSB1T3, PSB1K3, PSB1C3, PSB1A3) into individual 1.5mL tubes
* Add 80µL Binding Buffer 2 (B2) to each sample (4x volume) - pipette to mix

+ B2 is used for purifying PCR fragments

* Load each solution into individual spin column collection tubes. Centrifuge at 10,000g for 1 min. Discard flow through and re-insert the column into the collection tube.
* Add 650 µL wash buffer (W1) (ethanol had already been added). Centrifuge at 10,000g for 1 min. Discard flow-through and re-insert the column into the collection tube.
* Re-centrifuge at max speed (16,100) for 3 min.
* Using a clean collection tube, add 50µL Elution Buffer (E1) to column. Let sit for 1 min, then centrifuge at max speed for 2 min.

+ The purified PCR fragments are in the collection tube.

Products were nanodropped:

* PSB1T3: 20.1ng/µL
* PSB1K3: 7.8ng/µL
* PSB1C3: 21.1ng/µL
* PSB1A3: 23.3ng/µL

#### June 27, 2018

#### JM109 Overnight Culture

#### Shannon

* SOB was not coming to room temp. Fast enough, so it was put in the warm room for about 10 min.

+ Used at about 21.1C

* Put 5mL SOB into 2 round-bottom tubes and used a non-filtered pipette tip to inoculate the tubes with 2 colonies
* Put in downstairs shaker overnight
* Note: forgot to do this in the hood!

#### June 27, 2018

#### PCR of phz from Genomic DNA (1F-1R, 2F-3R, 4F-4R, 6F-6R)

#### Peter and Liz

#### Thermocycler Settings:

| Cycle | Time | Temp. (C) |
| --- | --- | --- |
| Initial denaturation | 30s | 98 |
| 35 Cycles | 10s | 98 |
|  | 30s | 64.5 |
|  | 2F-3R: 54 s  1F-1R, 4F-4R, 6F-6R: 25s | 72 |
| Final extension | 10 min | 72 |
| Hold | Indefinitely | 4 |

Notes:

* 1F-1R: 0.5 kb
* 2F-3R: 1.8 kb
* 4F-4R: 0.9 kb
* 6F-6R: 1 kb

#### Add to PCR tubes:

| Component | Amount (µL) |
| --- | --- |
| Nuclease-free water | 11.97 (20 - sum of other components) |
| Phusion buffer (HF) | 4 |
| 10mM dNTPs | 0.4 |
| 10mM forward primer | 1 |
| 10mM reverse primer | 1 |
| Template Genomic DNA (Pseudomonas aeruginosa) | 0.83 (40 ng - stock is 48.3 ng/ µL) |
| Phusion polymerase (add last) | 0.2 |
| DMSO | 0.6 |

Note: Started machine 2 on a 50 µL reaction for ~3 seconds and then stopped it and within 10 seconds started again for a 20 µL reaction

#### June 27, 2018

#### PCR purification and gel of backbones and phz

#### Liz

Made a gel for PCR products

* 25mL 1x TAE buffer and 0.25g agarose in flask and microwaved until dissolved
* Let flask cool until it could be picked up
* Added 2.5µL SYBR Safe
* Poured into mold, put in comb, and let harden under ice bin to protect SYBR Safe from light

Purified PCR products (PureLink PCR Purification Kit)

* Pipet 20µL PCR sample (1F/1R, 2F/3R, 4F/4R, 6F/6R) into individual 1.5mL tubes
* Add 80µL Binding Buffer 2 (B2) to each sample (4x volume) - pipette to mix

+ B2 is used for purifying PCR fragments

* Load each solution into individual spin column collection tubes
* Centrifuge at 11,000g for 1 min and discard flow through
* Add 650 µL wash buffer (W1) (ethanol had already been added)
* Centrifuge at 11,000g for 1 min and discard flow-through
* Centrifuge at max speed (16,100) for 3 min.
* Add 50µL Elution Buffer (E1) to column and let sit for 1 min
* Centrifuge at max speed (16,100) for 2 min.

+ The purified PCR fragments are in the collection tube

Nanodrop (blanked with elution buffer)

* 1F/1R: 5.5 ng/µL
* 2F/3R: 11.5 ng/µL
* 4F/4R: 6.4 ng/µL
* 6F/6R: 6.3 ng/µL
* Readings were very inconsistent (can we trust the nanodrop?)

Gel electrophoresis

* For each sample mixed 4µL water, 1µL loading dye, and 1µL PCR product
* Filled rig with 1x TAE buffer then loaded

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Ladder (2-log) | pSB1A3 | pSB1C3 | pSB1K3 | pSB1T3 | 1F/1R | 2F/3R | 4F/4R | 6F/6R | Ladder (2-log) |

* Filled rig with 1x TAE buffer then loaded

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Ladder (2-log) | pSB1A3 | pSB1C3 | pSB1K3 | pSB1T3 | 1F/1R | 2F/3R | 4F/4R | 6F/6R | Ladder (2-log) |

Gel did not come out

![](https://static.igem.org/mediawiki/2018/b/bd/T--UConn--Gels-June-27.jpg)

#### June 28, 2018

#### PCR troubleshooting

#### Liz and Peter

Redid PCR protocol for fadD and phz 2F/3R but used new dNTPs

| Cycle | Time |  | Temp. (C) |  |
| --- | --- | --- | --- | --- |
|  | fadD | phz 2F/3R | fadD | phz 2F/3R |
|  | 30s | 30s | 98 | 98 |
|  | 10s | 10s | 98 | 98 |
|  | 40s | 54s | 72 | 72 |
|  | 10 min | 10 min | 72 | 72 |
|  | infinite | infinite | 4 | 4 |

Annealing temperatures from NEB annealing temperature calculator

Set to 20µL reaction

Labeled PCR tubes fadD and phz 2F/3R

| Component | Amount (µL) |  |
| --- | --- | --- |
|  | fadD | phz 2F/3R |
| Nuclease free water | 12.2 | 11.97 |
| Template DNA (40ng) | 0.56\* | 0.83\* |
| Phusion buffer (HF) | 4 | 4 |
| dNTP stock | 0.4 | 0.4 |
| 10 mM forward primer | 1 | 1 |
| 10mM reverse primer | 1 | 1 |
| DMSO | 0.6 | 0.6 |
| Phusion polymerase (add last) | 0.2 | 0.2 |

#### Calculations

* fadD: 40ng x 1µL/71ng = 0.56µL
* phz 2F/3R: 40ng x 1µL/48.3ng = 0.83µL

New dNTP stock diluted (0.2µL each dNTP and 7.2µL dH2O) because old one had been thawed/refrozen too many times

Made a gel for PCR products

* 12.5mL 1x TAE buffer and ~0.125g agarose in flask and microwaved until dissolved

+ Can’t tell if gel is 1% because scale measures to nearest 0.05g

* Let flask cool until it could be picked up
* Added 1.75µL SYBR Safe
* Poured into mold, put in comb, and let harden under ice bin to protect SYBR Safe from light

#### June 28, 2018

#### PCR troubleshooting purification/gel

#### Liz and Peter

Purified PCR products (PureLink PCR Purification Kit)

* Pipet 20µL PCR sample (fadD and phz 2F/3R) into individual 1.5mL tubes
* Add 80µL Binding Buffer 2 (B2) to each sample (4x volume) - pipette to mix

+ B2 is used for purifying PCR fragments

* Load each solution into individual spin column collection tubes
* Centrifuge at 11,000g for 1 min and discard flow through
* Add 650 µL wash buffer (W1) (ethanol had already been added)
* Centrifuge at 11,000g for 1 min and discard flow-through
* Centrifuge at max speed (16,100) for 3 min
* Add 50µL Elution Buffer (E1) to column and let sit for 1 min
* Centrifuge at max speed (16,100) for 2 min

+ The purified PCR fragments are in the collection tube

Nanodrop (blanked with elution buffer)

* fadD: 11.2 ng/µL
* phz 2F/3R: 11.3 ng/µL

Gel electrophoresis

* For each sample mixed 4µL water, 1µL loading dye, and 1µL PCR product
* Topped off rig with 1x TAE buffer then loaded
* Ran at 50V for 50 min

| 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- |
| Ladder (2-log) | empty | phz 2F/3R | empty | fadD |

![](https://static.igem.org/mediawiki/2018/b/b8/T--UConn--Gels-June-28-2.jpg)

#### June 29, 2018

#### Backbone and phz PCR

#### Liz and Peter

Parts to PCR (1 PCR tube per sample):

* phz 1F/1R (510bp)
* phz 4F/4R (946bp)
* phz 6F/6R (997bp)
* pSB1A3 (2155bp)
* pSB1C3 (2070bp)
* pSB1K3 (2204bp)
* pSB1T3 (2461bp)

#### Set thermocycler:

| Cycle | Time |  | Temp. (C) |  |
| --- | --- | --- | --- | --- |
|  | phz | backbones | phz | backbones |
|  | 30s | 30s | 98 | 98 |
|  | 10s | 10s | 98 | 98 |
|  | 30s | 30s | 64.5 | 69 |
|  | 25s | 50s | 72 | 72 |
|  | 10 min | 10 min | 72 | 72 |
|  | infinite | infinite | 4 | 4 |

#### Added to each tube:

| Component | Amount (µL) |  |
| --- | --- | --- |
|  | phz | backbones |
| Nuclease free water | 11.97 | 11.2 |
| Template DNA (40ng) | 0.83\* | 1.6\* |
| Phusion buffer (HF) | 4 | 4 |
| dNTP stock | 0.4 | 0.4 |
| 10 mM forward primer | 1 | 1 |
| 10mM reverse primer | 1 | 1 |
| DMSO | 0.6 | 0.6 |
| Phusion polymerase (add last) | 0.2 | 0.2 |

#### Calculations

* backbones: 40ng x 1µL/25ng = 0.56µL
* phz: 40ng x 1µL/48.3ng = 0.83µL

Tubes kept on ice and vortexed before putting into thermocycler

Gel electrophoresis

* For each sample mixed 4µL water, 1µL loading dye, and 1µL PCR product
* Filled rig with 1x TAE buffer then loaded

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Ladder (2-log) | phz 1F/1R | phz 4F/4R | phz 6F/6R | Ladder (2-log) | pSB1A3 | pSB1C3 | pSB1K3 | pSB1T3 | Ladder (2-log) |

Gel didn’t get imaged correctly first time so redid gel on 7/2/2018:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
 Ladder (2-log) | pSB1A3 | pSB1C3 | pSB1K3 | pSB1T3 | phz 4F/4R | phz 6F/6R | phz 1F/1R | Ladder (2-log) |

Ran at 50V for 30+20 min (time in between because didn’t know current stops when timer does)

![](https://static.igem.org/mediawiki/2018/1/10/T--UConn--Gels-June-29.jpg)

### July

Week 8: 7/2/2018

#### July 2, 2018

#### iGEM Competent Cell Prep, Day 1

#### Liz

Going to try with 1 tube JM109 cells and 1 tube NEB Gibson cells

* Need to use all detergent-free glassware and rinsed plasticware

+ Autoclaved glassware filled ¾ of way with dH2O

* Put 250mL SOB media into each of two 500mL flasks (labeled JM109 and NEB cells)

+ Measured/poured in hood because no antibiotics in SOB
+ Put media into warm room about 20 minutes before pouring, not sure if this is necessary

* Put 1 tube thawed JM109 cells into JM109 flask and 1 tube thawed NEB Gibson cells into NEB flask and covered with foil

+ Thawed tubes in hand
+ Pipetted cells into media (not competent yet so shouldn’t kill cells)

* Put ice in bottom of ice bucket and left flasks of cells in ice bucket overnight

+ Want OD600 of 0.3 (should be about 16 hrs at 20 degrees C)
+ Start at 5:00pm

#### July 3, 2018

#### iGEM Competent Cell Prep, Day 1

#### Liz, Shannon

Pre-chilled 2 flat-bottomed centrifuge bottles and preset centrifuge for 4 degrees C

Checked OD600 of overnights (want at 0.3):

| Time | 7:38 | 8:03 | 8:39 | 9:15 | 9:56 | 10:30 | 11:18 | 11:52 | 12:34 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| JM109 | 0.005 | 0.006 | 0.017 | 0.015 | 0.026 | 0.054 | 0.113 | 0.179 | 0.294 |
| NEB cells | 0.015 | 0.016 | 0.022 | 0.020 | 0.024 | 0.028 | 0.039 | 0.055 | 0.081 |

* After first measurement, kept in warm room
* Gave up on NEB cells (not growing)

* Put equal amounts JM109 culture into two flat-bottomed centrifuge bottles
* Centrifuge for 3000g at 4 degrees C for 10 min
* Decant supernatent
* Resuspend in 80mL ice cold CCMB80 buffer

+ 40mL each tube
+ Resuspend in 20mL then pipet other 20mL against walls

* Incubate on ice 20 min
* Centrifuge at 3000g at 4 degrees C for 10 min
* Decant supernatent
* Resuspend pellet in 10mL ice cold CCMB80 (10mL is total, put 5mL in each bottle then combined bottles)
* Was supposed to check OD of 800μL SOC and 200μL culture using spectrophotometer (blank with 800μL SOC and 200μLCCMB80) then adjust to 1.0-1.5 using CCMB80 but OD started at 0.532 so just skipped this step
* Incubated on ice (was supposed to be 20 minutes but ended up being much longer because was trying to figure out the protocol)
* Aliquotted 50μL per tube over liquid nitrogen (suspended over top to be cold but not too cold like flash freezing)

+ Made two batches because liquid nitrogen evaporated

* Stored in -80 freezer

#### July 3, 2018

#### fadK PCR

#### Liz

Resuspended and diluted primers

* 1F: added 471μL to resuspend, to dilute to 10mM added 1μL resuspended primer and 9μL milliQ water
* 1R: added 339μL to resuspend, to dilute to 10mM added 1μL resuspended primer and 9μL milliQ water

#### Thermocycler Settings

| Cycle | Time | Temperature (degrees Celsius) |
| --- | --- | --- |
| Initial denaturation | 30s | 98 |
| 35 cycles | 10s | 98 |
|  | 30s | 63 |
|  | 40s | 72 |
| Final extension | 10 min | 72 |
| Hold | Infinite | 4 |

#### Added to PCR tube:

| Component | Amount (µL) |
| --- | --- |
| Nuclease-free water | 12.2 (20 - sum of other components) |
| Phusion buffer (HF) | 4 |
| DMSO | 0.6 |
| 10mM forward primer | 1 |
| 10mM reverse primer | 1 |
| dNTPs | 0.4 |
| Template (E. coli) DNA | 0.56 |
| Phusion polymerase (add last) | 0.2 |

#### July 5, 2018

#### fadK PCR purification and Nanodrops

#### Liz, Sricharan

Used protocol from 6/28 (1 PCR tube for fadK)

* Ran 10 Nanodrops (blanked with elution buffer (Buffer E1) from Invitrogen Purif Kit) of PCR purif (4 phz parts, fadD, fadK, 4 linear plasmid backbones)
* All ~10-15 ng/µL, good characteristic curves

| Sample Name | Conc. (ng/µL) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| phz1F-1R Purif | 13.31 | 2.04 | 1.75 |
| phz2F-3R Purif | 11.83 | 1.89 | 1.53 |
| phz4F-4R Purif | 15.53 | 1.97 | 1.77 |
| phz 6F-6R Purif | 10.65 | 2.13 | 1.46 |
| fadD Purif | 8.03 | 1.79 | 2.49 |
| fadK Purif | 14.49 | 1.69 | 1.84 |
| pSB1C3 Purif | 15.01 | 2.01 | 1.72 |
| pSB1T3 Purif | 15.56 | 1.91 | 1.70 |
| pSB1K3 Purif | 12.88 | 1.94 | 1.99 |
| pSB1A3 Purif | 10.55 | 2.11 | 1.78 |

#### July 5, 2018

#### resuspension of phenazine gBlocks

#### Liz

Needed to resuspend phz4-5 and start of phz for gibson assembly

Centrifuged for 5s to make sure all of product is in bottom of tube

Added 1x TE buffer to get concentration to 10ng/μL

* Phz4-5 (1000ng)
  1000ng x 1μL/10ng = 100μL
* Start of phz (500ng)
  500ng x 1μL/10ng = 50μL

Vortexed

Incubated in hat block at 50 degrees C for 20 min

Vortexed and centrifuged

#### July 5, 2018

#### phz gibson PCR and fadD/fadK secondary PCR

#### Liz, Sricharan

#### Thermocycler Settings

|  | Phz gibson |  | fad |  |
| --- | --- | --- | --- | --- |
|  | Time | Temperature (℃) | Time | Temperature (℃) |
| Initial denaturation | 30s | 98 | 30s | 98 |
| 35 cycles | 10s | 98 | 10s | 98 |
|  | 30s | 61 | 30s | 72 |
|  | 60s | 72 | 40s | 72 |
| Final extension | 10 min | 72 | 10 min | 72 |
| Hold | infinite | 4 | infinite | 4 |

#### Add to PCR tubes:

| Component | Amount (µL) |  |  |
| --- | --- | --- | --- |
|  | Phz gibson | fadD | fadK |
| Nuclease-free water | 11.3 (20 - sum of other components) | 7.8 (20 - sum of other components) | 10.04 (20 - sum of other components) |
| Phusion buffer (HF) | 4 | 4 | 4 |
| DMSO | 0.6 | 0.6 | 0.6 |
| 10mM forward primer | 1 (1F) | 1 (fadD bad start) | 1 (fadD bad start) |
| 10mM reverse primer | 1 (1R) | 1 (fadD bad end) | 1 (fadD bad end) |
| dNTPs | 0.4 | 0.4 | 0.4 |
| Template (E. coli) DNA | 1.5 | 5.0 | 2.76 |
| Phusion polymerase (add last) | 0.2 | 0.2 | 0.2 |

Gibson Assembly using the NEB kit … wanted 0.2pmol of DNA total (kit specifies 0.2-1pmol for 4-6 pieces)

* Start of phz (gBlock)

+ 0.05pmol x 50µL/1.950pmol = 1.3µL

* phz 1F-1R Purif

+ 0.05pmol x 157,303pg/1pmol x 1ng/1000pg x 1µL/13.3ng = 0.59µL

* phz 4-5 (gBlock)

+ 0.05pmol x 100µL/2.057pmol = 2.4µL

* phz 2F-3R Purif

+ 0.05pmol x 561,368pg/1pmol x 1ng/1000pg x 1µL/11.8ng = 2.4µL

For Gibson mix, mixing total volume of 6.69 µL fragments + 10 µL NEB Gibson Master Mix + 3.31 µL dH20

Above solution incubated at 50°C for 60 min then at 4°C for infinite hold.

Week 9: 7/9/2018

#### July 9, 2018

#### Resuspending G-Blocks

#### Shannon

* Resuspended phz 1-2, cymA, mtr start, and tesA using TB
* Centrifuge briefly to make sure all products were at the bottom of the tube

+ Note: Forgot to do this for phz 1-2 (ex. Phz 1-2 had 500ng in original tube, so 500ng x 1µL/10ng = 50µL)

| G-Block | Amount of TB (µL) |
| --- | --- |
| Phz 1-2 | 50 |
| cymA | 100 |
| Mtr start | 25 |
| tesA | 100 |

* Vortex briefly
* Incubate at 50C for 20 min in heat block

+ Note: forgot to pre-heat heat block so they sat at room temp for a few minutes

* Briefly vortex and centrifuge
* Stored at -20C

#### July 9, 2018

#### Restriction Digest

#### Shannon

* Used benchling protocol to digest fadD, tesA, cymA, psb1C3, and mtr start
* All enzymes checked on nebcloner.neb.com/#!/redigest for incubation temperatures and “time-saver” (they can be incubated at 37C for 10 minutes rather than 1hr) and to verify that CutSmart could be used as the buffer
* Add reagents in the order: water → buffer → DNA → enzymes
* fadD/tesA/cymA digested with ecoRI and pstI

| Component | Amount (µL) |
| --- | --- |
| ecoRI | 1 |
| pstI | 1 |
| CutSmart | 2 |
| DNA | 5.8 µL for fadD |
|  | 10 µL for tesA and cymA |
| dH2O | 10.2 µL for fadD  6µL for tesA and cymA |

* Calculations:

+ For water: 20 µL - volume of all other components
+ DNA: wanted 100ng, so calculated volume ex. 100ng tesA x 1µL/10ng = 5.8µL

#### Psb1C3 digested with dpnI, ecoRI, and pstI

| Component | Amount (µL) |
| --- | --- |
| dpnI | 1 |
| ecoRI | 1 |
| pstI | 1 |
| CutSmart | 2 |
| DNA | 4 |
| dH2O | 13 |

#### Mtr start digested with ecoRI, bspHI

| Component | Amount (µL) |
| --- | --- |
| ecoRI | 1 |
| bspHI | 1 |
| CutSmart | 2 |
| DNA | 10 |
| dH2O | 6 |

* Centrifuge briefly
* Heat block at 37C for 10min
* Heat block at 80C for 15min (heat-kill enzymes)
* Store at 20C

#### July 9, 2018

#### fadK, phz gibson PCR (try 2), phz Beg PCR Assembly

#### Liz, Sricharan

No bands on gel for fadK and phz gibson 1 (7/15) so redid PCR

#### Notes/changes:

* Did two tubes for phz (one uses HF buffer and other uses GC buffer)
* Phz 35 cycles time 3 changed to 45s because doing PCR assembly at the same time

Also tried to use PCR assembly instead of Gibson for phz Beg

* Just add phz 1F-1R, phz 4-5, and phz 2F-3R to PCR mix and they should assemble together in theory, Phusion does all the gaps
* Need 40ng total template (later realized this should be less, 40ng is for genomic DNA)
* Since construct is 300bp, this is ~0.02 pmol - using quantities from 7/5/2018

Followed PCR purification protocol from 6/28

Nanodropped pure products

* pure\_fadD: 17.24 ng/µL
* pure\_fadK: 3.57 ng/µL
* pure\_fadK\_try2: 4.81 ng/µL
* pure\_phzgibson1: 17.04 ng/µL

| Lane | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- |
| Sample | Ladder | fadD PCR Purif | fadK PCR Purif | Phz Beg Gibson PCR |  |

On a 1% agarose gel in TAE with SYBRSafe, as usual, run at 50V for 50min

![](https://static.igem.org/mediawiki/2018/7/7e/T--UConn--Gels-July-9.jpg)

#### July 10, 2018

#### Ligation of cymA, tesA, and fadD

#### Shannon and Toni

* Did a ligation to insert cymA, tesA, and fadD, separately, into psB1C3 backbones
* Using ligation protocol on benchling, calculated ng of insert dna needed via [link](https://nebiocalculator.neb.com/#!/ligation)

| Sample | Insert DNA Length | Vector DNA Length | Vector DNA Mass | Insert DNA Mass | Ratio |
| --- | --- | --- | --- | --- | --- |
| tesA | 800kb | 2070kb | 30ng | 34.78ng | 3:1 |
| Fad D | 1713kb | 2070kb | 30ng | 49.65ng | 2:1 |
| cymA | 773kb | 2070kb | 30ng | 33.61ng | 3:1 |

| tesA | fadD | cymA | Added |
| --- | --- | --- | --- |
| 4 µL | 1.1 µL | 4.3 µL | dH2O |
| 2 µL | 2 µL | 2 µL | T4 buffer |
| 6 µL | 6 µL | 6 µL | psB1C3 backbone |
| 7 µL | 9.9 µL | 6.7 µL | DNA insert |
| 1 µL | 1 µL | 1 µL | T4 ligase |

* Total volume of soln: 20ul; 100ng/20ul (20ng/ul) of stock vector/inserts
* All reagents kept on ice and added in the order listed
* Incubated at RT for 10 min
* Incubated at 65C for 20 min
* Placed immediately into -20C

#### July 10, 2018

#### PCR purification

#### Liz

#### Samples (4):

* Phz gibson 1 product

+ With HF buffer
+ With GC buffer

* fadK (secondary PCR)
* Phz assembly (PCR assembly)

Followed protocol from 6/28

Nanodropped purified product

* Phz gibson HF: 5.93 ng/µL
* Phz gibson GC: 10.77 ng/µL
* fadK: 9.28 ng/µL
* Phz assembly: 20.11 ng/µL

Ran gel at 50V for 42 min

| 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- |
| ladder | Phz gibson GC | Phz gibson HF | fadK | Phz PCA |

![](https://static.igem.org/mediawiki/2018/6/69/T--UConn--Gels-July-10.jpg)

#### July 10, 2018

#### MIT Competent Cells

#### Liz, Sricharan, Shannon, Toni

[Using protocol given to us at NEGEM by Brian Teague of MIT](https://tanet.mit.edu/drupal7/node/839)

2 5mL OCs of JM109 were put in 2xYT the day before

Put 800µL JM109 overnight cultures into 400mL LB broth and put into shaker (37 degrees C, 250rpm) at 9:30am

#### Making transformation buffers (TfbI, 150mL; TfbII, 20mL):

|  | TfbI Needed | TfbI Actual | TfbII Needed | TfbII Actual |
| --- | --- | --- | --- | --- |
| RbCl | 1.8g | 1.802g | 0.024g | 0.0235g |
| MnCl2 | 1.5g | 1.508g | - | - |
| CaCl2 | 0.166g | 0.171g | 0.166g | 0.166g |
| KOAc | 0.442g | 0.448g |  |  |
| MOPS | - | - | 0.042g | - |
| Glycerol | 22.4mL | Measured w/grad cyl | 3.0mL | Measured with serological pipet |

#### Checked OD:

|  |  |  |
| --- | --- | --- |
| Time | 11:51 | 13:15 |
| OD600 | 0.156 | >1 |

Failure - try again tomorrow; using TfbI and TfbII made today, which is not ideal but we’re also running out of RbCl

#### July 11, 2018

#### MIT Competent Cells

#### Liz, Sricharan, Peter

* 2 5mL OCs of JM109 were put in LB the day before
* Put 800µL JM109 overnight cultures into 380mL LB broth and put into shaker (37 degrees C, 250rpm) at 7:30am

+ Want OD600 of 0.5-0.7

#### Checked OD:

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Time | 9:25 | 10:02 | 10:22 | 10:41 | 10:46 | 10:54 |
| OD600 | 0.056 | 0.164 | 0.289 | 0.416 | 0.506 | 0.565 |

#### July 11, 2018

#### Transformation of Competent Cells

#### Peter and Shannon

* Did a transformation (iGEM protocol) with GFP to test that the cells are competent
* Pre-chill 3 1.5mL tubes of 50µL competent cells - kept on ice while labeling/pipetting
* Add 10, 50, and 100 pg/µL of RFP construct to tubes
* Incubate on ice for 30 min
* Heat shock for 45 sec in 37C water bath
* Incubate 5 min on ice
* Add 950 pre-warmed 2YT to each tube
* Incubate 1 hr at 37C on heat block
* Note: forgot to do a control - will do tomorrow

#### July 12, 2018

#### PCR of FadK using GC buffer

#### Shannon

#### Thermocycler Settings

| Phase | Time (sec) | Temp. (C) |
| --- | --- | --- |
| Initial denaturation | 30 | 98 |
| 35 cycles | 10 | 98 |
|  | 30 | 72 |
|  | 40 | 72 |
| Final extension | 10 | 72 |
| Hold | ∞ | 4 |

#### Add to tubes:

| Component | Amount (µL) |
| --- | --- |
| dH2O | 10 |
| GC buffer | 4 |
| DMSO | 0.6 |
| 10mM forward primer (bad) | 1 |
| 10mM reverse primer (bad) | 1 |
| dNTP mix | 0.4 |
| fadK purified PCR (template) | 2.76 |
| phusion | 0.2 |

* All components were kept on ice

#### July 12, 2018

#### iGEM Transformations

#### Liz, Sricharan

Tubes:

* cmmA-H (biobrick, resuspended in 10µL milliQ water and stored in Registry Parts 2018 box in -20C)
* RFP 25s HS
* RFP 45s HS
* RFP 60s HS
* RFP 90s HS

* Thawed competent cells on ice (~6 min)
* Put 1µL DNA into each corresponding tube
* Incubated on ice for 30 min

Heat shocked in 42C water

* RFP 25s HS: 25s
* RFP 45s HS, ccmA-H: 45s
* RFP 60s HS: 60s
* RFP 90s HS: 90s

* Incubated on ice for 5 min
* Added 450µL SOC media to each tube
* Incubated in shaker (37C, 250rpm) for 1 hr
* Spun down cells at 6800g for 3 min and discarded ~350-400mL
* Resuspended cells and plated remaining 100µL in hood using pipet tip to spread
* Incubated overnight at 37C (put in ~4:15)
* Took plates out ~9:00 7/13 - it worked (only for ccmA-H)!!

#### July 12, 2018

#### Gibson of phz 1-2, 4F-4R, 6F-6R

#### Peter, Ryan

Set up the following reaction on ice:

* phz 1-2 (gBlock): 0.045pmol x 50µL/1.180pmol = 1.91µL
* phz 4F-4R Purif: 0.045pmol x 614,900pg/1pmol x 1ng/1000pg x 1µL/15.53ng = 1.78µL
* phz 6F-6R Purif: 0.045pmol x 584,050pg/1pmol x 1ng/1000pg x 1µL/10.65ng = 2.74µL

For Gibson mix, mixing total volume of 6.43 µL fragments + 10 µL NEB Gibson Master Mix + 3.57 µL dH20

Above solution incubated at 50°C for 60 min then at 4°C for infinite hold.

#### July 12, 2018

#### PCR Cleanup of FadK

#### Shannon

* Combine 80µL B2 buffer (4x the amount of PCR sample) to 20µL PCR sample
* Transfer to collection tube, centrifuge at 10,000 rpm for 1 min. Discard flow-through. Re-insert column into collection tube.
* Add 650µL wash buffer with ethanol. Centrifuge at 10,000 rpm for 1 min. Discard flow-through. Re-insert column into collection tube.
* Centrifuge at max speed (16,100rpm) for 3 min. Discard flow-through. Insert column into a clean 1.7mL elution tube.
* Add 50µL elution buffer to center of column.
* Incubate at room temperature for 1 min.
* Centrifuge at max speed for 2 min.
* The elution tube now contains purified PCR product!
* Store at -20C.

#### July 13, 2018

#### PCR of Gibson assembly from 7/12 (Phz 4F-4R, Phz 1-2, Phz 6F-6R)

#### Peter, Ryan

#### Thermocycler Settings

| Phase | Time (sec) | Temp. (C) |
| --- | --- | --- |
| Initial denaturation | 30 | 98 |
| 35 Cycles | 10 | 98 |
|  | 30 | 67 |
|  | 78 | 72 |
| Final Extension | 10 | 72 |
| Hold | ∞ | 4 |

| Component | Amount (µL) |
| --- | --- |
| dH2O | 10.15 |
| GC buffer | 4 |
| DMSO | 0.6 |
| 10mM forward primer (bad) | 1 |
| 10mM reverse primer (bad) | 1 |
| dNTP mix | 0.4 |
| DNA Template from Gibson Assembly of | 2.65 |
| phusion | 0.2 |

* All components were kept on ice

#### July 13, 2018

#### Gel Electrophoresis of Purified FadK PCR

#### Shannon

* Prepare gel

+ Combine 15 mL 1x TAE and 0.125g agarose (weigh between 0.10 and 0.15 because our scale isn’t that accurate) in a small erlenmeyer flask
+ Microwave until dissolved
+ Cover with foil, let cool until flask isn’t too hot to handle
+ Add 1.25µL Sybr Safe, swirl
+ Pour into a small mold, add comb
+ Let harden

#### July 13, 2018

#### LB Broth

#### Liz

#### 500mL Broth

| Component | Amount (g) | Measured (g) |
| --- | --- | --- |
| tryptone | 5 | 5.05 |
| Yeast extract | 2.5 | 2.50 |
| NaCl | 5 | 5.00 |

* QSed to just under 500mL
* Put in stir bar and stirred/heated for 15 min
* QSed to 500mL in graduated cylinder
* Autoclaved on liquids 30 then stored in 4C

#### July 13, 2018

#### PCR Cleanup of Phz(4F-6R Gibson)

#### Ryan and Peter

* Combine 80µL B2 buffer (4x the amount of PCR sample) to 20µL PCR sample
* Transfer to collection tube, centrifuge at 10,000 rpm for 1 min. Discard flow-through. Re-insert column into collection tube.
* Add 650µL wash buffer with ethanol. Centrifuge at 10,000 rpm for 1 min. Discard flow-through. Re-insert column into collection tube.
* Centrifuge at max speed (16,100rpm) for 3 min. Discard flow-through. Insert column into a clean 1.7mL elution tube.
* Add 50µL elution buffer to center of column.
* Incubate at room temperature for 1 min.
* Centrifuge at max speed for 2 min.
* The elution tube now contains purified PCR product!
* Store at -20C.
* Nanodroped @ 16.7 ng/µL

#### July 13, 2018

#### Gel of PCR’s for Phz 4F-6R, fadK Primary and Secondary PCR’s

#### Peter, Ryan

#### Wells

| 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- |
| 2 Log Ladder | PHz 4F-6R (Gibson) purif. | fadK Primary purif. (7/5/18) | fadK secondary purif. (7/9/18) | fadK secondary purif. (7/12/18) |
| -6 µL ladder | -2 µL purif. PCR  -3 µL MilliQ DH2O  -1 µL Loading Dye | -5 µL purif. PCR  -1 µL Loading Dye | -5 µL purif. PCR  -1 µL Loading Dye | -5 µL purif. PCR  -1 µL Loading Dye |

* Gibson worked… two visible bands, one a little less than 3 kb according to the ladder (full gibson should be 2.588 Kb) and one band that is probably both 4F-4R and 6F-6R that didn’t assemble are about both ~ 1 Kb
* Secondary fadK most likely didn’t work because PCR used ~ 40 ng from Primary PCR so that is probably what is visible in this gel (also same length as primary PCR)

![](https://static.igem.org/mediawiki/2018/4/46/T--UConn--Gels-July-13.jpg)

Week 10: 7/16/2018

#### July 16, 2018

#### Secondary PCR of fadD and fadK

#### Ryan, Shannon

#### Set thermocycler:

| Phase | Time (sec) | Temp. (C) |
| --- | --- | --- |
| Initial denaturation | 30 | 98 |
| 35 cycles | 10 | 98 |
|  | 30 | 67 |
|  | 40 | 72 |
| Final extension | 10 minutes | 72 |
| Hold | ∞ | 4 |

#### Add to tubes:

| Component | fadD Amount (µL) | fadK Amount (µL) |
| --- | --- | --- |
| dH2O | 12.6 | 12.5 |
| Phusion buffer (HF) | 4 | 4 |
| DMSO | 0.6 | .6 |
| 10mM forward primer (bad) | 1 | 1 |
| 10mM reverse primer (bad) | 1 | 1 |
| dNTP mix | 0.4 | .4 |
| Template DNA | .2 | .3 |
| phusion | 0.2 | .2 |

* All components were kept on ice

#### July 16, 2018

#### Restriction Digest of Secondary fadD and fadK PCRs

#### Liz

#### fadD and fadK:

| Component | Amount |
| --- | --- |
| EcoRI (HF) | 1µL |
| PstI (HF) | 1µL |
| CutSmart | 2µL |
| DNA template | 100ng |
|  | fadD: 100ng x 1µL/14.3ng = 6.99µL |
|  | fadK: 100ng x 1µL/19.9ng = 5.02µL |
| Water | To 20µL |
|  | fadD: 9.00µL |
|  | fadK: 10.97µL |

* Order: water, buffer, DNA, enzymes
* Spin down in tabletop centrifuge
* Heat blocked at 37C for 25 min
* Heat blocked at 65C for 15 min

#### Backbone:

| Component | Amount |
| --- | --- |
| BpsHI | 1µL |
| EcoRI (HF) | 1µL |
| PstI (HF) | 1µL |
| CutSmart | 2µL |
| DNA template | 100ng |
|  | fadD: 100ng x 1µL/14.3ng = 6.99µL |
|  | fadK: 100ng x 1µL/19.9ng = 5.02µL |
| Water | To 20µL |
|  | fadD: 9.00µL |
|  | fadK: 10.97µL |

* Order: water, buffer, DNA, enzymes
* Spin down in tabletop centrifuge
* Heat blocked at 37C for 25 min
* Heat blocked at 80C for 15 min
* This digest was wrong because DnpI should have been used for the backbone instead of BpsHI

#### July 16, 2018

#### Gel Electrophoresis of Purified FadK, FadD PCR

#### Ryan

Prepare gel

* Combine 15 mL 1x TAE and 0.125g agarose (weigh between 0.10 and 0.15 because our scale isn’t that accurate) in a small erlenmeyer flask
* Microwave until dissolved
* Cover with foil, let cool until flask isn’t too hot to handle
* Add 1.25µL Sybr Safe, swirl
* Pour into a small mold, add comb
* Let harden

#### Wells:

| 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- |
| 2 Log Ladder | fadD PCR2 | fadD PCR0 | fadK PCR2 | fadK PCR0 |
| -6 µL ladder | -1 µL purif. PCR  -4 µL MilliQ DH2O  -1 µL Loading Dye | -1 µL purif. PCR  -4 µL MilliQ DH2O  -1 µL Loading Dye | -1 µL purif. PCR  -4 µL MilliQ DH2O  -1 µL Loading Dye | -1 µL purif. PCR  -4 µL MilliQ DH2O  -1 µL Loading Dye |

#### July 16, 2018

#### Transformation with RbCl JM109 Cells

#### Sricharan, Peter

#### Transforming:

BBa\_J04450 (RFP), BBa\_B0015 (DoubleTerminator), BBa\_K590025 (PetroBrick), BBa\_K1433005 (Lambda Red), BBa\_I718008 (Inducible Cre-generator), BBa\_K1316012 (Mtr + T7 Lac O.), TesA (from ligation), CymA (from ligation), BBa\_K917006 (ccmA-H)

* Grab 50μL aliquots of RbCl Uber-Competent Cells from -80° C freezer, let thaw on ice for ~3-4 minutes.
* Add 1μL of plasmid from iGEM kit (or 2 μL of plasmid from ligation product) to the tube with cells.
* Incubate cells on ice for 30 minutes.
* Heat shock tubes at 42° C for 45s.
* Incubate on ice again for 5 minutes.
* Add 450μL SOC media to each tube (9 x volume) and incubate on the shaker at 37° C and 250rpm for 1 hour.
* Spin down cells at ~6800rpm for 3 minutes, remove 370μL of supernatant, resuspend cell pellet in the remaining media, and pipet that onto the plate
* Use P200 to spread cells across plate
* Leave plates upside-down (with agar on top) in incubator (at 37° C) overnight for 16-18 hours.

#### Notes:

* Added 2 μL pf DNA for TesA & CymA because from ligation reaction so not as much DNA as a prepared BioBrick
* Heat-shocked ccmA-H (BBa\_K917006) in 5 different tubes for 30, 45, 60, 75, and 90 seconds (one time per tube)
* Added 900 μL of soc media for tube 4 because was 100 μL aliquot of cells
* Used Amp plate for BBa\_I718008 because plasmid has Amp resistance

#### Results (7/17/18):

| Part/ Vector | Transformation Result |
| --- | --- |
| BBa\_J04450 (RFP) | -No growth on plate |
| BBa\_B0015 (Double Terminator) | -No observable cell pellet when spun down  -No growth on plate |
| BBa\_K590025 (PetroBrick) | -Good growth (do O.C. on 7/17) |
| BBa\_K1433005 (Lambda Red) | -Good growth (do O.C. on 7/17) |
| BBa\_I718008 (Inducible Cre-generator) | -Good growth (do O.C. on 7/17) |
| BBa\_K1316012 (Mtr + T7 Lac O.) | -No observable cell pellet when spun down  -No growth on plate |
| TesA (from ligation) | -No growth on plate (Ligation enzymes can be a problem) |
| CymA (from ligation) | -No growth on plate (Ligation enzymes can be a problem) |
| BBa\_K917006 (ccmA-H) | -30 sec: works best  -45 sec: works well  -60 sec: works okay  -75 sec: no visible pellet but grew a few colonies  -90 sec: no visible pellet or growth on plate (probably heat-killed) |

#### July 17, 2018

#### Ligation of fadD and fadK

#### Liz

* \*didn’t work because digested backbone wrong
* fadD:
* Insert DNA: 1689 bp
* Vector length: 2070 bp
* Vector mass: 25 ng
* NEB Ligation Calculator: 2:1 → 40.80ng x 20L

#### July 17, 2018

#### Gel Electrophoresis of Purified FadK, FadD PCR

#### Ryan

Prepare gel

* Combine 15 mL 1x TAE and 0.125g agarose (weigh between 0.10 and 0.15 because our scale isn’t that accurate) in a small erlenmeyer flask
* Microwave until dissolved
* Cover with foil, let cool until flask isn’t too hot to handle
* Add 1.25µL Sybr Safe, swirl
* Pour into a small mold, add comb
* Let harden

#### Wells:

| 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- |
| 2 Log Ladder | fadD PCR2 | fadD PCR0 | fadK PCR2 | fadK PCR0 |
| -6 µL ladder | -1 µL purif. PCR  -4 µL MilliQ DH2O  -1 µL Loading Dye | -1 µL purif. PCR  -4 µL MilliQ DH2O  -1 µL Loading Dye | -1 µL purif. PCR  -4 µL MilliQ DH2O  -1 µL Loading Dye | -1 µL purif. PCR  -4 µL MilliQ DH2O  -1 µL Loading Dye |

* No bands visible on any part of the gel:

![](https://static.igem.org/mediawiki/2018/1/13/T--UConn--Gels-July-17.jpg)

#### July 17, 2018

#### Gel Extraction of phz 4F-6R

#### Sricharan

#### Onto a 1% agarose gel with SYBR Safe, loaded (with 6X Purple Loading Dye from NEB)

| 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- |
| 2-log ladder | phz 1F-3R PCR (GC Buffer) | ‘tesA(L109P) ligation | phz 4F-6R PCR | Same as Lane 4 |
| 6µL ladder | 5µL item  1µL dye | 1µL item  4µL dH2O  1µL dye | 15µL item  3µL dH2O |  |

Note, wells 2 and 3 had blue stain from tape (we tried to tape together these two wells to do gel extraction but it didn’t work very well).

![](https://static.igem.org/mediawiki/2018/2/22/T--UConn--Gels-July-17-2.jpg)

* Cut bands at 2.6kb on last two lanes out using tape (marked with arrows delineating top of gel, bottom of gel, and location of band to be excised) and wax paper + razor
* Weighed cut bands: 0.0840g, used Zymoclean Gel DNA Recovery Kit to clean up DNA. Only deviation from protocol was centrifuged ~16K rpm to elute 30µL buffer from Invitrogen PCR Pure Kit.

Nanodrop Results:

* Conc: 2.8 ng/µL
* 260/280: 1.23
* 260/230: 0.05

#### July 17, 2018

#### LB + Chloramphenicol Plates Preparation

#### Ryan

Preparing Chloramphenicol LB plates (24)

* Collected and measured out ingredients for LB media and added to bottle
* Q/s’d with dH20 to 400 and shook bottle to dissolve ingredients
* Stirred in beaker for 30 min
* Poured into grad cylinder to finish Q/s to 500 mL
* Poured back into bottle and stirred for ~ a minute
* Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
* Added 0.5 mL of chloramphenicol (1000x, 35 mg/mL) to solution
* Put them in the hood with the plates half open w/ agar side on the bottom to get rid of condensation

\*\*OOPS put in 35 microliters instead of .5 mL

#### July 17, 2018

#### Transformation with RbCl JM109 Cells

#### Liz, Peter

\*\*See Protocol from 7/16/18\*\*

#### Transforming failed transformations from 7/16:

BBa\_J04450 (RFP), BBa\_B0015 (DoubleTerminator), BBa\_K1316012 (Mtr + T7 Lac O.), TesA (from ligation + now purified w/ PCR purif), CymA (from ligation + now purified w/ PCR purif), BBa\_K917006 (ccmA-H)

\*\*\*See protocol from 7/16\*\*\*

#### Notes/ Deviations:

* CymA and TesA have been purified w/ PCR purif.
* Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube (only one tube for the blank)
* Heat-shocked for 30 seconds based on results from transformations of ccmA-H
* After resuspending, pooled secondary tube w/ primary tube if any cells (next time pool before spinning)
* All except blank had one tube w/ obvious pellet after first spin
* BBa\_J04450 (RFP), BBa\_B0015 (Double terminator), and CymA secondary tubes w/ no original pellet had slightly visible pellet after second spin. Were pooled with primary tube after resuspension
* Not pellet for 6 so didn’t bother plating

#### Results (7/18/18):

| Part/ Vector | Transformation Result |
| --- | --- |
| BBa\_J04450 (RFP) | -No growth on plate |
| BBa\_B0015 (Double Terminator) | -One colony (Do O.C. on 7/18) |
| BBa\_K1316012 (Mtr + T7 Lac O.) | -No growth on plate |
| TesA (from ligation + now purified w/ PCR purif) | -No growth on plate |
| CymA (from ligation + now purified w/ PCR purif) | -One colony (Do O.C. on 7/18) |

#### July 17, 2018

#### Overnight Cultures of successful transformations from 7/16/18

#### Peter, Shannon

#### Overnight Cultures:

* BBa\_K590025 (PetroBrick)
* BBa\_K1433005 (Lambda Red)
* BBa\_I718008 (Inducible Cre-generator)
* BBa\_K917006 (ccmA-H)

#### Results (7/18/18):

* They worked: proceed to mini-prep and glycerol stocking

#### July 18, 2018

#### Nanodrops and a Gel: Miniprepped Plasmids, Gel Extraction PCR of 4F-6R, Gel Extraction of 1F-3R

#### Sricharan

Some OCs were grown - Shannon miniprepped and Liz made glycerol stocks; I Nanodropped

| Item | Concentration (ng/µL) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| I718008 MP | 85.7 | 1.75 | 1.28 |
| K590025 MP | 51.7 | 1.73 | 0.85 |
| K1433005 MP | 53.9 | 1.74 | 1.37 |
| K917006 MP | 175.1 | 1.88 | 2.15 |
| Phz 4F-6R GelExtr PCR | 30.9 | 1.39 | -0.82 |

#### Ran a gel:

| Lane | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- |
| Item | 2-log ladder | Poked hole | K143305 MP | I718008 MP | K917006 MP | PCR of Gel Extr of phz 4F-6R |
| Amt (µL) | 6 | N/A | 5+1 dye | 5+1 dye | 5+1 dye | 2+3 dH2O+1 dye |

| 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- |
| 2-log ladder | phz 1F-3R GC PCR | phz 1F-3R GC PCR | empty | K590025 MP | 2-log ladder |
| 6 | 15 + 3 dye | 15 + 3 dye |  | 5 + 1 dye | 6 |

* Note, items in lanes 3, 4, 5, 11 digested with PstI.
* Dye is 6X Purple Loading Dye from NEB.
* Liz gel extracted bands 8 and 9 at desired size (~3kb)

#### PCR of these bands: To PCR tube on ice:

|  |  |
| --- | --- |
| dH2O | 11.8 µL |
| Phusion Buffer (GC) | 4 µL |
| DMSO | 0.6 µL |
| 10µM forward primer (1F) | 1 µL |
| 10µM reverse primer (3R) | 1 µL |
| 10µM dNTPs | 0.4 µL |
| Template DNA | 1 µL |
| Phusion polymerase | 0.2 µL |

#### Cycle is based on Phusion polymerase protocol:

|  | Time (s) | Temp (°C) |
| --- | --- | --- |
| Initial Denaturation | 30 | 98 |
| 35 Cycles | 10 | 98 |
|  | 30 | 61 |
|  | 60 | 72 |
| Final Extension | 600 | 72 |
| Hold | Infinite Hold | 4 |

![](https://static.igem.org/mediawiki/2018/d/d8/T--UConn--Gels-July-18.jpg)

#### July 18, 2018

#### Miniprep of overnight cultures from 7/17/2018

#### Shannon

* Used Zyppy Miniprep Kit and instructions from the manufacturer’s website
* Overnight cultures of: K590025, K1433005, I718008, and K917006
* Add 600µL of each bacterial culture to separate 1.5mL tubes
* Add 100µL 7x lysis buffer, mix by inverting about 6 times

+ Solution should turn from opaque blue to clear blue

* Add 350µL cold neutralization buffer (yellow), mix by inverting

+ Solution should turn yellow

* Centrifuge at 13,000g for 3 min
* Transfer supernatant (about 900µL) into column, place column in collection tube
* Centrifuge for 15 seconds
* Discard flow-through, put column back into the same collection tube
* Add 200µL endo wash buffer, centrifuge 20 seconds
* Add 400µL Zippy wash buffer to column. Centrifuge 1 min
* Transfer the column to clean 1.5 mL tube, add 30µL elution buffer directly to column. Let stand for 1 min
* Centrifuge 30 seconds to elute DNA

#### July 18, 2018

#### Transformation with RbCl JM109 Cells

#### Liz, Peter

\*\*See Protocol from 7/16/18\*\*

#### Transforming failed transformations from 7/17 + more:

BBa\_J04450 (RFP), BBa\_K1316012 (Mtr + T7 Lac O.) from 2017 and 2018 kit, TesA (from ligation + now purified w/ PCR purif), FadK (from ligation + now purified w/ PCR purif), FadD (from ligation + now purified w/ PCR purif), Blank

#### Notes/Deviations:

* Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube (only one tube for the blank)
* Heat-shocked for 30 seconds based on results from transformations of ccmA-H (done on 7/16)
* Pooled like tubes (same vector) after 1 hour incubation

#### Results (7/19/18):

| Part/ Vector | Transformation Result |
| --- | --- |
| BBa\_J04450 (RFP) | 1 colony (O.C. on 7/19) |
| BBa\_K1316012 (Mtr + T7 Lac O.) 2017 kit | No growth |
| BBa\_K1316012 (Mtr + T7 Lac O.) 2018 kit | No growth |
| TesA (from ligation + now purified w/ PCR purif) | 1 colony (O.C. on 7/19) |
| FadK (from ligation + now purified w/ PCR purif) | 1 colony (O.C. on 7/19) |
| FadK (from ligation + now purified w/ PCR purif) | No growth |
| FadD (from ligation + now purified w/ PCR purif) | No growth |
| Blank | No growth |

#### July 18, 2018

#### LB + Chloramphenicol Plates Preparation

#### Ryan

Preparing Chloramphenicol LB plates (24)

* Collected and measured out ingredients for LB media and added to bottle
* Q/s’d with dH20 to 400 and shook bottle to dissolve ingredients
* Stirred in beaker for 30 min
* Poured into grad cylinder to finish Q/s to 500 mL
* Poured back into bottle and stirred for ~ a minute
* Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
* Added 0.5 mL of chloramphenicol (1000x, 35 mg/mL) to solution
* Put them in the hood with the plates half open w/ agar side on the bottom to get rid of condensation

#### July 19, 2018

#### Miniprep and glycerol stocks of cymA ligation and B0015

#### Shannon

* Used Zyppy Miniprep Kit and instructions from the manufacturer’s website
* Add 600µL of each bacterial culture to separate 1.5mL tubes
* Add 100µL 7x lysis buffer, mix by inverting
* Add 350µL cold neutralization buffer (yellow), mix by inverting
* Centrifuge at 13,000g for 3 min
* Transfer supernatant (about 900µL) into column, place column in collection tube
* Centrifuge for 15 seconds
* Discard flow-through, put column back into the same collection tube
* Add 200µL endo wash buffer, centrifuge 20 seconds
* Add 400µL Zippy wash buffer to column. Centrifuge 1 min
* Transfer the column to clean 1.5 mL tube, add 30µL elution buffer directly to column. Let stand for 1 min
* Centrifuge 30 seconds to elute DNA
* Made glycerol stocks by combining 0.75mL of 40% glycerol and 0.75mL overnight culture, stored at -80C

#### July 19, 2018

#### PCR Purification of fadD and fadK

#### Shannon

* Using Lambda Biotech Column Pure PCR Cleanup Kit
* Mix 20µL PCR reaction with 100µL binding buffer
* Load onto spin column, centrifuge at full speed (16,100g) for 1 min
* Discard flow-through
* Add 700µL wash buffer, centrifuge 1 min
* Discard flow-through, centrifuge again 1 min
* Transfer column to clean 1.5mL tube
* Add 30µL elution buffer, let sit 1 min, centrifuge 1 min to elute DNA

#### July 19, 2018

#### Restriction Digest of CymA and B0015

#### Peter

#### B0015 (Terminator):

| Component | Amount |
| --- | --- |
| PstI (HF) | 1µL |
| CutSmart | 1µL (should have done 2µL) |
| DNA template | 100ng x 1µL/53.8ng = 1.86µL |
| Water | 16.14µL (To 20µL) |

* Order: water, buffer, DNA, enzymes
* Spin down in tabletop centrifuge
* Heat blocked at 37C for 10 min
* Heat-killed on block at 80C for 15 min

#### CymA:

| Component | Amount |
| --- | --- |
| PstI (HF) | 1µL |
| CutSmart | 1µL (should have done 2µL) |
| DNA template | 5µL (20 ng/µL) -wanted a lot of it) |
| Water | 13 µL (To 20µL) |

* Order: water, buffer, DNA, enzymes
* Spin down in tabletop centrifuge
* Heat blocked at 37C for 10 min
* Heat-killed on block at 80C for 15 min

#### MtrCAB (2017 or 2018?):

| Component | Amount |
| --- | --- |
| PstI (HF) | 1µL |
| CutSmart | 1µL (should have done 2µL) |
| DNA template | 100ng x 1µL/112.5ng = 0.89µL |
| Water | 17.11 µL (To 20µL) |

* Order: water, buffer, DNA, enzymes
* Spin down in tabletop centrifuge
* Heat blocked at 37C for 10 min
* Heat-killed on block at 80C for 15 min

#### July 19, 2018

#### PCR Purification of phz 1F-3R Gel Extraction PCR and a Gel

#### Sricharan

* PCR purified product from yesterday (phz 1F-3R gel extraction PCR)
* Used Lambda Biotech Kit protocol, eluted with 30µL buffer

#### Agarose gel (1%, large 35mL)

| Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Item | 2-log ladder | phz 1F-3R Gel Extraction PCR | cymA MP | B0015 MP | K1316012 plasmid (from 2017 Kit) | fadD PCR2 with correct annealing temp | fadD PCR0 diluted to 40ng/30µL | fadK PCR2 with correct annealing temp | fadK PCR0 diluted to 40ng/30µL | 2-log ladder |
| Amt (µL) | 5 | 3+2 dH2O+1 dye | 5+1 dye | 5+1 dye | 5+1 dye | 5+1 dye | 5+1 dye | 5+1 dye | 5+1 dye | 6 |

* Note that lanes 3-5 are digested with PstI

![](https://static.igem.org/mediawiki/2018/9/91/T--UConn--Gels-July-19.jpg)

* Seems that fadD PCR of the coding region (“PCR2”) is successful since it is significantly amplified past that of template.
* Need to redo fadK PCR2
* Also cymA and B0015 might be around the same size, but hard to tell here, need to retry the gel

#### July 19, 2018

#### Overnight Cultures of TesA and J04450 (RFP)

#### Peter

#### Overnight Cultures:

* TesA
* BBa\_J04450 (RFP)

#### Results (7/20/18):

* They worked: proceed to miniprep and glycerol stocking

#### July 20, 2018

#### PCR2 of FadK and PCR of ‘TesA(L109P) Basic Part from miniprep (7/18/18)

#### Peter, Sricharan

#### FadK PCR2:

Set thermocycler:

| Phase | Time (sec) | Temp. (C) |
| --- | --- | --- |
| Initial denaturation | 30 | 98 |
| 35 cycles | 10 | 98 |
|  | 30 | 72 (from NEB calculator) |
|  | 48 | 72 |
| Final extension | 10 minutes | 72 |
| Hold | ∞ | 4 |

#### Add to tubes in this order:

| Component | Amount (µL) |
| --- | --- |
| dH2O | 12.11 |
| HF buffer | 4 |
| dNTP mix | 0.4 |
| 10mM forward primer (bad) | 1 |
| 10mM reverse primer (bad) | 1 |
| DMSO | 0.6 |
| Template DNA (FadK PCR0) | 0.69 (10 ng) |
| phusion | 0.2 |

* All components were kept on ice

#### ‘TesA (L109P):

Set thermocycler:

| Phase | Time (sec) | Temp. (C) |
| --- | --- | --- |
| Initial denaturation | 30 | 98 |
| 35 cycles | 10 | 98 |
|  | 30 | 72 (from NEB calculator) |
|  | 26 | 72 |
| Final extension | 10 minutes | 72 |
| Hold | ∞ | 4 |

#### Add to tubes in this order:

| Component | Amount (µL) |
| --- | --- |
| dH2O | 12.54 |
| HF buffer | 4 |
| dNTP mix | 0.4 |
| 10mM forward primer (bad) | 1 |
| 10mM reverse primer (bad) | 1 |
| DMSO | 0.6 |
| Template DNA (‘TesA L109P from miniprep) | 0.26 (10 ng) |
| phusion | 0.2 |

* All components were kept on ice

#### July 20, 2018

#### Gel of FadK PCR2, ‘TesA (L109P) basic part, and other stuff (see Sricharan’s notebook)

#### Peter, Ryan

#### Wells:

| 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- |
| 2 Log Ladder | fadK PCR2 HF | fadK PCR0 diluted to 0.69µL/30µL | fadK PCR2 GC | ‘tesA(L109P) PCR Basic | K1316012 (mtrCAB) 2017 Kit Digest w/ PstI |

| 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- |
| 2 Log Ladder | ‘tesA(L109P) Miniprep Digest w/PstI | Kit 7, Well 2H 2018 Digest w/PstI | J04450 Miniprep Digest 3x | See lane 10 | See Lane 1 |

* Note that 6µL of ladder added to lanes 1, 7, 12; 5µL of item and 1µL of loading dye added to all other lanes

#### Results:

| 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |

| 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |

![](https://static.igem.org/mediawiki/2018/7/77/T--UConn--Gels-July-20.jpg)

#### July 20, 2018

#### Restriction 5 things

#### Peter

Digest of J04450 (RFP), ‘TesA (L109P), FadD PCR2, Mtr 2016 (K1316012), Kit 7 Well 2H

#### J04450 (RFP):

| Component | Amount |
| --- | --- |
| XBaI | 1µL |
| SpeI | 1µL |
| DpNI | 1µL |
| CutSmart | 1µL (should have done 2µL) |
| DNA template | 10µL |
| dH2O | 6µL (To 20µL) |

* Order: water, buffer, DNA, enzymes
* Spin down in tabletop centrifuge
* Heat blocked at 37C for 10 min
* Heat-killed on block at 80C for 15 min

#### CymA:

| Component | Amount |
| --- | --- |
| PstI (HF) | 1µL |
| CutSmart | 1µL (should have done 2µL) |
| DNA template | 5µL (20 ng/µL) -wanted a lot of it) |
| Water | 13 µL (To 20µL) |

* Order: water, buffer, DNA, enzymes
* Spin down in tabletop centrifuge
* Heat blocked at 37C for 10 min
* Heat-killed on block at 80C for 15 min

#### MtrCAB (2017 or 2018?):

| Component | Amount |
| --- | --- |
| PstI (HF) | 1µL |
| CutSmart | 1µL (should have done 2µL) |
| DNA template | 100ng x 1µL/112.5ng = 0.89µL |
| Water | 17.11 µL (To 20µL) |

* Order: water, buffer, DNA, enzymes
* Spin down in tabletop centrifuge
* Heat blocked at 37C for 10 min
* Heat-killed on block at 80C for 15 min

Week 11: 7/23/2018

#### July 23, 2018

#### LB + Cam, LB + Kan Plates Preparation

#### Ryan

Preparing Chloramphenicol LB plates (18):

* Collected and measured out ingredients for LB media and added to bottle
* Q/s’d with dH20 to 350 mL and shook bottle to dissolve ingredients
* Stirred in beaker for 30 min
* Poured into grad cylinder to finish Q/s to 375 mL
* Poured back into bottle and stirred for ~ a minute
* Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
* Split solution in half such that 9 plates are for Cam and 9 plates are for Kan
* Added 0.1875 mL of chloramphenicol (1000x, 35 mg/mL) to solution in one half, 0.1875 mL of kanamycin (1000x, 35 mg/mL) to other
* Let plates cool for an hour on lab bench

#### July 23, 2018

#### Digest of J04450 and psb1C3

#### Peter and Shannon

* Add reagents in order listed
* Centrifuge briefly
* Heat block at 37C (45 min for psb1C3, 10 min for the rest)
* Heat block at 80C for 15 min to heat kill enzymes

#### psb1C3, I718008, K1433005, K1316012

| Component | Amount (µL) |
| --- | --- |
| Water (milliQ) | psb1C3: 12 |
|  | 1718008: 14.83 |
|  | K1433005: 14.14 |
|  | K1316012: 15 |
| CutSmart | 2 |
| DNA | psb1C3: 4 |
|  | 1718008: 1.17 |
|  | K1433005: 1.86 |
|  | K1316012:1 |
| EcoRI | 1 |
| PstI | 1 |

#### J04450

| Component | Amount (µL) |
| --- | --- |
| Water (milliQ) | 15.65 |
| CutSmart | 2 |
| DNA | 0.35 |
| SpaI-HF | 1 |
| XbaI | 1 |

#### July 23, 2018

#### Reviving Glycerol Stocks

#### Shannon

* Put glycerol stocks on ice (try not to let them thaw too much)
* Stab into the glycerol stock with a p20 pipette tip
* Use pipette tip to t-streak on a place with the correct antibiotic
* Incubate overnight at 37C
* Revived: J23114, J23110, J23100, and cymA. Was going to revive K864400 but could not find it in the -80C freezer
* Note: There were three tubes labelled J23110, and one tube that had the label wiped off. These may have been mislabelled. I believe two are J23110 and two are J23100. The two that have more color should be the stronger promoter, and the two with less color should be the weaker promoter. I labelled one plate “S” and one “W” for strong and weak.
* Note: For the tube labelled J23110, I don’t know which of the three “J23110” tubes I used. I did this one before realizing the tubes may have been mislabelled.

#### July 23, 2018

#### Agarose Gel for Extraction and Checking stuff

#### Sricharan

* Another 1% agarose gel
* Liz gel extracted lanes 2 and 3 at pSB1C3 band (~2kb)

| 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- |
| 2-log ladder | J04450 Digest | J04450 Digest | K143305 Digest 7/23 | I718008 Digest 7/23 | K14316012 Digest 7/23 |

* Note that 6µL of ladder to lane 1; 5µL item and 1µL dye to every other well. Lanes 2 - 5 are sourced from minipreps. Lane 6 is just DNA from the distribution kit we wanted to test.

![](https://static.igem.org/mediawiki/2018/d/d1/T--UConn--Gels-July-23.jpg)

#### July 23, 2018

#### Gel Extraction of J04450 3x Digest

#### Shannon

* Excise DNA from gel, add to 1.5mL tube. 184mg were excised.
* Add 3x volume ADB. 555mL were added.
* Incubate at 45C for 5-10 min until dissolved
* Transfer to spin column
* Centrifuge 60 sec at 10,000g. Discard flow-through.
* Add 200µL wash buffer, centrifuge 30 sec. Discard flow through. Repeat.
* Add 10µL elution buffer directly onto column. Let sit for 1 min.
* Place column in clean 1.5mL tube. Centrifuge 60 sec.
* Store at -20C.
* Note: the elution buffer was from the invitrogen kit.

#### July 23, 2018

#### Gel Extraction of J04450 3x Digest

#### Shannon

* Excise DNA from gel, add to 1.5mL tube. 184mg were excised.
* Add 3x volume ADB. 555mL were added.
* Incubate at 45C for 5-10 min until dissolved
* Transfer to spin column
* Centrifuge 60 sec at 10,000g. Discard flow-through.
* Add 200µL wash buffer, centrifuge 30 sec. Discard flow through. Repeat.
* Add 10µL elution buffer directly onto column. Let sit for 1 min.
* Place column in clean 1.5mL tube. Centrifuge 60 sec.
* Store at -20C.
* Note: the elution buffer was from the invitrogen kit.

#### July 23, 2018

#### J04450 Digest

#### Liz

Set heat blocks to 37C and 80C

| Components (in order) | Amount (µL) |
| --- | --- |
| milliQ water | 6 |
| Cutsmart | 2 |
| DNA (J04450 miniprep SK 7/20, 287ng/µL) | 10 |
| SpeI-HF | 1 |
| XbaI | 1 |

* Incubated at 37C for 10 min
* Heat shocked at 15 min

#### July 24, 2018

#### PCR of PSB1C3 Template from RFP mini-prep

#### Peter, Sricharan

#### Set thermocycler:

| Phase | Time (sec) | Temp. (C) |
| --- | --- | --- |
| Initial denaturation | 30 | 98 |
| 35 cycles | 10 | 98 |
|  | 30 | 71 (from NEB calculator) |
|  | 60 | 72 |
| Final extension | 10 minutes | 72 |
| Hold | ∞ | 4 |

* \*set to 50 µL reaction

#### Add to tubes in this order:

| Component | Amount (µL) |
| --- | --- |
| dH2O | 31.65 |
| HF buffer | 10 |
| dNTP mix | 1 |
| 10mM forward primer (bad) | 2.5 |
| 10mM reverse primer (bad) | 2.5 |
| DMSO | 1.5 |
| Template DNA (FadK PCR0) | 0.348 (5 ng - from 10x dilute of miniprep) |
| phusion | 0.2 |

* All components were kept on ice
* Used lambda PCR cleanup kit
* Nano-dropped @ 48.8 ng/µL
* Did gel extract for linearized plasmid for Gibsons (no longer using for Gibsons)

#### July 24, 2018

#### Overnight Cultures

#### Shannon

* Did overnight cultures of J23114, J23110, J23100, and cymA
* Combine 5mL LB and 5µL antibiotic (ampicilin for J23114, J23110, and J23100 and chloramphenicol for cymA)
* Put in shaker at 4:20pm
* Note: “S” and “W” are either J23110 or J23100. “S” should be the stronger promoter, “w” should be the weaker promoter.
* Note: The tube labelled “J23110” may be J23100. Check color against “S” and “W”?

#### July 24, 2018

#### Digest of psB1C3, acr 1, acr 3, fadD, fadK, B0015, cymA, pSB1K3, ccmA-H

#### Ryan and Liz

* Add reagents in order listed
* Centrifuge briefly
* Heat block at 37C (45 min for psb1C3, 10 min for the rest)
* Heat block at 80C for 15 min to heat kill enzymes

#### fadD, fadK, cymA, pSB1K3

| Component | Amount (µL) |
| --- | --- |
| Water (milliQ) | fadD: 11.7 |
|  | fadD: 9 |
|  | cymA: 11 |
|  | pSB1K3: 12 |
| CutSmart | 2 |
| DNA | fadD: 4.3 |
|  | fadK: 7 |
|  | cymA: 5 |
|  | pSB1K3: 4 |
| EcoRI | 1 |
| Pstl | 1 |

#### pSB1C3

| Component | Amount (µL) |
| --- | --- |
| CutSmart | .5 |
| DNA | 4 |
| EcoRI | .25 |
| PstI | .25 |

#### Acr 1

| Component | Amount (µL) |
| --- | --- |
| CutSmart | 1.25 |
| DNA | 10 |
| Pstl | 1.25 |

#### Acr 3

| Component | Amount (µL) |
| --- | --- |
| CutSmart | 1.25 |
| DNA | 10 |
| Pstl | 1.25 |

#### B0015

| Component | Amount (µL) |
| --- | --- |
| Water (milliQ) | 14.1 |
| CutSmart | 2 |
| DNA | 1.86 |
| Xbal | 1 |
| Pst1 | 1 |

#### ccmA-H

| Component | Amount (µL) |
| --- | --- |
| Water (milliQ) | 15.4 |
| CutSmart | 2 |
| DNA | 0.57 |
| EcoRI | 1 |
| Spel | 1 |

#### July 24, 2018

#### Gibson Reactons, Circular Version

#### Sricharan

Planning Gibson reactions to assemble acr. We realized the overlaps between our acr inserts and the pSB1C3 backbone isn’t great. We decided on a couple reactions to see if we can use these inserts

For 50ng pSB1C3, or 0.037pmol of backbone, using the NEB kit, we want about 2x excess of inserts. Using NEB calculator to figure out mass of insert required:

|  |  |  |
| --- | --- | --- |
| acr Synthesis 1 | 501bp | 26ng |
| acr Synthesis 2 | 529bp | 27.5ng |
| acr Synthesis 3 | 642bp | 33.4ng |

#### Gibson 1: Negative Control Reaction

| Item | Amount (µL) |
| --- | --- |
| pSB1C3 from J04450 (XbaI and SpeI digested) | 1.43 |
| dH2O | 9.41 |
| NEB Gibson Master Mix | 10.8 |

#### Gibson 2: acr Assembly

To have adequate overlap between acr inserts and pSB1C3, we’d need to digest something with pSB1C3 backbone with XbaI and SpeI (poorly designed inserts on our part). We did this to J04450 and gel extracted this band. However, this means that there’s a risk of a) contamination with J04450 plasmid and b) religation of the sticky ends of XbaI and SpeI. That’s why we’re running the negative control above and are considering reordering some gBlocks to make Gibsons go smoother. Other option would be to get new primers to amplify pSB1C3 with the overlaps designed into the primers.

| Item | Concentration (ng/µL) | Amount (µL) |
| --- | --- | --- |
| pSB1C3 from J04450 (XbaI and SpeI digested) | 35 | 1.43 |
| acr Synthesis 1 (EcoRI digested) | 100/12.5 | 3.01 |
| acr Synthesis 2 | 10 | 2.54 |
| acr Synthesis 3 (PstI digested) | 100/12.5 | 3.86 |
| NEB Gibson Master Mix |  | 10.8 |

#### Gibson 3: acr Assembly Alternate

* Strategy - ligating pSB1C3 cut with EcoRI and PstI, acr Synthesis 1 cut with EcoRI, acr Synthesis 3 cut with PstI
* Then this ligation is added to the Gibson reaction with acr Synthesis 2
* Back calculated approx amts of each component required so that concentrations of digest/ligation is high as possible

For 70ng digested pSB1C3, or 0.052 pmol, a 1:1 excess of insert means for the ligation:

| Item | Concentration (ng/µL) | Amount (µL) |
| --- | --- | --- |
| pSB1C3 Linear Backbone (EcoRI and PstI digested) | 20 | 3.5 |
| acr Synthesis 1 (EcoRI digested) | 100/12.5 | 2.12 |
| acr Synthesis 3 (PstI digested) | 100/12.5 | 2.12 |
| T4 Ligase | 100/12.5 | 0.46 |
| T4 Ligation Buffer |  | 0.91 |

* (Coordinated on digest concentrations with Liz, used NEB calculator to obtain ng amounts of each insert)
* Ligation was incubated at 10 min, RT; Heat killed at 65 °C for 20 min
* Added 7µL of this ligation (0.04pmol, theoretical max of 78ng ligated product) to 3µL of acr Synthesis 2 (0.088pmol or 30ng)

#### July 25, 2018

#### Gibson Reactons, Circular Version, Day 2

#### Sricharan

* Finished Gibson 2’ (same as 2 but redid since we were unsure about digests), 3 as described yesterday
* Next day, got only 1 colony from all Gibsons, from Strategy 2 … later realized this was probably religated plasmid

#### July 25, 2018

#### Miniprep of J23114, “J23110”, “S”, “W”, and cymA

#### Shannon

* “J23110” could be J23110 or J23100. “S” should be J23110, and “W” should be J23100.
* Spin down 1.5mL of overnight culture, discard supernatant, add another 1.5mL, spin down, discard supernatant, and rehydrate the pellet in 600µL dH2O.

+ Spun at 16,100g for 1 min each time

* Add 100µL lysis buffer, invert to mix
* Add 200µL cold neutralization buffer (kept on ice), invert to mix
* Centrifuge at 13,000 for 3 min
* Transfer supernatant to column (about 900µL)
* Place column in collection tube. Centrifuge 15 sec.
* Discard flow-through, put back into collection tube
* Add 200µL endo-wash buffer, centrifuge 30 sec.
* Add 400µL wash buffer, centrifuge 1 min
* Transfer column to clean 1.5mL tube. Add 30µL elution buffer. Let stand 1 min.
* Centrifuge 30 sec to elute DNA

#### July 25, 2018

#### Transformation with RbCl JM109 Cells

#### Sricharan and Peter

\*\*See Protocol from 7/16/18\*\*

#### Transforming:

BBa\_K1316012 (Mtr + T7 Lac O.) from 2016 kit w/ Strataclone Commercial Cells and our JM109 cells, FadD (from ligation + now purified w/ PCR purif), FadK (from ligation + now purified w/ PCR purif), BBa\_J23100 (Strong Anderson Promoter), BBa\_J23110 (Medium Anderson Promoter), BBa\_K864400 (pTac promoter), ACR Gibson 2 (7/24), Gibson control (7/24), ccmA-H 3A (7/24), ACR Gibson 2’ (7/25), ACR Gibson 3 (7/25)

#### Notes/Deviations

* Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube (Gibsons each get 1 μL per tube instead of 0.5, ligations should have maybe gotten 1 μL per tube instead of 0.5, 1 μL for both Mtr tubes of different cells)
* Took DNA from diluted Gibson solutions
* Heat-shocked for 30 seconds based on results from transformations of ccmA-H (done on 7/16)
* Pooled like tubes (same vector) after 1 hour incubation
* Amp plates for Anderson Promoters and Kanamycin plate for ccmA-H 3A

#### Results (7/26/18):

| Part/ Vector | Transformation Result |
| --- | --- |
| BBa\_K1316012 (Mtr + T7 Lac O.) 2016 kit Strataclone | 1 colony (O.C. on 7/26) |
| BBa\_K1316012 (Mtr + T7 Lac O.) 2018 kit Our JM109 Cells | No growth |
| FadD (from ligation + now purified w/ PCR purif) | No growth |
| FadK (from ligation + now purified w/ PCR purif) | 1 colony (O.C. on 7/26) |
| BBa\_J23100 (Anderson Strong) | Good growth (O.C. on 7/26) |
| BBa\_J23110 (Anderson Medium) | Good growth (O.C. on 7/26) |
| BBa\_K864400 (pTac) | No growth |
| ACR Gibson 2 (7/24) | 1 colony - could be plasmid backbone ligated to itself (O.C. on 7/26) |
| Gibson Control (7/24) | 1 colony - shows that plasmid backbone can ligate to itself (O.C. on 7/26) |
| ccmA-H 3A (7/24) | Good growth (O.C. on 7/26) |
| ACR Gibson 2’ (7/25) | No growth |
| ACR Gibson 3 (7/25) | No growth? (Might not have carried out this transformation) |

#### July 26, 2018

#### Resuspension of phz G-Blocks (end and 3) for Gibson

#### Peter

* Centrifuge tube for 3-5 seconds on table-top centrifuge
* Add TE to reach a final concentration of 10 ng/µL

+ Phz end 500ng / 50µL = 10 ng/µL
+ Phz 3 250ng/ 25µL = 10 ng/µL

* Vortex briefly
* Incubate @ 50 C for 20 minutes
* Vortex and briefly centrifuge
* Store in -20 C

#### July 26, 2018

#### Diagnostic Gel of B0015 and CymA and Gel Extract of PSB1C3

#### Peter and Liz

#### Wells:

| 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- |
| 2 Log Ladder | CymA | B0015 | PSB1C3 Extract | PSB1C3 Extract |
| -6 µL ladder | -5 µL Double Digest  -1 µL Loading Dye | -5 µL Double Digest  -1 µL Loading Dye | -5 µL Digest  -1 µL Loading Dye | -5 µL Digest  -1 µL Loading Dye |

![](https://static.igem.org/mediawiki/2018/b/b1/T--UConn--Gels-July-26.jpg)

Gel Extract (Zyppy gel extraction kit):

* Extracted last 2 wells of gel above to get pSB1C3 (brightest band)

+ Other DNA in the band is RFP and ‘tesA (length of ‘tesA is questionable but we don’t really care about that since we just want backbone)

* Pice of gel extracted: 0.1268 = 126.8mg → added 126.8 x 3 = 380.4µL ADP
* Incubated at 42C in heat block until agarose dissolved
* Put into spin column and centrifuged 1 min at 16,100g then pipetted out flow-through
* Added 200µL wash buffer and centrifuged 30s at 16,100g (did this step 2x)
* Added 6µL elution buffer (from invitrogen PCR purification kit), let sit for 1 min, then centrifuged 1 min at 16,100g

#### July 26, 2018

#### 3A Assembly of cymA and Anderson promoters into psb1K3

#### Shannon

* Need a 1:1 molarity of backbone to inserts, so use 8.768ng cymA and 0.397ng J23110
* Dilute promoter solutions

+ We need 0.397ng of each. If undiluted, you would need to small an amount to pipette
+ Add 1µL of the J23100/J23110 digestion (concentration = 57.48 ng/µL and 57.81ng/µL) to 29µL dH2O (new concentration = 1.916ng/µL and 1.927ng/µL).
+ Add 1µL of the J23114 digestion (concentration = 469.18ng/µL) to 249µL dH2O (new concentration = 1.88ng/µL)

| Component | Amount (µL) |
| --- | --- |
| Backbone (psb1K3) | 5 |
| cymA | 1.75 |
| Promoter | 0.2 |
| T4 ligase buffer | 2.5 |
| T4 enzyme | 1.25 |
| dH2O | 14.3 |

* Combine components in order listed
* Ligate at 16C for 30min
* Heat kill at 80 C for 20 min
* Note: accidently put 1.2µL of J23100 into that reaction.
* Note: I also forgot to dilute the promoters. Will have to re-do this tomorrow.

#### July 26, 2018

#### Some Planning

#### Sricharan

* Planned out a Gibson for phz, but realized we don’t have enough of 1F-3R and 4F-6R fragments PCRed
* Toni will run PCR of above two fragments again

#### July 26, 2018

#### Agarose Gel of Minipreps & PCR of Gibson 1F-3R and 4F-6R

#### Sricharan

| Lane | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- |
| Item | fadK MP Digest | ccmA-H + B0015 MP Digest | J23110 MP Digest | J23100 MP Digest | Phz Gibson 2 MP Digest | K1316012 MP Digest |

| 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- |
| Ladder (2-log) | Gibson Ctrl MP Digest | pSB1C3 PCR Digest | phz 1F-3R Gibson PCR | phz 4F-6R Gibson PCR | Nothing |

* All items digested w/PSTI except pSB1C3 (w/ EcoRI, PstI, DpnI - concentrations are off so might not work)

5µL item, 1µL loading dye, Dated 7/27/2018

![](https://static.igem.org/mediawiki/2018/9/90/T--UConn--Gels-July-26-2.jpg)

#### July 26, 2018

#### Restriction Digests for phz Gibson and CymA 3A

#### Liz

#### Start of phz: (adjusted so no water for higher concentration)

| Component | Amount (µL) |
| --- | --- |
| Water | 0 |
| CutSmart | 1.25 |
| DNA (rehydrated start of phz gBlock) | 10 (100ng x 1µL/10ng = 10µL → 10/4 = 2.5µL enzymes/water) |
| EcoRI | 1.25 |

#### End of phz: (adjusted so no water for higher concentration)

| Component | Amount (µL) |
| --- | --- |
| Water | 0 |
| CutSmart | 1.25 |
| DNA (rehydrated start of phz gBlock) | 10 (100ng x 1µL/10ng = 10µL → 10/4 = 2.5µL enzymes/water) |
| EcoRI | 1.25 |

#### Medium Anderson Promoter (J23110):

| Component | Amount (µL) |
| --- | --- |
| Water | 14.27 |
| CutSmart | 2 |
| DNA (J23110 miniprep) | 1.73 (100ng x 1µL/57.81ng = 1.73µL) |
| EcoRI | 1 |
| Spel | 1 |

#### cymA:

| Component | Amount (µL) |
| --- | --- |
| Water | 15.27 |
| CutSmart | 2 |
| DNA (cymA miniprep) | 0.83 (100ng x 1µL/120.6ng = 0.83µL) |
| XbaI | 1 |
| PstI | 1 |

#### Strong Anderson Promoter (J23100):

| Component | Amount (µL) |
| --- | --- |
| Water | 14.26 |
| CutSmart | 2 |
| DNA (J23100 miniprep) | 1.74 (100ng x 1µL/57.48ng = 1.74µL) |
| EcoRI | 1 |
| Spel | 1 |

#### Weak Anderson Promoter (J23114):

| Component | Amount (µL) |
| --- | --- |
| Water | 15.79 |
| CutSmart | 2 |
| DNA (rehydrated start of phz gBlock) | 0.213 (100ng x 1µL/469.18ng = 0.213µL) |
| EcoRI | 1 |
| Spel | 1 |

#### RFP miniprep (J04450):

| Component | Amount (µL) |
| --- | --- |
| Water | 6 |
| CutSmart | 2 |
| DNA | 10 |
| XbaI | 1 |
| Spel | 1 |

* For DNA used all of remaining miniprep and then another random miniprep (‘tesA?) to try to get to 10µL to maximize backbone
* Heat blocked at 37C for 1 hr then 80C for 15 min (put in thermocycler at 5µL reaction)

#### July 26, 2018

#### Transformations

#### Liz

Used 2 tubes of RbCl JM109 cells for each sample

* Gibson control (1µL DNA per tube)
* acr gibson 2 (1µL DNA per tube)
* acr gibson 2’ (1µL DNA per tube)
* K864400 (0.5µL DNA per tube)
* fadD ligation (1µL DNA per tube)

Followed transformation protocol (7/16/2018)

#### July 27, 2018

#### Restriction Digests

#### Shannon

#### FadK, J23110, J23100, gibson 2 transformation, gibson control

| Component | Amount (µL) |
| --- | --- |
| Water (milliQ) | For fadK: 16.57 |
|  | For J23110:16.26 |
|  | For J23100: 16.80 |
|  | For gibson 2 transformation: 16.56 |
|  | For gibson control: 16.54 |
| CutSmart | 2 |
| DNA | For fadK: 0.43 |
|  | For J23110: 0.74 |
|  | For J23100: 0.20 |
|  | For gibson 2 transformation: 0.44 |
|  | For gibson control: 0.46 |
| Pstl | 1 |

#### ccmA-H + B0015

| Component | Amount (µL) |
| --- | --- |
| Water (milliQ) | 14.97 |
| CutSmart | 2 |
| DNA | 1.03 |
| PstI | 1 |
| XbaI | 1 |

#### Stratoclone K1316012

| Component | Amount (µL) |
| --- | --- |
| Water (milliQ) | 15.79 |
| CutSmart | 2 |
| DNA | 0.21 |
| PstI | 1 |
| BspHI | 1 |

#### psb1K3

| Component | Amount (µL) |
| --- | --- |
| Water (milliQ) | 12 |
| CutSmart | 2 |
| DNA | 4 |
| PstI | 1 |
| DpnI | 1 |

#### psb1C3

| Component | Amount (µL) |
| --- | --- |
| Water (milliQ) | 24.4 |
| CutSmart | 9.76 |
| DNA | 10 |
| EcoRI | 4.88 |
| PstI | 4.88 |
| DpnI | 4.88 |

* Add reagents in order listed
* Centrifuge briefly
* Heat block at 37C

+ 10 min for all except psb1K3 and psb1C3. 45 min for those.

* Heat block at 80C for 15 min.
* Note: shouldn’t have increased the volume for the components in psb1C3. Did not need to scale.

#### August 2, 2018

#### Resuspending G-Blocks

#### Shannon

* Resuspended phz start, phz end, acr1, acr3, pmt1231.1, pmt1231.2
* 500ng of each g-block was deliverd, so 50µL TE was added to each for a final concentration of 10ng/µL.
* Centrifuge tubes for 3-5 sec on tabletop centrifuge
* Add TE
* Vortex briefly
* Incubate at 50C for 20min
* Briefly vortex and cetrifuge
* Store at -20C

#### July 27, 2018

#### Making SOC Medium

#### Peter and Ryan

* Bottle 1: Will be SOC Medium
* Bottle 2: 2 M MgCl2
* Bottle 3: 1 M Glucose

#### Add to Bottle 1:

| Component | Amount |
| --- | --- |
| Water (dH2O) | 950 mL |
| Bacto-tryptone | 20 g |
| Bacto-yeast extract | 5 g |
| NaCl | 0.5 g |
| KCl | 0.1863775 g  10 ml (0.01 L) of 250 mM solution (0.25 M solution)  0.01 L x 0.25mol/1L x 74.551g/1mol = 0.1863775g |

* Shake and used stir bar to dissolve components
* pH to 7.0 w/ 5 N NaOH (~ 0.2 ml) -- didn’t have any so used 1 N NaOH
* Adjust volume to 1 liter w/ dH2O (mark 1 liter point on bottle before adding components)
* Autoclave with Bottle 2

#### Add to Bottle 2:

| Component | Amount |
| --- | --- |
| Water (dH2O) | 90mL |
| MgCl2 | 19 g |

* After components are dissolved, adjust volume to 100 mL w/ dH2O (mark 100 mL point on bottle before adding components)
* Autoclave with Bottle 1
* Add 5 mL to Bottle 1 AFTER AUTOCLAVING

#### Bottle 3:

| Component | Amount |
| --- | --- |
| Water (dH2O) | 90mL |
| Glucose | 18g |

* After Glucose is dissolved, adjust volume to 100 mL w/ dH2O (mark 100 mL point on bottle before adding components)
* Sterilize by filtration through 0.22-micron filter
* Add 20 mL to Bottle 1 AFTER AUTOCLAVING

#### July 27, 2018

#### Minipreps

#### Liz

Miniprepped overnight cultures using zyppy miniprep kit

* fadK ligation
* ccmA-H + B0015 3A
* J23110
* J23100
* Gibson acr 2 transformation
* Gibson control
* Strataclone K1316012

* Used 3mL each overnight, spun down, and rehydrated in 600µL water
* Eluted in 30µL elution buffer

Week 12: 7/30/2018

#### July 30, 2018

#### 3 Digests and a Ligation (And a gel)

#### Sricharan and Liz

* Digesting 13µL of mtrCAB (K1316012) with BspHI and PstI to be ligated to start and assembled to pSB1K3
* Digesting 15µL of pSB1C3 (Peter’s PCR) and pSB1K3 (Linearized backbones kit) with EcoRI and PstI and DpnI (ligations in the future)
* 14µL ccmA-H + B0015 diagnostic digest with EcoRI to check on a gel
* 6µL of Mtr Start (gBlock) digested with EcoRI and BspHI to be ligated to rest of mtrCAB and assembled to
* 13µL of ccmA-H (K917006) also digested with EcoRI and SpeI for future 3A assembly
* Using standard digestion protocol (all 20µL digests)

#### Ligation: mtrCAB (TU Delft part) + synthesized mtr start (rehydrated gBlock) + pSB1K3

| Component | Amount (µL) |
| --- | --- |
| water | 3.24 |
| T4 ligase buffer | 0.67 |
| Backbone (pSB1K3 from digestion above) | 1.33 (25ng x 20µL/375ng = 1.33) |
| Digested mtr start (equimolar to backbone) | 0.28 (1.418ng x 20µL/100ng = 0.28) |
| Digested mtrCAB (equimolar to backbone) | 0.82 (58.87ng x 20µL/1437.36ng = 0.82) |
| T4 ligase | 0.33 |

* Scaled to 6.67µL reaction based on 25ng plasmid backbone

| Lane | 1 | 2 | 3 |
| --- | --- | --- | --- |
| Item | 2-log ladder | ccmA-H + B0015 cut with EcoRI (7/30), 5µL | pSB1C3 cut w/ EcoRI, PstI, DpnI (7/30), 1µL |

![](https://static.igem.org/mediawiki/2018/c/c4/T--UConn--Gels-July-30.jpg)

#### July 30, 2018

#### Transformation with RbCl JM109 Cells

#### Sricharan and Peter

\*\*See Protocol from 7/16/18\*\*

#### Transforming:

FadD (from ligation + now purified w/ PCR purif), mtrCAB 3A (mtrCAB + New Start + PSBIC3), BBa\_K864400 (pTac promoter)

#### Notes/Deviations:

* Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube.
* Ligation gets1 μL per tube instead of 0.5
* Took DNA from diluted Gibson solutions
* Heat-shocked for 30 seconds
* Pooled like tubes (same vector) after 1 hour incubation
* Kanamycin plate for mtrCAB 3A new start

#### Results (7/31/18):

| Part/Vector | Transformation Result |
| --- | --- |
| FadD (from ligation + now purified w/ PCR purif) | No growth |
| mtrCAB 3A (mtrCAB + New Start + PSBIC3) | Good growth (O.C. on 7/31) |
| BBa\_K864400 (pTac) | Good growth (O.C. on 7/31) |

#### July 31, 2018

#### PCR of phz 4f-6R, ‘tesA (L109P), Basic cymA

#### Peter

#### Notes:

* phz 4F-6R: 2.588 kb
* Basic ‘tesA (L109P): 0.57 kb
* Basic cymA: 0.595 kb

#### Set thermocycler:

| Phase | Time (sec) | Temp. (C) |
| --- | --- | --- |
| Initial denaturation | 30 | 98 |
| 35 cycles | 10 | 98 |
|  | 30 | Phz: 67 |
|  | Phz: 77.6s | ‘tesA & CymA: 72 (from NEB calculator) |
|  | ‘tesA & CymA: 17.3 | 72 |
| Final Extension | 10 minutes | 72 |
| Hold | ∞ | 4 |

* \*set to 50 µL reaction for phz 4F-6R
* \*set to 20 µL reaction for ‘tesA and cymA

#### Phz 4F-6R Tube:

| Component | Amount (µL) |
| --- | --- |
| dH2O | 29 |
| HF buffer | 10 |
| dNTP mix | 1 |
| 10mM forward primer (bad) | 2.5 |
| 10mM reverse primer (bad) | 2.5 |
| DMSO | 1.5 |
| Template DNA (Gibson of 4F-6R) | 3 (10ng x 1µL/3.785ng = 2.642µL) -rounding up to 3 µL |
| phusion | 0.5 |

#### ‘tesA (L109P):

| Component | Amount (µL) |
| --- | --- |
| dH2O | 12.6 |
| HF buffer | 4 |
| dNTP mix | 0.4 |
| 10mM forward primer (bad) | 1 |
| 10mM reverse primer (bad) | 1 |
| DMSO | 0.6 |
| Template DNA | 0.211 (10ng x 1µL/47.5ng = 0.211µL) -from 4x dilute of miniprep |
| phusion | 0.2 |

#### Cyma (Basic) Tube:

| Component | Amount (µL) |
| --- | --- |
| dH2O | 12.6 |
| HF buffer | 4 |
| dNTP mix | 0.4 |
| 10mM forward primer (bad) | 1 |
| 10mM reverse primer (bad) | 1 |
| DMSO | 0.6 |
| Template DNA (FadK PCR0) | 0.249 (10ng x 1µL/120.6ng = 0.249µL) -from 3x dilute of miniprep |
| phusion | 0.2 |

* All components were kept on ice
* Used lambda PCR cleanup kit

#### August 1, 2018

#### Gel

#### Peter

| Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Item | K86440 0(pTac) | Basic CymA (pcr purif.) | Basic ‘tesA (L109P) | 2-log ladder | Failed Lane | Failed Lane | phz 4F-6R (pcr purif.) | mtrCAB new st. 3A |

![](https://static.igem.org/mediawiki/2018/6/6c/T--UConn--Gels-August-1.jpg)

#### August 1, 2018

#### So Many Things

#### Sricharan

* Miniprepping Peter’s OCs of MtrCAB (3A Assembled w/ new start) ->3 colonies picked, 3 OCs
* Minipreps done with Zymo Plasmid MP Kit
* BBa\_K864400 (pTac) is 1 OC

* 250µL of each on ice, ready to add glycerol once we know which ones are good size… added glycerol

#### Lots of Digests (all in CutSmart buffer, all volumes in µL):

| Sample | mtrCAB new st (1-3) | K864400 (4) | ccmA-H (5) | fadK (6) | cymA Basic (7) | ‘tesA Basic (8) |
| --- | --- | --- | --- | --- | --- | --- |
| dH20 | 0 | 0 | 11 | 11 | 0 | 0 |
| Buffer | 2 | 2 | 2 | 2 | 2 | 2 |
| DNA | 16 | 16 | 5 | 5 | 16 | 16 |
| Enzyme | 1 XbaI  1 PstI | 1 EcoRI  1 SpeI | 1 EcoRI  1 SpeI | 1 XbaI  1 PstI | 1 EcoRI  1 PstI | 1 EcoRI  1 PstI |

* 1-4 by PC, 5-8 by SK

3A Assemblies … Using NEB Ligation Calculator for amounts, 1:1:1 molar ratios

#### 1) Anderson (J23110) + cymA + pSB1K3

| Component | Mass (ng) | Volume (µL) | Notes |
| --- | --- | --- | --- |
| pSB1K3 (SK 7/30) | 25 | 1.33 | Digested 7/30 |
| J23110 Digest (LCC 7/26) | 33.84 | 6.77 | 2983 bp |
| cymA Digest (LCC 7/26) | 32.25 | 6.45 | 2843 bp |
| T4 Ligase Buffer | - | 2 | - |
| T4 Ligase | - | 1 | - |
| dH2O | - | 2.45 | - |

#### 2) Anderson (J23110) + mtrCAB w/new st + pSB1C3

| Component | Mass (ng) | Volume (µL) | Notes |
| --- | --- | --- | --- |
| pSB1C3 (SK 7/30) | 25 | 0.68 | Digested 7/30 |
| J23110 Digest (LCC 7/26) | 36.03 | 7.21 | - |
| mtrCAB w/new st Digest (8/1) | 89.23 | 2.85 | > 388 bp |
| T4 Ligase Buffer | - | 2 | - |
| T4 Ligase | - | 1 | - |
| dH2O | - | 6.26 | - |

#### 3) ccmA-H (K917006) + B0015 + pSB1K3 … 10µL rxn

| Component | Mass (ng) | Volume (µL) | Notes |
| --- | --- | --- | --- |
| pSB1K3 (SK 7/30) | 25 | 1.33 | 2204 bp |
| B0015 Digest (LCC 7/24) | 24.94 | 4.99 | 2199 bp |
| ccmA-H Digest (SK 8/1) | 96.84 | 2.2 | 8537 bp |
| T4 Ligase Buffer | - | 1 | - |
| T4 Ligase | - | 0.5 | - |
| dH2O | - | 0 | - |

#### pTac (K864400) + fadK + pSB1K3 … 10µL rxn

| Component | Mass (ng) | Volume (µL) | Notes |
| --- | --- | --- | --- |
| pSB1K3 (SK 7/30) | 25 | 1.33 | 2204 bp |
| pTac Digest (PC 8/1) | 24.17 | 0.27 | 2131 bp |
| fadK Digest (SK 8/1) | 43 | 0.73 | 3717 bp |
| T4 Ligase Buffer | - | 1 | - |
| T4 Ligase | - | 0.5 | - |
| dH2O | - | 6.17 | - |

#### August 2, 2018

#### Planning Gibsons for Tomorrow

#### Sricharan

* phz (2x insert excess)
* Note for all the Gibsons we’re using new gBlocks that have revised short and end sequences designed to work well w/ pSB1C3 … using NEB calculator.

#### Fragments with identifying attributes and amounts listed below:

| pSB1C3 (SK 7/30) | phz Start 7.27.2018 | 1F-3R (7/27) | phz3 | 4F-6R (8/1) | phz End 7.27.2018 |
| --- | --- | --- | --- | --- | --- |
| 50ng | gBlock, 10 ng/µL | 24.6 ng/µL | gBlock, 10ng/µL | 95.3 ng/µL | gBlock, 10ng/µL |
| 20µL/375ng | 426 bp | 3072 bp | 222 bp | 2588 bp | 277 bp |
| 2.66µL | 20.6ng | 148 ng | 10.7ng | 125 ng | 13.4 ng |
| 0.037pmol | 2.06µL | 6.02µL | 1.07µL | 1.31 µL | 1.34 µL |

* 14.46µL total, add equivalent amt of NEB Gibson Master Mix

* We’ll also do a ctrl w/ 50ng plasmid only w/ rest dH2O and NEB MMix
* acr (2x insert excess)

#### Fragments with identifying attributes and amounts listed below:

| pSB1C3 (SK 7/30) | acr1 7.27.2018 | acr2 | acr3 7.27.2018 |
| --- | --- | --- | --- |
| 25ng | gBlock, 10ng/µL | gBlock, 10ng/µL | gBlock, 10ng/µL |
| 1.33µL | 512 bp | 529 bp | 653 bp |
|  | 12.4ng | 12.8ng | 15.8ng |
|  | 1.24µL | 1.28µL | 1.58µL |

* 5.43µL total, add equivalent amt of SGI Gibson MMix

pmt1231

#### Fragments with identifying attributes and amounts listed below:

| pSB1C3 (SK 7/30) | pmt1231.1 | pmt1231.2 |
| --- | --- | --- |
| 25ng | gBlock, 10ng/µL | gBlock, 10ng/µL |
| 1.33µL | 532 bp | 512 bp |
|  | 12.9ng | 12.4ng |
|  | 1.29µL | 1.24µL |

* 3.84µL total, add 1.14µL dH2O and 5µL SGI Gibson MMix

* Also doing control with 25ng plamsid only and rest dH2O and SGI MMix
* (1.33µL pSB1C3, 3.67µL dH2O, 5µL SGI MMix)

#### August 2, 2018

#### Transformation (RbCl JM109) of 3A’s from 8/1

#### Peter

\*\*See Protocol from 7/16/18\*\*

#### Transforming:

* CymA + J23110 + PSB1K3 (8/1 SK)
* mtrCAB w/ new st. + J23110 + PSB1C3 (8/1 SK)
* ccmA-H + B0015 + PSB1K3 (8/1 SK)
* fadK + pTac + PSB1K3 (8/1 SK)
* fadD ligation to PSB1C3 (7/24 LCC)

#### Notes/ Deviations:

* Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube. Exceptions: 2 x 1μL for CymA and Mtr (from 20 μL 3A so more dilute than std 10 μL 3A); 2 x 1μL for fadD (hasn’t worked yet so adding a lot of DNA)
* Heat-shocked for 30 seconds
* Pooled like tubes (same vector) after 1 hour incubation
* Plated with spinner and flame

#### Results (8/3/18):

| Part/Vector | Transformation Result |
| --- | --- |
| CymA + J23110 + PSB1K3 (8/1 SK) | Good growth (O.C. on 8/3) |
| mtrCAB w/ new st. + J23110 + PSB1C3 (8/1 SK) | No growth |
| ccmA-H + B0015 + PSB1K3 (8/1 SK) | Good growth (O.C. on 8/3) |
| fadK + pTac + PSB1K3 (8/1 SK) | ~32 colonies (O.C. on 8/3) |
| FadD (from ligation + now purified w/ PCR purif) | 3 colonies (O.C. on 8/3) |

#### August 3, 2018

#### Gibsons

#### Sricharan

* Set up Gibsons as detailed 8/2/2018
* Set @ 50°C 1 hr

+ 4°C infinite hold

* In thermal cycler (2 different cyclers for differing rxn volumes)

### August

Week 13: 8/6/2018

#### August 2, 2018

#### Transformation (RbCl JM109) of 3A’s from 8/1

#### Peter

\*\*See Protocol from 7/16/18\*\*

#### Transforming:

* mtrCAB w/ new st. + J23110 + PSB1C3 (8/1 SK)
* Phz Gibson (NEB) w/ redesigned start and end (8/3 SK)
* Acr Gibson (SGI) w/ redesigned start and end (8/3 SK)
* Pmt1231 Gibson (SGI) w/ redesigned start and end (8/3 SK)
* NEB PSB1C3 ctrl
* SGI PSB1C3 ctrl

Notes/ Deviations:

* Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube. Exceptions: 2 x 1μL for Mtr (from 20 μL 3A so more dilute than std 10 μL 3A)
* Heat-shocked for 30 seconds
* Pooled like tubes (same vector) after 1 hour incubation
* CAM plates for all

#### Results (8/7/18):

| Part/Vector | Transformation Result |
| --- | --- |
| mtrCAB w/ new st. + J23110 + PSB1C3 (8/1 SK) | No growth |
| Phz Gibson (NEB) w/ redesigned start and end (8/3 SK) | No growth |
| Acr Gibson (NEB) w/ redesigned start and end (8/3 SK) | No growth |
| Pmt1231 Gibson (SGI) w/ redesigned start and end (8/3 SK) | 5-10 colonies (O.C. on 8/8) |
| NEB PSB1C3 ctrl | No growth |
| SGI PSB1C3 ctrl | No growth |

#### August 7, 2018

#### Minipreps of 3As and fadD + Gel

#### Sricharan

Using Genscript Kit miniprepped:

* tac+fadK 3A
* cymA 3A (w/ med Anderson)
* B0015+ccmA-H 3A
* fadD

#### Nanodropped, results below:

| Sample Name | Concentration (ng/μL) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| tac+fadK | 4.08 | 1.51 | 1.16 |
| cymA 3A | 41.13 | 1.58 | 0.69 |
| B0015+ccmA-H | 59.52 | 1.61 | 0.77 |
| fadD | 156.54 | 1.82 | 1.90 |

#### Diagnostic digests of these items, with

|  |  |
| --- | --- |
| dH2O | 0μL |
| CutSmart | 2μL |
| DNA | 17μL |
| EcoRI | 1μL |

#### August 7, 2018

#### Gel of a bunch of things

#### Liz

Made a 1% agarose gel

| 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- |
| Ladder (6μL) | Tac + FadK EcoRI digest (5μL sample + 1μL digest) | cymA 3A EcorI digest (5μL sample + 1μL digest) | ccmA-H + B0015 3A EcoRI digest ((5μL sample + 1μL digest) | fadD EcoRI digest (5μL sample + 1μL digest) |

Only lane 5 (fadD) had a band

![](https://static.igem.org/mediawiki/2018/8/86/T--UConn--Gels-August-7.jpg)

#### August 8, 2018

#### Digests for 3A Assemblies

#### Liz, Peter

Double digests of pSB1A3 and J23114

#### pSB1A3:

|  |  |
| --- | --- |
| cutsmart | 2μL |
| DNA | 15μL |
| EcoRI | 1μL |
| PstI | 1μL |
| DpnI | 1μL |

DNA from iGEM linearized plasmid backbone

In retrospect this is bad because need water in the digestion

#### J23114:

|  |  |
| --- | --- |
| cutsmart | 2μL |
| DNA (J23114 miniprep SD 7/25) | 10μL |
| EcoRI | 1μL |
| SpeI | 1μL |
| water | 6μL |

* Heat blocked at 37C for 15 min
* Heat blocked at 80C for 15 min
* Assembly part is in Peter’s notebook

#### August 8, 2018

#### 3A Assemblies

#### Peter

\*\*\*Using NEB Ligation Calculator for amounts, 1:1:1 molar ratios\*\*\*

#### Protocol:

* Add components in following order: water, buffer, DNA, enzymes
* Let sit at room temp for 10 minutes
* Heat block @ 65 C for 20 minutes

#### mtrCAB w/ new st. + Anderson (J23114) + pSB1C3

| Component | Mass (ng) | Volume (µL) | Notes |
| --- | --- | --- | --- |
| pSB1C3 | 25 | 0.68 | (7/30 SK) |
| J23114 Digest | 36.03 | 0.21 (should have been 0.33) | (8/8 LCC) |
| mtrCAB w/ new st. | 89.58 | 2.86 | (8/1 SK) |
| T4 Ligase Buffer | - | 2 | - |
| T4 Ligase | - | 1 | - |
| dH2O | - | 3.26 | - |

#### mtrCAB w/ new st. + pTac (K864400) + pSB1A3

| Component | Mass (ng) | Volume (µL) | Notes |
| --- | --- | --- | --- |
| pSB1A3 | 25 | 1.25 | (8/8 LCC) |
| pTac (K864400) | 24.72 | 0.27 | (SK 8/1) |
| mtrCAB w/ new st | 2.74 | 2.86 | (SK 8/1) |
| T4 Ligase Buffer | - | 2 | - |
| T4 Ligase | - | 1 | - |
| dH2O | - | 2.74 | - |

#### August 8, 2018

#### Transformations of Gibsons and 3A Assemblies

#### Liz and Sricharan

| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| --- | --- | --- | --- | --- | --- | --- |
| NEB control gibson pSB1C3 (SK 8/3) | SGI control gibson pSB1C3 (SK 8/3) | Acr Gibson (SK 8/3) | Phz Gibson (SK 8/3) | mtrCAB with new start and J23114 pSB1C3 (PC 8/8) | mtrCAB with new start and pTac (K864400) in pSB1A3 (PC 8/8) | mtrCAB with new start and J23110 in pSB1C3 (SK 8/1) |

* 2 tubes per sample, 1μL DNA in each tube
* Followed transformation protocol (iGEM)

#### August 8, 2018

#### Colony PCR Checks of 3A Assemblies

#### Liz and Sricharan

1st Thing: Setting up PCR mixes … Fprimer = VF2, Rprimer = VR @ 10μM

#### Taq PCRs

| Item | GoTaq (stuff < 3kb) … 100μL rxn |
| --- | --- |
| 5x Buffer | 20μL |
| 10mM dNTPs | 2μL |
| F Primer | 5μL |
| R Primer | 5μL |
| dH2O | 62.5μL |
| GoTaq Polymerase | 0.5μL |
| Colony Suspensionr | 5μL |

|  | Temperature (°C) | Time |
| --- | --- | --- |
| Initial Denaturation | 95 | 5min |
| 35 Cycles | 95 | 45sec |
|  | 65 | 45sec |
|  | 72 | 2.5min |
| Final Extension | 72 | 5min |
| Soak | 4 | Infinite |

#### Phusion PCRs

| Item | NEB Phusion (ccm) … 50μL |
| --- | --- |
| 5x Buffer | 10μL |
| 10mM dNTPs | 1μL |
| F Primer | 2.5μL |
| R Primer | 2.5μL |
| dH2O | 31μL |
| GoTaq Polymerase | 0.5μL |
| Colony Suspension | 2.5μL |

|  | Temperature (°C) | Time |
| --- | --- | --- |
| Initial Denaturation | 98 | 5min |
| 35 Cycles | 98 | 10sec |
|  | 63 | 30sec |
|  | 72 | 2:10min |
| Final Extension | 72 | 10min |
| Soak | 4 | Infinite |

Picked 4 colonies of each 3A assembly (B0015+ccmA-H, tac+fadK, med Anderson+cymA)

Put tip in 20μL dH2O, pipetted up and down, spotted 3μL onto marked plate (Cam), incubated overnight … Later realized that these plasmids are kanamycin resistant

Put 0.5μL w/9.5μL of master mix above … ran cycler

Will run gel to check which ones are right

#### August 8, 2018

#### Colony PCR Checks of 3A Assemblies

#### Sricharan and Liz and Shannon

| Lane | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- |
| Item | 2-log ladder | ccm colony 1 | ccm colony 2 | ccm colony 3 | Phusion colony ctrl |

* Run @ 100V, 25min
* No visible bands on this gel (?)

| Lane | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- |
| Item | 2-log ladder | fadK colony 1 | fadK colony 2 | fadK colony 3 | fadK colony 4 |

| Lane | 6 | 7 | 8 | 9 | 10 |
| --- | --- | --- | --- | --- | --- |
| Item | cymA colony 1 | cymA colony 2 | cymA colony 3 | cymA colony 4 | GoTaq colony control |

Run @ 100V, 25min

![](https://static.igem.org/mediawiki/2018/f/f5/T--UConn--Gels-August-8.jpg)

#### August 9, 2018

#### Transformations & OCs

#### Sricharan and Peter

Usual PC protocol

* acr Gibson 8/3 (Strataclone, 1 tube, 1)
* phz Gibson 8/3 (Strataclone, 1 tube 1)
* weak Anderson + mtrCAB revised 8/8
* acr Gibson 8/3 (RbCl cells)
* phz Gibson 8/3 (RbCl cells)
* pTac + mtrCAB revised 8/8

3, 4, 5, 6 - 2 tubes RbCl cells (50μL aliquots), 1μL DNA each tube

OCs of following in Cam:

* tac+fadK 3A colony PCR (8/8)
* pmt1231 Gibson (8/6)
* med Anderson + mtrCAB w/ new start (8/8)

Week 14: 8/13/2018

#### August 13, 2018

#### Transformation of anderson, araC, and pBad/ araC promoters

#### Peter

\*\*See Protocol from 7/16/18\*\*

#### Transforming:

* BBa\_J23112 (amp)
* BBa\_J23103 (amp)
* BBa\_J23109 (amp)
* BBa\_J23117 (amp)
* BBa\_R0080 (inducible araC)
* BBa\_I0500 (inducible pBad/ araC)

#### Notes/Deviations:

* Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube
* Heat-shocked for 30 seconds
* Pooled like tubes (same vector) after 1 hour incubation

#### August 13, 2018

#### OCs

#### Sricharan

* OCs of pmt1231, acr, phz Gibsons - 2 colonies each
* cymA, tac+fadK 3As - 3 colonies each
* All OCs grew after 18 hours

#### August 14, 2018

#### Making Ampicillin Plates + Diagnostic Digests

#### Sricharan

From LCC’s MPs - Nanodropped - 3As are low concentration (~10-20ng/μL, Gibsons high concentration ~100ng/μL)

#### For digests:

| Item | 3A Assemblies | Gibsons |
| --- | --- | --- |
| dH2O | 0μL | 12μL |
| CutSmart | 2μL | 2μL |
| DNA | 17μL | 5μL |
| EcoRI-HF | 1μL | 1μL |

#### August 14, 2018

#### Lots of minipreps

#### Liz

Did 12 minipreps to digest and run on a gel

* Pmt1231 (1 and 2)
* Acr gibson (1 and 2)
* Phz gibson
* Tac and fadK 2A (1, 2, 3)
* cymA 3A

Used GenScript kit and eluted in 30μL elution buffer

Minipreps were digested and run on a gel

Only pmt1231 had a logical band

* Made a glycerol stock with 0.75mL 40% glycerol and 0.75mL pmt overnight culture

#### August 15, 2018

#### more minipreps

#### Liz

Did minipreps of overnight cultures

* BBa\_R0080
* BBa\_J23103

Used GenScript kit

* Eluted in 30μL elution buffer

Nanodropped:

* BBa\_R0080: 113.97 ng/μL
* BBa\_J23103: 307.66 ng/μL

#### August 15, 2018

#### Colony PCR Gel

#### Sricharan and Liz

More colony PCRs using same protocol as some weeks before, this time using the correct antibiotic selection plate.

#### August 16, 2018

#### Colony PCR Gel

#### Sricharan and Liz

All samples had 5μL DNA and 1μL loading dye

#### Gel 1:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| ladder | Mtr 1 | Mtr 2 | Mtr 3 | Mtr 4 | Mtr 5 | Mtr 6 | phz1 | phz2 | phz3 | ladder |
| 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| phz4 | phz5 | phz6 | ccm1 | ccm2 | ccm3 | ccm4 | ccm5 | ccm6 | Kan phusion ctrl | ladder |

![](https://static.igem.org/mediawiki/2018/a/aa/T--UConn--Gels-August-16.jpg)

#### Gel 2:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| ladder | fadK1 | fadK2 | fadK3 | fadK4 | fadK5 | fadK6 | acr1 | acr2 | Kan ctrl gotaq | ladder |
| 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| Cam ctrl gotaq | empty | cymA1 | cymA2 | cymA3 | cymA4 | cymA5 | cymA6 | Cam ctrl phusion | ladder | empty |

![](https://static.igem.org/mediawiki/2018/8/81/T--UConn--Gels-August-16-2.jpg)

#### August 16, 2018

#### phz Linear Gibson PCR

#### Liz

#### Thermocycler

|  | time | Temperature (C) |
| --- | --- | --- |
| Initial denaturation | 30 s | 98 |
| 35 cycles | 10 s | 98 |
|  | 20 s | 64 |
|  | 2:10 (min:sec) | 72 |
| Final extension | 10 min | 72 |
| hold | infinite | 4 |

#### PCR tube

|  |  |
| --- | --- |
| Phusion HF buffer | 4μL |
| DMSO | 0.6μL |
| 10mM 1F primer | 1μL |
| 10mM 6F primer | 1μL |
| dNTPs | 0.4μL |
| Template DNA (used SK linear gibson) | 1μL |
| phusion | 0.2μL |

Week 15: 8/20/2018

#### August 24, 2018

#### Restriction Digests for 3A Assemblies and 3A Assembly

#### Liz

cymA: cymA + J23110 (medium Anderson promoter) + pSB1K3

#### J23110

|  |  |
| --- | --- |
| water | 13.79μL |
| CutSmart | 2μL |
| DNA (J23110 miniprep 7/27 LCC) | 300ng = 2.21μL |
| EcoRI | 1μL |
| SpeI | 1μL |

#### cymA

|  |  |
| --- | --- |
| water | 13.51μL |
| CutSmart | 2μL |
| DNA (cymA miniprep SD 7/25) | 300ng = 2.49μL |
| EcoRI | 1μL |
| PstI | 1μL |

#### pSB1K3

|  |  |
| --- | --- |
| water | 4μL |
| CutSmart | 2μL |
| DNA (J23110 miniprep 7/27 LCC) | 300ng = 12μL |
| EcoRI | 1μL |
| PstI | 1μL |

* Put in heat block at 37C for 15 min
* Heat killed at 80C for 15 min

Forgot that backbone also needs to be digested with DpnI so cooled, added 1μL DpnI to pSB1K3, heatblocked at 37C for 15 min, and heat killed at 80C for 15 min

#### Used digestions in 3A assembly: cymA + J23110 + pSB1K3 (used open wetware protocol)

|  |  |
| --- | --- |
| water | 2.17μL |
| Digested pSB1K3 | 10ng = 0.7μL |
| Digested cymA | 37.02ng = 2.45μL |
| Digested J23110 | 47.75ng = 3.18μL |
| T4 ligase buffer | 1μL |
| T4 ligase | 0.5μL |

Thermocycler:

* 22.5C for 30 min
* 65C for 10 min

#### August 24, 2018

#### A lot of colony PCR fails and Linear Gibson

#### Sricharan

A new Linear Gibson of phz 1F-6R

|  |  |
| --- | --- |
| 5μL | NEB MMix |
| 3.01μL | 1F-3R (7/27) |
| 0.53μL | phz3 (gBlock) |
| 0.66μL | 4F-6R (8/1) |
| 0.80μL | dH2O |

Running PCR on this Gibson (Phusion) using usual protocol, and using 1μL of template. Annealing temperature is 64 degrees C, extension time is 2min,30sec.

Week 16: 8/27/2018

#### August 27, 2018

#### cymA 3A gel

#### Sricharan and Peter

Changing some items to potentially have success in Gibson reactions:

Also redesigned inserts so that there are correct overlaps with pSB1C3

#### Using 0.05pmol backbone, equimolar inserts

| pSB1C3 (SK 7/30) | acr1 7.27.2018 | acr2 | acr3 7.27.2018 |
| --- | --- | --- | --- |
| 3.57μL | gBlock (532bp) | gBlock (529bp) | gBlock (653bp) |
| 67ng | 16.57ng | 17.12ng | 21.14ng |
| 2070bp | 1.66μL | 1.71μL | 2.11μL |

| pSB1C3 (SK 7/30) | phz Start 7.27.2018 | phz 1F-6R | phz End 7.27.2018 |
| --- | --- | --- | --- |
| 3.57μL | gBlock (426bp) | gBlock (5882bp) | gBlock (277bp) |
| 67ng | 13.79ng | 190.4ng | 8.97ng |
| 2070bp | 1.38μL | 2.73μL | 0.90μL |

For actual reactions:

* Cutting reaction volume in half (5μL Master mix, 5μL DNA + water)

#### August 27, 2018

#### phz 1F-6R Gel

#### Liz

Made a 1% agarose gel for linear phz 1F-6R gibson PCR

Purified linear 1F-6R gibson PCR (SK 8/24) using lambda kit

Nanodropped: 69.7 ng/μL

#### Gel:

| 1 | 2 | 3 | 4 |
| --- | --- | --- | --- |
| ladder | Phz gibson pure PCR | pSB1C3 A | pSB1C3 B |
|  | 5μL sample + 1μL dye | 1μL dye+ 1μL sample + 4μL water |  |

![](https://static.igem.org/mediawiki/2018/3/34/T--UConn--Gels-August-27.jpg)

#### August 28, 2018

#### Linear Gibson PCR + Making more Backbone

#### Sricharan

* Running Phusion PCR on phz 1F-6R (before the PCR was ~1kb, should have been ~6kb), pSB1C3

* Usual protocol: for phz used 0.4μL sample in 20μL rxn, annealing at 64 degrees C, extension 2 minutes. Did PCR in HF and GC Buffers
* For pSB1C3, used 0.2μL of Peter’s diluted J04450 MP (7/24) to 0.14ng/μL (100x). This was a 50μL rxn, annealing at 71 degrees C, 40s extension.

* PCR purified on 8/28 by Sricharan

#### Nanodrop

|  | phz HF | phz GC | pSB1C3 HF |
| --- | --- | --- | --- |
| Conc (ng/μL) | 15.1 | 21.7 | 39.5 |
| 260/280 | 1.43 | 1.63 | 1.71 |
| 260/230 | -0.27 | -0.48 | -0.49 |

* Curves look like junk for all 3
* Peter has gel on pg 49

#### August 28, 2018

#### Gel of Phz (1F-6R) pcr’s and pSB1C3

#### Peter

| Lane | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| Item | 2-log ladder | - 3 μL Phz (HF) pcr  -2 μL dH2O  -1 μL loading dye | Phz (GC) pcr  -2 μL dH2O  -1 μL loading dye | pSB1C3 (HF)  -2 μL dH2O  -1 μL loading dye |

![](https://static.igem.org/mediawiki/2018/c/cc/T--UConn--Gels-August-28.jpg)

#### August 28, 2018

#### Transformation (RbCl JM109) of 3A’s from 8/1

#### Peter

\*\*See Protocol from 7/16/18\*\*

#### Transforming:

* Acr circular gibson (8/27 SK)
* CymA + J23110 + PSB1K3 3A (8/24 LCC)

#### Notes/Deviations

* Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube. Exceptions: 2 x 1μL for Mtr (from 20 μL 3A so more dilute than std 10 μL 3A)
* Heat-shocked for 30 seconds
* Pooled like tubes (same vector) after 1 hour incubation
* CAM for Acr
* Kan for CymA

#### August 30, 2018

#### Overnight Cultures of cymA 3A Assemblies

#### Liz

* Overnights of cymA 3A assemblies
* Took 3 colonies from “RbCl transformation cymA + J23110 + pSB1K3 3A PC 8/28” plate

+ 3A done by LCC 8/24

* 5mL LB broth + 5μL kanamycin
* Put in 37C shaker overnight

#### August 31, 2018

#### Miniprep of Cultures Liz Picked up this Morning

#### Sricharan

MP of cymA + J23110 + pSB1K3 by LCC … Genscript Kit, eluted 30μL and glycerol stocks

### September

Week 17: 9/3/2018

#### September 4, 2018

#### cymA 3A gel

#### Liz

Made 1% agarose gel

Nanodropped SK’s cymA minipreps 1-3 (8/31)

* Miniprep 1: 21.05 ng/μL
* Miniprep 2: 17.49 ng/μL
* Miniprep 3: 29.24 ng/μL

#### Gel:

| 1 | 2 | 3 | 4 |
| --- | --- | --- | --- |
| Ladder (6μL) | cymA miniprep 1 | cymA miniprep 2 | cymA miniprep 3 |

Samples loaded with 5μL sample and 1μL loading dye

![](https://static.igem.org/mediawiki/2018/4/44/T--UConn--Gels-September-4.jpg)

Didn’t digest before running gel--ran again next day

#### September 5, 2018

#### cymA 3A gel 2.0

#### Liz

Digested cymA minipreps with EcoRI to run on gel

|  |  |
| --- | --- |
| CutSmart | 2μL |
| DNA (cymA minipreps 1-3 SK 8/31) | 12μL |
| EcoRI | 1μL |
| water | 5μL |

Heat blocked at 37C for 15 min

Heat killed at 65C for 15 min

Ran 1% agarose gel (same setup as previous day)

![](https://static.igem.org/mediawiki/2018/1/1f/T--UConn--Gels-September-5.jpg)

#### September 5, 2018

#### Gel extraction of phz

#### Peter

#### Notes:

* Following Azady’s advice on getting greater concentration of pieces for gibson

#### Gel:

| 1 | 2 |
| --- | --- |
| Ladder (6μL) | 10 μL phz 4F-6R pure pcr (PC 7/13) |

![](https://static.igem.org/mediawiki/2018/3/3d/T--UConn--Gels-September-5-2.jpg)

Extraction:

* Bio basic kit
* 400 μL buffer \* (0.048 g/100 mg) \* (1000 mg/1 g) = 192 μL buffer used

#### September 7, 2018

#### phz 4F-6R PCRs - Aiming for high purity

#### Sricharan

Using

* Original Gibson (7/12)
* Old gel extract
* PC new gel extract (9/5)

Usual Phusion PCR, 1μL template DNA, 67 degrees C annealing, 1:18 extension time. All put into 1.5mL tubes

Week 18: 9/10/2018

#### September 10, 2018

#### PCR purification, Nanodrop, Gel

#### Ryan

Started off by preparing a small gel, .125g agarose, 12.5 mL TAE, mixed and microwaved for 30 seconds. Added 1.25 uL SYBR Safe and poured into mold and let sit

PCR purification of Charan’s 3 PCR products from 9/3/18, phz 4F-6R

* Volume of PCR product was 20 uL, so added 100 ul Binding Buffer, and loaded it into Spin column and centrifuged for 1 min, @10,000 rpm
* Discard flow-through and add 700 uL Wash Buffer, centrifuge again (same settings)
* Discard flow-through, centrifuge again
* Transfer top of spin column into a new 1.5 mL tube for collection
* Add 40 uL Elution Buffer and let sit for 1 min
* Centrifuge again

I labelled the 3 PCR products 1, 2, 3 and ran a nanodrop

* 1 was found to be 22.8 ng/uL
* 2 was found to be 19.1 ng/uL
* 3 was found to be 16.5 ng/uL

Put gel into the electrophoresis machine and added 6 uL 2-log Ladder into lane 1

* 3 uL of PCR 1 + 2uL water + 1 uL loading dye into lane 2
* 3 uL of PCR 2 + 2uL water + 1 uL loading dye into lane 3
* 3 uL of PCR 3 + 2uL water + 1 uL loading dye into lane 4

And ran it for 30 minutes at 100V \*23 minutes dye was near end of gel

#### September 10, 2018

#### Azady PCRs acr3 and pXB1C3

#### Liz

#### Acr

|  |  |
| --- | --- |
| HF buffer | 4uL |
| 10mM dNTPs | 0.4uL |
| F primer (Azady acr 3F) | 1uL |
| R primer (Azady acr 3R) | 1uL |
| Template DNA (acr 3 gBlock) | 0.2uL |
| DMSO | 0.6uL |
| phusion | 0.2uL |
| Water | 12.6uL |

#### pSB1C3

|  |  |
| --- | --- |
| HF buffer | 4uL |
| 10mM dNTPs | 0.4uL |
| F primer (SB-prep-3P-1-mutated) | 1uL |
| R primer (SB-prep-2Ea) | 1uL |
| Template DNA (BBa\_R0080 diluted 10x) | 0.2uL |
| DMSO | 0.6uL |
| phusion | 0.2uL |
| Water | 12.6uL |

#### Thermocycler

|  |  |  |
| --- | --- | --- |
| Initial denaturation | 98C | 30s |
| 35 cycles | 98C | 10s |
|  | 67C | 30s |
|  | 72C | 35s |
| Final extension | 72C | 10 min |
| hold | 4C | infinite |

#### September 13, 2018

#### Azady PCRs, acr 1 & 2

#### Sricharan

PCRing parts of acr to remove hairpins and amplify gBlocks, for better Gibson results

Resuspended primers in TE

* spun down dry tubes
* to 100µM in tubes, let sit 10 min
* aliquot and dilute to 10µM

* Usual Phusion HF Buffer
* For acr 1, Primers: Azady acr 1F + Azady acr 1R, Anneal 68°C
* For acr 2, Primers: Azady acr 2F + Azady acr 2R, Anneal 67°C
* Usual cycle, with 15s extension time for both

* Transferred into 1.5mL tubes

#### September 13, 2018

#### PCR Purifs + Gel of acr 1-3 + pSB1C3 mut

#### Sricharan

* PCR purifying acr 1-3 gBlock PCRs & pSB1C3 mut (adapted for Gibson)
* Lambda Kit, eluting with 30µL … Nanodrop and gel by PC

#### September 14, 2018

#### Gel of Acr g-block PSB1C3 pcr’s

#### Peter

#### Notes:

* Following Azady’s advice to pcr the g-blocks and backbone before gibson
* Backbone is mutated (using primers with one nucleotide difference) so can overlap with new acr 1 & 3 g-blocks and not form hairpin

#### Gel:

| 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- |
| Ladder (6μL) | Acr 1 pcr purif. (9/14 SK)  -512 bp | Acr 2 pcr purif. (9/14 SK)  -529 bp | Acr 3 pcr purif. (9/14 SK)  -653 bp | Mutated pSB1C3 pcr purif. (SK 9/14) |

![](https://static.igem.org/mediawiki/2018/7/78/T--UConn--Gels-September-14.jpg)

Week 19: 9/17/2018

#### September 17, 2018

#### Azady PCR acr 1 and 2 (more DNA)

#### Liz

|  |  |
| --- | --- |
| HF buffer | 4uL |
| 10mM dNTPs | 0.4uL |
| F primer (Azady acr 1F/2F) | 1uL |
| R primer (Azady acr 1R/2R) | 1uL |
| Template DNA (acr 1, 2 gBlock) | 2uL |
| DMSO | 0.6uL |
| phusion | 0.2uL |
| Water | 10.8uL |

Temperatures for thermocycler from Sricharan’s same PCR last week

![](https://static.igem.org/mediawiki/2018/c/c3/T--UConn--Gels-September-17.jpg)

#### September 18, 2018

#### Zymo DNA concentrator planning (acr)

#### Peter

#### Notes:

* Concentrating fragments before gibson as suggested by Azady
* Wanted 50 ng of acr 3 and calculated equimolar amounts of other fragments (acr 1 & 2)
* Used 2:1 fragment to backbone ratio
* Need to add at least 6 μL elution buffer
* Added total of 10 μL of elution buffer to get 8.7 μL final product
* Below is initial amounts added to column, see zymo kit for further details on the next steps and buffers to add
* Centrifuging done at 15500 g

| Component | Amount (μL) |
| --- | --- |
| Acr 3 pcr purif. (9/14 SK) | 5.62 |
| Acr 1 pcr purif. (9/14 SK) | 3.25 |
| Acr 2 pcr purif. (9/14 SK) | 2.57 |
| Mutated pSB1C3 pcr purif. (9/14 SK) | 7.86 |
| Binding Buffer | 96.5 = 19.3 \* 5 (total volume DNA mult. 5) |

#### September 18, 2018

#### Gibson of acr (circular)

#### Peter

| Component | Amount (μL) |
| --- | --- |
| Zymo pcr fragments product | 8.7 |
| SGI GA HiFi Master Mix | 8.7 |

* -run in thermo cycler @ 50 C for 1 hour

#### September 19, 2018

#### Transformation of acr Gibson (by PC)

#### Sricharan

We’ve now PCRed parts for acr Gibson and backbone (some mutations to remove secondary structure)

* Peter concentrated DNA and ran Gibson yesterday
* Note: (By accident) we forgot to restriction digest pSB1C3 mut
* Will this affect product?
* Also no - control with backbone … will do - control with water

* Transformation of the usual RbCl2 cells with 1μL in each 50μL cells tube

* Acr - transformed with 1μL acr Gibson each 50μL cells
* Ctrl - transformed with 1μL dH2O each 50μL cells … 1 tube was forgotten so only transformed tube

* Plates labeled PC since Peter helped

#### September 19, 2018

#### Azady PCR: acr 3 and pSB1C3 (2nd time)

#### Sricharan

* Acr 3: Phusion HF PCR with 67°C annealing, 15 sec extension, primers Azady acr 3F and azady acr 3R
* pSB1C3: Phusion HF PCR with 66°C annealing, 40 sec extension, primers SB-prep-3P-1-mut and SB-prep-2-Ea

Week 20: 9/24/2018

#### September 24, 2018

#### Acr 1-3 PCR cleanup and gel

#### Liz

Made 1% agarose gel (small wells)

PCR purified acr 1-3 and pSB1C3

* Acr 1-2 LCC 9/17
* Acr 3 and pSB1C3 mut SK 9/21
* Lambda kit
* Eluted in 30μL

Nanodropped:

* Acr 1: 12.68 ng/μL
* Acr 2: 19.82 ng/μL
* Acr 3 HF: 33.59 ng/μL
* Acr 3 GC: 9.76 ng/μL
* pSB1C3: 78.88 ng/μL

#### Gel:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| ladder | Acr 1 | Acr 2 | Acr 3 HF | Acr 3 GC | pSB1C3 | ladder | Acr 1 |
| 6μL | 5μL sample, 1μL dye |  |  |  |  | 6μL | 1μL dye,  1.5μL water,  3.5μL sample |

![](https://static.igem.org/mediawiki/2018/6/69/T--UConn--Gels-September-24.jpg)

#### September 26, 2018

#### Transforming acr

#### Sricharan

Trying again to transform Gibson acr from 9/17

* 1 tube (50μL) Strataclone - 4μL acr Gibson (9/17)
* 2 tubes (100μL) RbCl cells - 8μL acr Gibson (9/17)

* 1 tube (50μL) RbCl cells - 4μL dH2O (ctrl)

* Usual protocol for RbCl cells
* For Strataclone
* 20 min on ice
* 45 sec heat shock
* 2 min on ice
* 250μL SOC - Incubate slightly >1hr
* Plate 100μL or so

* Tubes started 1 hr incubation at 2:45pm
* End 3:45 pm

3 plates waiting on bench for Peter to plate - not in warm room since it seems humid there

#### September 27, 2018

#### acr OCs

#### Sricharan

* Picked up transformations from yesterday
* 1 colony RbCl acr
* Many (~20 colonies) Strataclone acr

OC’ed 1 RbCl and 6 Stratclone colonies

* LB from Kate downstairs (says it was made 2013 on bottle?)
* Accidentally forgot to add Cam so added after tips were submerged in tubes
* In at 4:38pm, shaker downstairs

#### September 28, 2018

#### acr Miniprep/Diagnostic

#### Liz

Made 1% agarose gel

Minipreps of strataclone acr 1-6 and RbCl acr (SK 9/27)

* Used genscript kit
* Eluted in 30μL elution buffer

#### Single digest for diagnostic gel

|  |  |
| --- | --- |
| CutSmart | 2μL |
| water | 7μL |
| Miniprep DNA (str acr 1-6 and RbCl acr) | 10μL |
| EcoRI | 1μL |

Thermocycler:

* 37C for 15 minutes
* 65C for 15 min
* 4C hold

#### Gel:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| ladder | Acr str 1 | Acr str 2 | Acr str 3 | Acr str 4 | Acr str 5 | Acr str 6 | RbCl acr |

5μL sample, 1μL loading dye

![](https://static.igem.org/mediawiki/2018/f/fa/T--UConn--Gels-September-28.jpg)

## Protocols

Note: Embedded PDFs not viewable in Safari. Click/tap "Download" to view them.

[Download](https://static.igem.org/mediawiki/2018/f/f4/T--UConn--3AAssembly.pdf)

### 3A Assembly

[Download](https://static.igem.org/mediawiki/2018/1/1d/T--UConn--Chloramphenicol-LB-Broth.pdf)

### Chloramphenicol LB Broth

[Download](https://static.igem.org/mediawiki/2018/f/f7/T--UConn--DNA-Gel-Extraction-%28EZ-10_Kit%29.pdf)

### DNA Gel Extraction (EZ-10 Kit)

[Download](https://static.igem.org/mediawiki/2018/a/a2/T--UConn--DNA-Ligation.pdf)

### DNA Ligation

[Download](https://static.igem.org/mediawiki/2018/5/5e/T--UConn--Enzyme-DNA-Digestion.pdf)

### Enzyme DNA Digestion

[Download](https://static.igem.org/mediawiki/2018/b/b3/T--UConn--GenScript-Miniprep.pdf)

### GenScript Miniprep

[Download](https://static.igem.org/mediawiki/2018/5/57/T--UConn--Gibson-Assembly.pdf)

### Gibson Assembly

[Download](https://static.igem.org/mediawiki/2018/5/5e/T--UConn--Lambda-PCR-Purification-Kit.pdf)

### Lambda PCR Purification Kit

[Download](https://static.igem.org/mediawiki/2018/9/99/T--UConn--LB-Agar-Plates-with-Antibiotic.pdf)

### LB Agar Plates with Antibiotic

[Download](https://static.igem.org/mediawiki/2018/8/8a/T--UConn--PCR-Protocol-for-Phusion-High-Fidelity-DNA-Polymerase.pdf)

### PCR Protocol for Phusion® High-Fidelity DNA Polymerase

[Download](https://static.igem.org/mediawiki/2018/c/c5/T--UConn--PureLink-PCR-Purification-Kit.pdf)

### PureLink PCR Purification Kit

[Download](https://static.igem.org/mediawiki/2018/5/53/T--UConn--RbCl-Competent-Cell-Transformation.pdf)

### RbCl Competent Cell Transformation

[Home](https://2018.igem.org/Team%3AUConn)

[Project](https://2018.igem.org/Team%3AUConn/Project)

* [Background](https://2018.igem.org/Team%3AUConn/Project#background)
* [Description](https://2018.igem.org/Team%3AUConn/Project#description)

[Notebook](https://2018.igem.org/Team%3AUConn/Notebook)

* [Interlab](https://2018.igem.org/Team%3AUConn/Notebook#interlab)
* [Lab Notebook](https://2018.igem.org/Team%3AUConn/Notebook#labnotebook)
* [Protocols](https://2018.igem.org/Team%3AUConn/Notebook#protocols)

[Parts](https://2018.igem.org/Team%3AUConn/Parts)

* [List of Parts](https://2018.igem.org/Team%3AUConn/Parts#listofparts)

[Human Practices](https://2018.igem.org/Team%3AUConn/HumanPractices)

* [Community Outreach](https://2018.igem.org/Team%3AUConn/HumanPractices#communityoutreach)
* [Collaborations](https://2018.igem.org/Team%3AUConn/HumanPractices#collaborations)

[Safety](https://2018.igem.org/Team%3AUConn/Safety)

[Team](https://2018.igem.org/Team%3AUConn/Team)

* [Attributions](https://2018.igem.org/Team%3AUConn/Notebook#attributions)

[![instagram](https://static.igem.org/mediawiki/2018/3/35/T--UConn--Instagram.svg)](https://www.instagram.com/uconnigem/)

[University of Connecticut (UConn), Storrs, Connecticut 06269, United States of America](https://goo.gl/maps/yexLUgoBazx)
Contact us atuconnigem@gmail.com


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

# Team:UConn/Experiments

![](https://static.igem.org/mediawiki/2018/4/4f/2018_igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/igem.org/3/3b/Menu_icon.svg)

[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3AUConn)
[HOME](https://2018.igem.org/Team%3AUConn)

TEAM

[Team Members](https://2018.igem.org/Team%3AUConn/Team)
[Collaborations](https://2018.igem.org/Team%3AUConn/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AUConn/Description)
[Design](https://2018.igem.org/Team%3AUConn/Design)
[Experiments](https://2018.igem.org/Team%3AUConn/Experiments)
[Notebook](https://2018.igem.org/Team%3AUConn/Notebook)
[InterLab](https://2018.igem.org/Team%3AUConn/InterLab)
[Model](https://2018.igem.org/Team%3AUConn/Model)
[Results](https://2018.igem.org/Team%3AUConn/Results)
[Demonstrate](https://2018.igem.org/Team%3AUConn/Demonstrate)
[Improve](https://2018.igem.org/Team%3AUConn/Improve)
[Attributions](https://2018.igem.org/Team%3AUConn/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AUConn/Parts)
[Basic Parts](https://2018.igem.org/Team%3AUConn/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AUConn/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AUConn/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AUConn/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AUConn/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUConn/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AUConn/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AUConn/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AUConn/Hardware)
[Measurement](https://2018.igem.org/Team%3AUConn/Measurement)
[Model](https://2018.igem.org/Team%3AUConn/Model)
[Plant](https://2018.igem.org/Team%3AUConn/Plant)
[Software](https://2018.igem.org/Team%3AUConn/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=UConn)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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

# Team:UConn/Results

[![](https://static.igem.org/mediawiki/2018/b/bf/T--UConn--Logo.svg)](https://2018.igem.org/Team%3AUConn)

* [![ ](https://static.igem.org/mediawiki/2018/9/95/T--UConn--ExperimentIcon.svg )](/Team%3AUConn/Description)

  ###### Project

  [Description](https://2018.igem.org/Team%3AUConn/Description%20)
  [Design](https://2018.igem.org/Team%3AUConn/Design%20)
  [Model](https://2018.igem.org/Team%3AUConn/Model%20)
  [Results](https://2018.igem.org/Team%3AUConn/Results%20)
* [![ ](https://static.igem.org/mediawiki/2018/f/f1/T--UConn--NotebookIcon.svg )](/Team%3AUConn/InterLab)

  ###### Notebook

  [InterLab](https://2018.igem.org/Team%3AUConn/InterLab%20)
  [Model](https://2018.igem.org/Team%3AUConn/Model%20)
  [Demonstrate](https://2018.igem.org/Team%3AUConn/Demonstrate%20)
  [Improve](https://2018.igem.org/Team%3AUConn/Improve%20)
  [Attributions](https://2018.igem.org/Team%3AUConn/Attributions%20)
* [![ ](https://static.igem.org/mediawiki/2018/2/2f/T--UConn--PartsIcon.svg )](/Team%3AUConn/Parts)

  ###### Parts

  [Parts Overview](https://2018.igem.org/Team%3AUConn/Parts%20)
  [Basic Parts](https://2018.igem.org/Team%3AUConn/Basic_Part%20)
  [Composite Parts](https://2018.igem.org/Team%3AUConn/Composite_Part%20)
  [Parts Collection](https://2018.igem.org/Team%3AUConn/Part_Collection%20)
* [![ ](https://static.igem.org/mediawiki/2018/3/3e/T--UConn--HumanIcon.svg )](/Team%3AUConn/Human_Practices)

  ###### Human Practices

  [Human Practices](https://2018.igem.org/Team%3AUConn/Human_Practices%20)
  [Education and Engagement](https://2018.igem.org/Team%3AUConn/Public_Engagement%20)
  [Collaborations](https://2018.igem.org/Team%3AUConn/Collaborations%20)
[![ ](https://static.igem.org/mediawiki/2018/0/02/T--UConn--SafetyIcon.svg )](/Team%3AUConn/Safety%20)
[![ ](https://static.igem.org/mediawiki/2018/1/1f/T--UConn--TeamIcon.svg )](https://2018.igem.org/Team%3AUConn/Team%20)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)