iGEM 2017 — Team Arizona_State

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# Team:Arizona State/Notebook

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# Arizona\_State

# Notebook:Amber Mani

## Friday 6/23/17 - Running colony PCR on the AUBr and the BTA3

From earlier this week the AUBr results showed that number 9 may be a match for the vector we needed but the sequencing was returned and it was an error. Redoing the AUBr today and running the PCR for the BTA3 to try for a result on at least one per receiver.

Since it is the weekend, instead of placing the new agar plate with the 24 samples of plated AUBr and BTA3 into the 37c incubator, leaving the sample out in room temperature as to not accelerate the growth too much over the weekend. See picture below:

![](https://static.igem.org/mediawiki/2017/3/3b/Amb_1.png)
![](https://static.igem.org/mediawiki/2017/6/6a/Amb_2.png)

* Defrost template, 2x Master Mix and primers
* Determine desired total working volume (~20uL per tube of 24 total/ each AUBr and BTA3 have 24 samples=520ul. The additional 40ul over the 480ul is to give 40ul (2 additional samples) over as precautionary buffer)
* Label reaction tubes
* Create below reaction mix
* Place mixture in thermal cycler at settings shown below

\* Used 24 PCR tubes and labeled 1-24 for each receiver

\* Thawed two AMP plate and labeled 24 spots on tray / AUB/AMBER/6/23/17 and BTA3/BRIANNA/6/23/2017

\* Used 260 μl of Go Taq Green - added to each 2 DNA tubes

\* Added 249.6 μl of H20, to each of the 2 DNA tubes

\* Added FP and RP 5.2 ul of each to DNA tubes,(1 μ molar as final concentration)

\* Pipetted 20 μl of newly mixed green solution into each PCR tube

### Colony Prep: See images at the beginning of todays entry for the new plated 24 samples of AUBr and BTA3

\* Next scraped one colony at a time (up to the 24 used) from the existing grown AUB plate and gently placed the colonly onto the new AUB plate with the labeled 24 spots. Disposed of each tip used into the cooresponding PCR tube labeled to match the placement number on the new AUB plate. Same protocol for the BTA3.

\* Used empty pipette to attach to each tip in the PCR tubes and pump up and down to mix and remove as much of the leftover bacteria from the tip as possible so it mixed into the PCR tube. Then disposed of the tips in dry waste.

\* Took 2 full sets of 24 PCR tubes to the thermocycler and had Stephan help with the settings since each setting is different for each type of bacteria or sample being run for PCR.

\* PCR set to take 2.5 hours/ in process now.

PCR finished up and running gels for both the AUBr and BTA3.

Running a gel to test the DNA vector concentration that we are looking for - AUBr has 24 samples with 20 μl in each PCR tube and BTA3 has 24 samples of 20 μl each PCR tube. Will run 2 gels, one for AUB and one for BTA3, each with with 5 μl of each sample in the gel. To fit everything we ran with 2 rows of the 14 wells.

\* The first gel to run is the AUBr: The first well is the KB+ ladder, the 2nd well is blank. Across the top row, in numberical order, starting in the 3rd well, are samples number 1-12. In the second row the first well is the KB+ ladder, the second well is blank and starting in the 3rd well are numbers 13-24. Ran the gel at 110V for 45 minutes.

Image of the AUBr gel setup:

![](”https://static.igem.org/mediawiki/2017/5/53/AMp3.png”)

\* The second gel to run is the BTA3: The first well is the KB+ ladder with the second well blank. Across the top row, in numerical order, starting in the 3rd well, are 5μl samples number 1-12. In the second row the first well is the KB+ ladder, the second well is blank and starting in the 3rd well are 5μl samples number 13-24. Ran the gel at 110V for 45 minutes.

Image of the BTA3 gel setup:

![](https://static.igem.org/mediawiki/2017/9/96/AMP4.png)

In the gel results for the AUBr there were ZERO/ 0 samples that were successful:

![](https://static.igem.org/mediawiki/2017/b/b7/AMP5.png)

The gel results for the BTA3 below showed that there was ZERO / 0 successful BTA3 samples.

![](https://static.igem.org/mediawiki/2017/8/87/AMP6.png)

After closer examination there were some good samples but the placement of the DNA in the loading wells was mixed up. Sequencing confirmed the fragments were what we were looking for and the error was corrected resulting in a successful experiment.

## Transformation of DH5alpha with PSB1C3 and PSB1A3 backbones

### Wednesday, 7/19

In preparation Alyssa and I rehydrated 2 backbones from iGEM, the PSB1C3 and PSB1A3. The C3 was from plate 7 location O,23 and the A3 was from plate 4 location H,2. Will let the plates grow overnight in the incubator and make MP samples, in triplicates of each, tomorrow.

DNA extraction from kit and rehydration protocol:

To use the DNA in the Distribution Kit, follow these instructions:

*Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/µL*

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic\* and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

*\* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.*

**7/20/2017 - something went wrong with the process, the plates to not show sufficient growth. Tests and plating will be redone today.**

![](https://static.igem.org/mediawiki/2017/6/61/Image_asu.png)
![](https://static.igem.org/mediawiki/2017/3/3f/Image_%281%29.png)

Gathered the available colonies and put in LB with (AMP or CHLOR) to grow overnight and replate for more usable growth to be MP. samples in the shaking incu overnight, was able to grab 3 colonies from the A3 and two from the C3. Put everything in the shaking at 345pm.

![](https://static.igem.org/mediawiki/2017/a/a6/Image_asu_2%281%29.png)
![](https://static.igem.org/mediawiki/2017/6/6d/Image_%283%29.png)
![](https://static.igem.org/mediawiki/2017/4/48/Image_%284%29.png)

Doing the MP on the A3 and C3 growth tubes. Concentrations:

* psb1c3: 80ng/ul
* psb1c3: 40ng/ul
* psb1a3: 50ng/ul
* psb1a3: 67ng/ul
* psb1a3: 75ng/ul

![](https://static.igem.org/mediawiki/2017/1/19/Image_%285%29.png)

# Notebook: Chris Connot

## AHL Degredation: Ethanol/ISO TraR

### Wednesday, 9/20

Notes: This is the second run of testing the degradation of AHLs via the treatment of 2-Propanol and ETOH. The first was with LasR and the next will be with 2620 (Lux) if the synthetic AHLs can be found. The first protocol is for preparing necessary parts for the plate reader which will have a protocol added below the Prep Protocol

Notes: This is the second run of testing the degradation of AHLs via the treatment of 2-Propanol and ETOH. The first was with LasR and the next will be with 2620 (Lux) if the synthetic AHLs can be found. The first protocol is for preparing necessary parts for the plate reader which will have a protocol added below the Prep Protocol.

### Prep Protocol:

1. Grabbed 6 15mL culture tubes
2. Labeled 2 for Neg Sender, 2 for TraR #2, and 2 for Neg Receiver
3. Using a serological tube, 25mL of LB/AMP was pipetted out, and approximately 4 mL was pipetted into each culture tube (more than 4 mL will cause for decreased growth capacity).
4. A seperate 250mL flask was used for additional supernatant of neg sender, and 50mL of LB/AMP was also pippetted into the flask.
5. Using the bacteria plates for neg sender/receiver (9/14/17) and TraR #2 (8/16/17) transformation (s). The cells were scraped off from each plate using a different tip each time (single colony) and ejected into the corresponding culture tubes.
6. The tubes were then properly closed and a foil was placed over the flask
7. The tubes and flask were then placed into the 37C shaking incubator for overnight growth.
8. After incubation, the OD600 is taken for the GFP+, -REC and the Sender
9. Calculated volumes of GFP, -REC and the Sender to give a concentration of .70 at a volume of 500 ul
10. The appropriate volume of supernatant is taken from the overnight cultures (after being spun down in the centrifuge at max speed for 1 minute) and placed in a total volume (including supernatant) of 500 ul of fresh LB/Amp
11. Where calculated volumes utilize the equation C1V1=C2V2
12. Sender is spun down till clear in 50 ml conical vial
13. a filter syringe is used to transport the supernatant into a new 50 ml conical vial
14. The receiver (TraR) is now spun down in a 50 ml conical vial (4 ml) and supernatant is discarded and the cells are resuspended in 20 mL of LB/AMP.
15. After all cells have been spun down, resuspended and transferred into new media it is time to prepare the treatment tubes of AHL (treated with chosen alcohol)
16. 1.5 ml tubes are used to create serial dilutions of the alcohol for concentrations 10^-1 to 10^-12 (choose range based on need)
17. Powdered synthetic AHL's are used to create the diluted 1.5ml concentrations
18. To each 1.5 ml tube (10^-1 to 10^-12) the chosen alcohol (i.e ETOH or 2-propanol) is added and the tubes are closed to allow treatment to take place over 15 minutes
19. The ETOH or 2-Propanol is added at an amount that maintains a total volume concentration of 70% ETOH or 2-propanol
20. Once the 15 minutes is over the tubes are opened and placed in a well ventilated incubator in order to assist in the evaporation of the alcohol and ethyl-acetate that the AHL's were suspended in
21. Once all components have evaporated the solid AHL's are resuspended in the original volume of ethyl-acetate that the dilutions were made in and placed on the vortex to ensure that the AHL's re-dissolve
22. Then the overnight induction plate is setup with the following layout
23. In the table below Well A4-A12 each have equal parts 150ul of the labeled product (i.e GFP+/-Sender supernatant) The wells were filled from 500ml stocks made for each different component.
24. For the wells from B1-F6 there was the following stocks made 500ul Receiver, 490 -Sender and 10ul AHL)
25. The wells themselves received 300 ul out of the stocks made from each concentration
26. For wells B1-C3 the 'U' stands for Untreated (AHLs were not treated with the alcohol),
    Wells C4-D12 the 'I' stands for Isopropanol and cells D13-F-6 the 'E' stands for ethyl alcohol (EtOH).
27. The well plate is then placed in the appropriate imaging machine and OD600 and GFP expression is measured over a 8-16 hr period depending on the length of data collection needed.
![](https://static.igem.org/mediawiki/2017/1/18/CC_graph.png)

## ESA Induction Plates Liquid/Plated

### Wednesday, 7/19

will be starting with 3 ul of solution for the liquid culture plates for each of the ESA/-REC/GFP+/2620

finale amount used was 9 ul

* Liquid culture made @ 9:42 am
* ESA Plated Culture: Plated at 10:08 am
* -Sender Plate (was improvised) and plated at 10:30 am
* Liquid culture checked @ 10:51 am
* ESA Plated Culture: Checked at 11:08 am
* -Sender Plate (was improvised) and Checked at 11:32 am
* Liquid culture checked @ 11:51 am
* ESA Plated Culture: Checked at 12:08 pm
* -Sender Plate (was improvised) and Checked at 12:32 pm
* Liquid culture checked @ 12:58 pm
* ESA Plated Culture: Checked at 12:59 pm
* -Sender Plate (was improvised) and Checked at 1:02 pm
* Liquid culture checked @ 1:58 pm
* ESA Plated Culture: Checked at 1:59 pm
* -Sender Plate (was improvised) and Checked at 2:02 pm
* Liquid culture checked @ 2:58 pm
* ESA Plated Culture: Checked at 2:59 pm - Induction has been started (minimum 4-5 hrs for ESAI)
* -Sender Plate (was improvised) and Checked at 3:08 pm
* Liquid culture checked @ 3:58 pm
* ESA Plated Culture: Checked at 3:59 pm
* -Sender Plate (was improvised) and Checked at 4:08 pm

(initial plates need to be analyzed from an 18hr incubation time and distance migrated)

### Thursday, 7/20

Plates were checked and expression was seen in all components and all of the cells that can produce GFP. Though the GFP+ is still lacking expression compared to the GFP+ only plate. The Plated Culture and the negative Sender plates had the greatest amount of GFP+ though this is not of much concern as it is a cell that is simply there to verify that fluorescence is able to be seen. Since the 2620 is well characterized and expresses in each run the lack of GFP+ expression hasn't been much of an issue. Another phenomena that was noticed was the variation in the machine's exposure. I was using set exposure settings in the manual capture mode along with the UV 6 and Epi UV setting. it seems that the camera is still auto exposing to the brightest emitted light. This means that the relative exposure of all samples is dependent on the brightest object being photographed. So If I only have the GFP+ in the frame then the machine captures more of its detail and shows a (possible) different picture than when the GFP+ is imaged next to the 2620 which is marginally brighter than the GFP+

Today I will be investigating over time lapse the progression of GFP expression in the 2620. I will be plating ESAI as the sender at 1.5 inches away from the 2620 receiver as well as at .25 inches away from 2620 and placing them side by side under the imager if possible in order to time lapse both of their progressions. It is my aim to have a more uniform spread for this aspect of the experiment and I will be looking for time points of when expression begins and progresses towards its maximum.

* Plated ESAI at 1:55 pm
* Plated LuxI at 2:00 pm
* Putting in Incubator at 2:20 pm till I leave the lab which should be around 5 pm.
* There may be an issue with the plates as the 2620 is auto-inducing
* Because 2620 was auto-inducing (and LUX was already induced.. possible auto-inducing also) I took another image at 5:22 pm and left the plates in the imaging device in order to gather 50 images over the course of 12.5 hours to see if the LUX/ESA/2620 will have a trend of induction at room temperature. While this will not show us the change from non-induced to induced and beyond... It will show us a possible trend of induction from where they started at 5:22pm on 7-20-17

## Induction Plates TraR, AubI, BjaI, CerI, LasI, LuxI, RpaI, EsaI

### Sunday, 10, 1

These induction plates will have all senders with TraR (AubI, BjaI, CerI, LasI, LuxI, RpaI, TraI, EsaI) and one sender with LuxR (Bja).
These plates will be setup the same as previous induction plate (GFP+, -Rec, Rec)

### Saturday, 10/7

The induction plates were supposed to be setup on 10/1/17 but the TraR receiver, after transformation did not have enough viable colonies in order to make induction plates with 7 other receivers. Plates were setup on 10/6 and were left overnight to incubate at 37 degrees celsius since TraR is notorious for being slow to induce.

Unfortunately after 24 hours of imaging (1st image was at 12 hours after incubation and last image was at 24 hrs) there was little to no evidence of induction on the plates. This may be due to either the the Receiver being 5 days old or that the senders were 5 days old. It seems that upon refrigeration and after extended periods of time on Amp agar, many of the cells lose the ability to send or possibly receive signals via AHL's. This might be due to the fact that when the cells multiply there is somatic recombination and the genes that control the AHL's production just get folded out. There is also the possibility that during the initial transformation of the TraR and related senders the intended insert was not taken up by the new cells.

# Notebook: Brianna Lopez

## Transformation of Negative Sender and LuxR

* These colonies will be used for overnight liquid culture growth, which will be used in 96-well plating for overnight growth curves.
* Transformation of a negative sender, and 2620 vector.
* Concentration of 2620 & Neg Sender:

-2620 = 135 ng/uL

-Neg Sender = 461 ng/uL (will have to do 1:10 dilution)

* Calculation Calculation to get concentration of 60 ng/uL each:
* Negative Sender:

- 461 ng/ul (1uL into 9uL H20) = 46.1 ng/uL

- 46.1 ng/uL \* x uL = 60 ng

- x = 1.3 uL

* 2620:

- 135 ng/uL \* x uL = 60 ng

- x = .44 uL

### Protocol:

Materials:

* Ice bucket
* 2620
* Negative Sender
* DI H20

### Dilution Procedure:

1. One 1.5mL tube was used to perform a dilution, and labeled dilution tube.
2. 9uL of DI H20 was pipetted into the dilution tube.
3. 1uL of negative sender was taken out of stock and pipetted into the dilution tube.
4. The volume was then pipetted up and down to mix and dilute the solution.

### Transformation Procedure:

1. One tube was labeled 2620, the other negative sender
2. 1.3uL of negative sender was pipetted from the dilution tube into the negative sender tube
3. 0.44uL of 2620 was pipetted from 2620 stock tube into the 2620 tube.
4. Tubes were then placed on ice for 2-5 minutes
5. The tubes were then placed on the hot plate of 42C for exactly 45 seconds then immediately placed on ice for 5 minutes.
6. 300uL of SOC was added to each tube
7. It was then placed in the 37C shaking incubator for 20-30 minutes
8. The tubes were then spun down at 3000 rpm for 1 minute
9. The liquid in the tubes were gently tapped out into liquid waste
10. 100uL of SOC was added to the tubes and gently pipetted up and down to resuspend the cells
11. 100uL of each tube were then plated onto LB/AMP plates
12. Then placed in 37̊C still incubator over night

## Serial Dilutions

* This is where the set range of dilution concentrations comes in handy.
* The dilutions for these sets of ranges is a 1:10 dilution. So, a 1-part AHL and 9-part ethyl acetate solution.
* No matter how large the volume of your dilutions, it will always be 1:10.* For this project, it is a 100uL volume, therefore 10uL will be AHL solution and 90uL ethyl acetate.
  * In some cases, the dilution mixture will be a 50uL volume with 5uL being AHL solution and 45uL will ethyl acetate. This is to conserve the longevity of the AHL when doing a single receiver at a time and plating with 2 AHLs.
  * For this particular example, however, a 100uL volume and its corresponding 1:10 dilution will be performed.

1. 100uL of the 1\*10^(-2)M stock solution is pipetted into a tube (for easy access and to prevent contamination of the stock solution)
2. 10uL of the stock solution is pipetted into 90uL of Ethyl Acetate to bring the concentration down to 1\*10^(-3)M
3. Mixing Each Tube:

1. Pipette up/down
2. Vortex
3. Spin down (using tabletop centrifuge)
4. Continue onto the next dilution

1. Steps 2- 3d will be repeated until the final lowest concentration is reached.
2. The dilutions will be set up as follows:

![](https://static.igem.org/mediawiki/2017/9/91/Ahl.png)

* For half dilutions, i.e. 5\*10^(-2), 5\*10^(-3) it is the same concept except they are 1:1 dilutions instead of 1:10.
* Two ways of doing this includes doing one 1:1 dilution with the stock solution, then doing a 1:10 dilution using the result from the 1:1 dilution. Or it can be done doing a 1:1 dilution for each individual concentration, i.e. 1\*10^(-2), 1\*10^(-3)

Note: When these solutions are plated, the concentration will be further diluted by 10^-2. For example, 1\*10^-2M will be pipetted to its corresponding well on the well-plate, and the AHL final concentration on the well will be 1\*10^-4M. This is where the 1\*10^-4M to 1\*10^-14M concentration range of AHL comes into effect.

## Dissolving AHL in Ethyl Acetate

* The first thing that must be done is to determine the molecular weight of the AHL, in this case the Lux AHL.
* Usually the synthetic AHLs are purchased from Sigma-Aldrich and depending on the volume of what is bought from the company, the milligrams it comes in is important when calculating how much must be dissolved.
* In the case of this project, the final stock concentration that was desired was 1\*10^(-2)M. In this project, the highest AHL concentration desired was 1\*10^(-4)M, but when plated the concentration is diluted down by 10^(-2). Therefore a starting concentrating of 1\*10^(-2)M is beneficial.
* Then Stoichiometry must be performed to determine the volume in which the AHL in milligrams will be dissolved in, then how much ethyl acetate must be added to get to the desired final stock concentration.

1. First is to determine the molecular weight and container size the AHL comes in.

1. 213.2 g/mol
2. 10 milligrams of AHL in the container

2. Then is to determine the desired stock concentration.

1. This depends upon your range of concentrations, and how high the range is.
2. Range is: 1\*10^(-4)M- 1\*10^(-14)M
3. Therefore stock solution concentration: 1\*10^(-2)M

3. Then determine how large of a stock solution is needed

1. Since it is difficult to measure out exact milligrams of the AHL and back into a container without losing any grams, a large enough stock solution would be the most beneficial and use the entire AHL in the container.
2. Desired Volume of Stock Solution: 500uL

4. Determine Concentration of AHL contained in the stock volume

1. Here is where stoichiometry comes into play. The milligrams per stock volume must be converted into standard SI molarity.
2. 10mg/500μL \* 1g/1\*103mg \* 1\*106L/1μL \* 1mol/213.2g = 0.094M
3. The concentration found is the stock solution concentration needed for dilutions.
4. Once done with the dilution to 1\*10^-2 M, immediately put inside a -80C freezer for long term storage.

5. With the concentration found above, it will be used in the C1V1=C2V2 equation.

1. C1 will be the concentration found above.
2. V1 is the amount of AHL from stock solution into ethyl acetate solution
3. C2 is the concentration of the stock solution
4. V2 is the volume of the solution that is going to be further diluted and used in plating
5. (0.094M)(V1)=(50uL)(1e-2M)
6. V1=5.32uL AHL

6. Subtract AHL volume from total volume

1. This volume of AHL will be used to find the volume of ethyl acetate to dilute the AHL into a 1\*10^-2M solution.
2. So, 500 – 5.32uL = 44.68uL of ethyl acetate

7. Add these two amounts together in a 1.5mL centrifuge tube

1. Mixing the Tube:

1. Pipette up/down
2. Vortex
3. Spin down (using tabletop centrifuge)

8. Immediately place dilution in the -20C freezer for immediate use of plating or for further dilutions.

# Notebook:Christina Smith

## Friday 9/22/17 - Running Gradient PCR on the BjaR

Materials Used for this experiment

* BjaR New Receiver G-Block
* BioBrick prefix Forward Primer
* BioBrick Suffix Reverse primer
* Phusion Polymerase
* dNTP 5 mM
* HF Buffer (5x)
* Distilled Water

There is no particular order to place these materials into an epindorph tube. However after these components are in the tube, needed to mix them thoroughly and spin them down. After, this mixture was divided into 8 PCR tubes with ~20 uL in each. A gradient PCR protocol was setup on the computer to run. The gradient annealing temperature would run from 70 ºC to 50 ºC.

Below is a chart to describe the particular quantities of the materials used:

| Materials | Quantity used in Lab’s Protocols (µL) | Quantity in Correct Ratio (µL) |
| --- | --- | --- |
| BjaR New Receiver | 0.2 | 1.6 |
| Forward primer | 0.2 | 1.6 |
| Reverse Primer | 0.2 | 1.6 |
| Distilled Water | 14.4 | 115.2 |
| Phusion Polymerase | 0.2 | 1.6 |
| dNTPs (5mM) | 0.8 | 6.4 |
| HF Buffer (5x) | 4 | 32 |

This particular PCR ran for 1 hour and 16 minutes. After this, I needed to run a agarose gel to verify if the DNA amplified or not. Once the gel was made, I inputted a kb+1 DNA ladder into the first slot of the gel. I skipped the second slot and proceeded to input 2 µL 6X Loading Dye + 4 µL of PCR into the remaining 8 wells. The gel was ran at 110 V for 45 minutes. The following are the wells that correspond to the annealing temperature

* A: 70 ºC
* B: 66.8 ºC
* C: 64 ºC
* D: 61.2 ºC
* E: 58.4 ºC
* F: 55.6 ºC
* G: 52.8 ºC
* H: 50ºC

This is the process to make an agarose gel:

* Weigh 0.6 grams of Agarose powder and place it into a 100 mL volumetric flask.
* Next, add 60 mL of TAE to the powder and place into microwave.
* Microwave for 1 minute. Take out the flask using heat protective gloves and swirl the flask to dissolve the particles
* Place the flask back into the microwave and run it for another 8 seconds
* Using heat protective gloves, take the flask out of microwave, swirl and add 6 µL of Syber Safe DNA Stain
* Pour liquid into gel mold with small dividers and let it sit for 15 minutes or until opaque

![Design Flowchart](https://static.igem.org/mediawiki/2017/6/65/BjaR_PCR.png)

## Tuesday 9/26/17 - PCR Cleanup of BjaR

### PCR Cleanup

The volume of this PCR is 128 uL. The following is the protocol for PCR cleanup in lab.

##### Binding

* Add 5x Volume of PB
* Add 1/10 X Volume of PCR results of NaAc pH 5.3
* Spin through PCR Cleanup tube at maximum speed for 1 minute and dump the flow through

##### Wash

* Add 750 uL of PE wash solution
* Spin through column at maximum speed for 1 minute
* Dump the flow through liquid into waster container

##### Spin Again

* Spin at maximum speed for 1 minute
* Dump flow through liquid

##### Transfer PCR Column to a new epindorph tube

* Add 35 uL of distilled water and let it sit for 1 minutes
* Spin to elute at maximum speed for 1 minute

##### Test the Concentration in (ng/uL)

The concentration of BjaR was 123.12 ng/uL

## Wednesday 9/27/17 - Double Digest of BjaR with EcoRI and PtsI

The following quantities were added to a edpindorph tube, mixed and spun down. They were then placed in a 37 degree incubator for 45 minutes. The order I placed them into the tube are as followed:

* Distilled Water
* 10x Fast Digest Buffer + Green Dye
* BajR New Receiver from PCR Cleanup
* EcoRI Restrictive Enzyme
* PtsI Restrictive Enzyme

| Materials | Quantity (µL) |
| --- | --- |
| BjaR New Receiver | 7.3 μL |
| 10x Fast Digest Buffer + Green Dye | 3 μL |
| Distilled Water | 17.7 μL |
| EcoRI Restrictive Enzyme | 1 μL |
| PtsI Restrictive Enzyme | 1 μL |

After the digest was completed, I ran an agarose gel to confirm the digestion. The results are displayed below. I realized after running the gel that it would be very hard to confirm whether or not the digest was successful. This is because the size that I digested out is less than 100 nucleotide difference between the original PCR cleanup and the digested version. The next step I need to take is to gel extract the top band, ligate it and then transform it in DH5(alpha) turbo cells.

![Design Flowchart](https://static.igem.org/mediawiki/2017/1/13/Bja_double_digest.png)

# Notebook: Xylaan Livingstone

### July 31st , 2017

### Mini Prep for backbone:

+ 3 15 mL tubes of the LB Cor C
+ 3 15 mL tubes of LB AMP A3

1. Centrifuge down for pellet at highest speeds
2. Each tube, I used 200 uL of resuspension fluid and transfer each to 2 mL tubes
3. Each tube added lysis (200 uL), invert the tubes 5 times and immediately added 350 ul of neutralization buffer
4. Cap each tube and centrifuge for 10 minutes at max speeds
5. While centrifuging, I got 6 microfuge tubes for column prep, Put 6 columns in each pipet 500 uL of column prep into each
6. Transfer the lysate from the 10-minute centrifuge to the labeled columns, then centrifuge the flow through
7. Dump the flow through after

~Proceeded with the same measurements from sigma mini prep

~ Error with the elution step, after water flow through on the last step. 2 PJ1A3 tubes DNA elution was poured out on accident, so a lower concentration for two b was expected

### Mini Preps Concentration

#### 1st Concentration results

| PSB1A3 | PSB1C3 |
| --- | --- |
| 16.068 ng/uL | 6.024 ng/uL |
| 41.607 ng/uL | 40.571 ng/uL |
| 12.99 ng/uL | 7.578 ng/uL |

## August 1, 2017

Yesterday’s concentration created low DNA concentration. Thus, we are doing a second batch.

2mL of PSB1C3

2mL of PS1A3 A3

+ All have fairly the same expression/ Approximately
+ Bl21 bacteria for both batches
+ Ran same mini prep procedure as yesterday, as well found in the Sigma mini prep procedure

~Side note: it’s possible I am getting a lot of expression but the bacteria is not taking the plasmid

### 2nd Concentrations Results:

| PSB1A3 | PSB1C3 |
| --- | --- |
| 16.158 ng/uL | 31.964 ng/uL |
| 37.435 ng/uL | 14.273 ng/uL |

Will talk to graduate/ higher up to see a different result or possible error in steps of mini prep.

## August 1st, 2017

Liquid Culture of

- 3ml Cor C3
- 3mL Amp A3

New liquid cultures overnight, two different tubes.

- PSB1A3 in AMP
- PSB1C3 in Cor
- Left in the incubator at 3:21 p.m.
- Coming in at 10:00 a.m. to finish

## August 2nd, 2017

![](https://static.igem.org/mediawiki/2017/3/3b/Mcherry.png)

- Took liquid culture out at 12:45 p.m.
- Ran mini-prep procedure. Instead of using neutralization in fridge, I am using a bottle at room temperature
- Lysis/ neutralization
- Accidently put 550 uL of lysis solution within tubes to counter balance mistake, I used 700 uL of neutralization solution to make up amount.

### Concentration results:

| PSB1A3 | PSB1C3 |
| --- | --- |
| 122.576 ng/uL | 98.408 ng/uL |

Placed in freezer 2:20 P.M.

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# Arizona\_State

# Protocols

## Christina Smith

## Ligation Protocol with T4 DNA Ligase

### Materials:

* 10X T4 DNA Ligase Reaction Buffer
* T4 DNA Ligase
* MRV Vector DNA
* Receiver Insert DNA
* Nuclease-free water

### Procedure

*\*Note:* T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. Use NEB calculator to calculate molar ratios.

1. Thaw the T4 DNA Ligase Buffer and resuspend at room temperature.
2. Set up the following reaction in a microcentrifuge tube on ice:

| Component | Volume (uL) |
| --- | --- |
| 10X T4 DNA Ligase Buffer | 2 |
| Vector DNA: 50 ng | X |
| Insert DNA : 37.5 ng | X |
| Nuclease Free Water | 17 |
| T4 DNA Ligase | 1 |
| Total | 20 |

3. Gently mix the reaction by pipetting up and down and microfuge briefly
4. For cohesive sticky ends, incubate at 16 C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 1 C overnight or room temperature for 2 hours
5. Heat inactivate at 65 C for 10 minutes
6. Chill on ice and transform 1-5 uL of the reaction into 50 uL of competent cells

## Vector Digest Protocol for Modular Receiver Vector

### Materials

+ Vector DNA
+ 10x Fast Digest Buffer + Green Dye
+ Restriction Enzymes
+ BbsI
+ SpeI
+ Deionized Water

### Procedure

1. Set up the following reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer (total reaction volume 50 µl).
2. Set up the following digest reaction on ice:

table, th, td {
border: 1px solid black;
border-collapse: collapse;
}

| Components | Volume (µL) |
| --- | --- |
| dH2O | 25 |
| 10X Fast Digest Buffer + Green Dye | 3 |
| MRV Vector | 10 |
| BbsI | 1 |
| SpeI | 1 |

1. Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.
2. Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.
3. Incubate

## Brianna Lopez

## Plate Reader Protocol for Synthetic AHL Liquid Culture Inductions

Purpose: To set up and run a plate of synthetic AHL liquid culture inductions for overnight growth curves in a plate reader.

**Materials:**

+ Use of 1 or 2 separate synthetic AHLs, in this example Las AHL and Lux AHL.
+ Use of 1 receiver that will interact with the synthetic AHLs, in this example LuxR.
+ Controls that will need to be transformed into colonies on LB/AMP plates:
o Negative Sender (negative control for synthetic AHL)
o Receiver
o GFP (positive control)
o Negative Receiver (negative control for the receiver)
o Sender Supernatant (in this example, LuxI supernatant from the natural AHL that a sender cell produces, is a positive control for the receiver to show it works)

**Procedure:**

For plating purposes in this example, Negative Receiver, GFP Postive, and Reciver liquid cultures must have a final OD600 of 0.4 once it enters the plate reader for overnight growth curve readings. This specific OD600 value can be set depending on what range on the curve is needing analysis.

Day 1:

1. Make liquid cultures of LuxR, Negative Sender, Negative Receiver, Positive GFP, and Receiver cells.
2. In 15mL culture tubes for each individual sample, put 3-4 mL of LB/AMP broth into the tubes.
3. Then picking colonies off of the transformed plates of LuxR, Negative Sender, Negative Receiver, Positive GFP, and Receiver cells into their corresponding culture tube containing LB/AMP broth.
4. Make sure there is enough liquid culture for the following day of plating, taking into account the evaporation of LB/AMP broth.
5. Place culture tubes into a shaking incubator at 37̊C for overnight growth.

Day 2:

1. Take samples out of the shaking incubator as they have grown overnight.
2. Take another culture tube and using the culture tube of receiver grown the previous night, reseed in fresh LB/AMP inside the culture tube.
3. This reseeding culture contains 10% overnight culture and the remaining volume containing LB/AMP.
4. Leave this to regrow for 5 hours before plating.
5. Check OD600 for the LuxR (receiver), Negative Receiver, and Positive GFP samples with an LB/AMP well as a control comparison.
6. These OD600 readings should be between 0.4 and 0.6 once put into the plate reader for curve readings at the end of day.
7. Then filter senders from cells in the culture tubes into supernatant. These senders include Negative Sender, and LuxI or the positive sender control for the receiver.

1. Begin by centrifuging the culture tubes until cells are at the bottom, and the remaining supernatant is of similar color to LB.
2. Filter this remaining supernatant through a syringe with a filter at the bottom into a new tube and label the type of sender.
3. Do this for both senders.

1. Begin plating all samples into 96-well plate.
2. Plate Reader Protocol:

1. Go to Protocol > Procedure > Check Temperature is set to 37̊C, Check A(600) and F(485,515) are set > Click Flourescence > Gain > Options > Make sure the automatic Gain Adjustment is set properly > scale to low wells > scale wells (this is where the three Negative Sender and Receiver wells are on the plate) > scale value 200

1. Save this plate reader data to place of choice on the computer, and begin to run the plate for overnight growth curves.

## Mini-Prep Protocol

#### Harvest Cell :

• Pellet 1-5 mL of an overnight recombinant E. coli culture by centrifugation.
-The optimal volume of culture to use depends upon the plasmid and culture density.

+ For best yields for E. coli grown in LB:
+ use 1-3 mL of culture for high copy plasmids
+ use 1-5 mL of culture for low copy plasmids
+ for best yields for E. coli grown in TB or 2X YT:
+ use only 1 mL of culture

1. Resuspend Cells (verify that appropriate volume RNase A solution was added to the Resuspension Solution

1. Completely resuspend the bacterial pellet with 200 µL of the Resuspension Solution.
2. Vortex or pipette up and down to thoroughly resuspend the cells until homogenous

2. Lyse Cells

1. Lyse the resuspended cells by adding 200 µL of the Lysis Solution
2. Mix the contents by gentle inversion (6-8 times) until the mixture becomes clear and viscous (Don’t vortex)
3. Do not allow the lysis reaction to exceed 5 minutes

3. Neutralize

1. Precipitate the cell debris by adding 350 µL of the Neutralization/Binding Solution
2. Gently invert the tube 4-6 times
3. Pellet the cell debris by centrifuging at ≥ 12,000 x g or maximum speed for 10 minutes
4. Cell debris, proteins, lipids, SDS, and chromosomal DNA should fall out as a cloudy, viscous precipitate.
5. If the supernatant contains a large amount of floating particulates after centrifugation, re-centrifuge the supernatant

4. Prepare Column

1. Insert a GenElute Miniprep Binding Column into a provided micro-centrifuge tube, if not already assembled
2. Add 500 µL of the Column Preparation Solution to each miniprep column and centrifuge at ≥ 12,000 x g for 30 seconds to 1 minute
3. Discard the flow-through liquid

5. Load cleared lysate

1. Transfer the cleared lysate from step 3 to the column prepared in step 4 and centrifuge at ≥ 12,000 x g for 30 seconds to 1 minute.
2. Discard the flow-through liquid

6. Wash Column (verify that ethanol has been added to the bottle of Wash Solution 2)

1. Add 750 µL of the diluted Wash Solution to the column
2. Centrifuge at ≥ 12,000 x g for 30 seconds to 1 minute
3. Discard the flow-through liquid and centrifuge again at maximum speed for 1-2 minutes without any additional Wash Solution to remove excess ethanol

7. Elute DNA

1. Transfer the column to a fresh collection tube. Add 100 µL of Elution Solution or molecular biology reagent water to the column
2. For DNA sequencing and other enzymatic application, use water or 5 Mm Tris-HCL, Ph 8.0, as an eluant
3. Centrifuge at ≥ 12,000 x g for 1 minute
4. Use immediately or store at -20˚C

## Amber Mani

## Plate reader protocol with Data Analysis for Liquid Culture Inductions

How to set up and run the plate reader for overnight growth curves.

### Materials:

+ (Your unique set of senders, receivers etc. that you want for testing) In this example set up, Lux I, Esa I, and RpaI were all used in BL21 and are shown to produce AHL
+ (Controls) Negative sender- missing the gene for synthase that produces AHL, used for
  control purposes
+ Receiver
+ GFP (+)
+ (-) Receiver

#### Procedure:

### Done over 2 days and plate reader protocol at the end:

You will want to know the concentrations of the samples you use so that you can see, based on the growth curves, which concentrations will yield the best results. In one example of the growth curve tests, we used concentrations of .05, .1, .2, .3, .5. after determining the OD600 concentrations. the actual growth reader will be on total volumes. We want our final OD600 to be 0.4.

### Day 1

Make liquid cultures of Lux I, EsaI, RpaI, (-)sender in LB/Amp
3- 5 mL of LB with plated senders
want bacteria in the stationary phase of bacterial growth to maximize AHL
Make liquid culture of 3 receivers: F2620, GFP(+), and (-) receiver
3-5 mL of LB with plated senders

### Day 2

Make another culture using the starter cultures and reseed them
get 5 mL of fresh LB/Amp media and make a 10% seed culture
5mL of LB with 600 uL of grown start
grow for 5 hours
wanting receivers at exponential phase of growth
can check this by checking their OD reading
between 0.4 and 0.6
GEN5>protocol of OD600> choose H8-11, H8 being LB well
Filter senders: (18, 19, 20 & 21:)
put in Big centrifuge
start 1 min at 1 RPM, then 2nd minute at 2 RPM, then 8 minutes at 3.6 RPM
extract the supernatant and put in syringe with a filter on the bottom, filer it into a tube with a
screw tab on it and label it
AHL stable at room temperature or in the freezer in the back room with all the plates

### Plate Reader Protocol - check settings as these are examples from a previous experiment measures relative GFP fluorescence in unit of RFU ReneRynaJiagi, overnight, OD GFP save experiment to your folder fix layout to say what rows you want to work with go to side tab protocol>procedure> make sure at 37C, A(600), F( 485,515), click on fluroescence > Gain > options > make sure setup at Automatic gain adjustment > scale to low wells > scale wells A1A3 > scale value 200 To analyze the data collected from the plate reader 1. Export the data to Excel from the plate reader, graph the data to view the OD and GFP expression curves and pick out the data that stands out. 2. Take the triplicates and use excel to get averages for each triplicate of data. 3. Use the averaged OD and GFP figures to graph GFP over OD to get the normalization curves. 4. Compare the graphs to see what data stands out, searching for orthogonality in combinations of senders (low GFP expression) meaning low interference and possible orthogonality. 5. Once you select the 'interesting' data, add error bars (standard deviations) to see the success of the samples. 6. Use the Hill equation to re-graph the data in a more professional display. Plasmid transformationIntroduction How to insert a plasmid into bacteria to transform it.Materials + Get how ever many 50ul tubes of the cells you need to to transform (in our case it was BL21 e. coli) + Get the plasmid tubes you need, for insertion into the cells. + Gather PCR tubes to be used for Plasmid DNA dilution, same number of tubes as cells and plasmids. + Keep everything on ice during preparation. ›Procedure Calculate the concentrations and transform the cells 1. check ng/ul concentrations of each plasmid DNA - want to add about 60ng of DNA to each of the cell tubes. 2. Dilute each DNA sample in PCR tube with water and do the math to know how many ul to add of the new dilution to get appx 60ng of DNA for each of the 50ul cell tubes. 3. Add the appropriate amount of the plasmid needed into each cell tube. 4. Place cell tubes (with plasmids added) on ice for about 2-5 minutes 5. Then place tubes on hot plate (42C) for appx 45 seconds 6. Finally place cell tubes back on ice for 5 minutes. Suspend and grow newly transformed cells 7. Add SOC (300ul) to each cell tube 8. Place in shaking incubator (37C) for 30 minutes 9. Spin down tubes, max speed for one minute 10. Gently tap out liquid and resuspend cells in 100ul SOC 11. Plate all 100ul onto petri plate (in our case, AMP plate) and place in regular incubator overnight 12. The next day you have access to the freshly transformed, plated and grown cells DNA Gel Extraction from Agarose GelIntroduction: How to extract specific DNA fragments from an agarose gel. Materials: - Sigma GenElute Gel Extraction Kit with included materials: - 15ml culture tubes - Scalpel - KB+ Ladder - GenElute Binding Column G tubes & 2ml collection tubes Procedure: \*\*\*\*\*\*\*\*\*\*\*\*\*1st do restriction digest protocol and run the agarose gel\*\*\*\*\*\*\*\*\*\*\*\*\*\* 1. Use the sample of cells you wish to extract DNA from AFTER completing the protocol for restriction digest on them. 2. Run the agarose gel with the digested DNA for 90 minutes at 90 V. 3. Use the appropriate sequencing map to 'map' out the length of the strand you want to extract by finding the 1st restriction enzyme used and measuring the length of the pairs to the 2nd restriction enzyme. Compare to the KB+ Ladder (#SM1331) and the plasmid guide. 4. Once gel has run use a gel imager to view the DNA strand lengths and identify the proper section of DNA you need to extract. Removing DNA band 5. While gel is on an imager (so you can use UV light to see the bands of DNA) excise the DNA fragment of interest from the agarose gel with a clean, sharp scalpel or razor blade. Trim excess gel away to minimize the amount of agarose. 6. Place gel into a culture tube and weigh the gel - remember to 1st weigh an empty culture tube and 'zero' the scale so the weight of the tube is not included. 7. Solubilize gel is needed for this step - included in the GenElute kit. Add 3 gel volumes of the gel solubilization solution to the gel tube. (for every 100mg of agarose gel add 300ul of the gel solubilization solution). 8. Incubate the gel mixture at 50-60C for 10 minutes. As needed for total gel dissolution, vortex briefly every 2-3 minutes to aid in gel dissolution. 9. Prepare the binding column: place GenElute binding column G into one of the provided 2ml collection tubes. Add 500ul of the column preparation solution to each of the binding columns, centrifuge for one minute and discard the flow through liquid. 10. Next go back to the dissolved gel and check the color, should be a yellowish color. 11. Add isopropanol: add 1 gel volume of 100% isopropanol and mix completely. 12. Bind the DNA: load the solubilized gel solution mixture from the culture tube into the binding column (may turn red at this point, this is normal). Centrifuge for one minute and discard the flow through liquid. 13. Wash column: add 700ul of wash solution (diluted from wash solution concentrate G as described under preparation instructions in manual). Centrifuge for one minute. Remove the binding column and discard the flow through liquid. replace binding column and centrifuge for one more minute and discard the flow through liquid again. Elute DNA:14. Transfer the binding column to a fresh collection tube and add 50ul of the (preheated to 65C) Elution Solution to the center of the membrane and incubate for one minute. Then centrifuge for one minute. What remains in the collection tube can be tested for DNA concentration and kept as the collection tube now contains the extracted DNA segments. Colony PCR and Colony Prep Materials and protocol for running the colony PCR. PCR is used to reproduce (amplify) selected sections of DNA or RNA for analysis. Materials: * 2x Master Mix * Forward and reverse primers * DNA template that will be used * Water * Agar plate(s) Procedure: Follow these steps: 1. Defrost template, 2x Master Mix and primers 2. Determine desired total working volume (~20-25uL per PCR tube) 3. Label reaction tubes 4. Create below reaction mix: 5. Use this template for the amounts and concentrations to use for making the master mix Next steps after you have the newly made Master Mix: 1. Pipette 20 μl of newly mixed green solution into each PCR tube 2. Colony Prep: to grow more of the sample that you took a colony from for the colony PCR. Can be used to have backup plates when you get a positive result from PCR gel. 3. Scraped one colony at a time from the existing plate and gently placed the colonly onto the new plate with the labeled spots. Disposed of each tip used into the cooresponding PCR tube labeled to match the placement number on the new plate. 4. Used empty pipette to attach to each tip in the PCR tubes and pump up and down to mix and remove as much of the leftover bacteria from the tip as possible so it mixed into the PCR tube. Then disposed of the tips in dry waste. 5. Place the new plates in the incubator and remove the next day to freeze. 6. Place PCR tubes with the new DNA samples in thermal cycler using the appropriate settings Growing samples in culture tubes for growth curvesThis is used for growing up colony samples overnight for use in the plate reader. Materials: * 15ml culture tubes * LB (AMP or CHLOR) * Grown colonies ready to use on petri dishes * going to use micropipette to grab one colony at a time and add to LB in culture tubes Procedure: Collecting colonies and prepping the culture tubes\*\*Best to do this in the evening so the cultures don't grow too long\*\* * Label each culture tube with the contents, date and your name * Add LB (AMP or CHLOR) to the culture tube. (general rule for how much LB - use about 1/4 the container volume. If tube is 15ml use about 3-4ml LB) * Take pipette and use a clean tip to collect ONE colony from the plate. Discard the tip INTO the culture tube with the colony on the tip. * Place the lid on the culture tube (not too tight so there is room for gas exchange) * Place the culture tubes into the shaking (37F) incubator for overnight growth. * In the morning you can use the Synergy plate reader to check the OD of the sample and dilute or add fresh LB as needed for the test you are running. Making and running a gelWhat you need and how much of it to make and run an agarose gel Materials: * \* 60 ml of TAE liquid into the flask for gel making. * \* 0.6g of the agarose powder to the flask and swirled * \* 6.0 μl of the Syber Safe DNA gel stain to the flask and swirled Procedure: How to make gel using ingredientsGel making/running protocol used: 1. Started with adding 60 ml of TAE liquid into the flask for gel making. 2. Add 0.6g of the agarose powder to the flask and swirled 3. Use the microwave to heat the mixture (1 min, swirl and repeat) till all pieces dissolved and was a pure clear liquid 4. Add 6 μl of the Syber Safe DNA gel stain to the flask and swirled 5. Carefully poured the mixture into a blank gel tray with spacers in place. 6. Once the gel is cooled, add the KB+ DNA ladder to the first slot, 2nd slot is left blank and each following slot is to be loaded with 5 μl of each sample. 7. Once the slots are filled place the gel (still inside the plastic gel holder) into the Galileo/ BioRad electric current machine. Run for approximately 45 minutes at 110V. 8. To image the gel once finished, use the SynGene gel imager machine. Chris Connot Plate Induction & Imaging ProtocolIntroduction This protocol will lead you through the steps of setting up induction plates for Sender/Receiver combos as well as the imaging of the agar plate. Materials * Agar plates * Sender/receiver bacteria on plates (suggested to be freshly transformed) * Positive/negative controls (GFP+/-Receiver) * MicroPipetter (05 ul to 200ul) * Pipette Tips * GeneSys Machine oProcedure**Induction Plate Setup** 1. Procure agar plates and warm to 37℃. 2. Procure plates with desired bacteria (senders,receivers,positive/negative control) 3. design/print out template (fig. 1) for induction test ![]( https://static.igem.org/mediawiki/2017/8/8e/Inc_Pic_1.png) **Fig 1: Induction Plate Layout**4. Using a micropipettor and a sterile tip, bend the tip on the back of the lid of the agar plate ![]( https://static.igem.org/mediawiki/2017/8/8c/Inc_Pic_2.png) **Fig 2: Bent Tip Method**5. Use the bent tip to scrape bacteria from any of the required plates (sender/receiver, pos/neg controls) 6. Paint the bacteria (liberally) on the plate in the designated sections. 7. Be sure to add enough bacteria so that there is a semi-thick layer that is continuous throughout the designated area. Imaging the Induction plates8. Put magnetic cover on top of UV light ( will have to adjust placement after imaging) 9. Take off Lid of Culture plate ![](https://static.igem.org/mediawiki/2017/7/7d/Inc_Pic_33.png) **Fig 3: Prepping Culture Plate for imaging** ![](https://static.igem.org/mediawiki/2017/e/e4/Inc_pic_44.png) **Fig 4: SynGene Settings** 10. Place culture plate on top of magnetic cover and close the tray. (Fig. 3) 11. Machine Settings: (Fig. 4) * Blots: Fluorescent Blot * Alexa 488 * Epi Mid Wave UV * UV06 12. Press Green arrow once machine is done calibrating 13. Press Capture 14. Save as .sgd in order to have the original data still available to be able to save as .tif 15. If 3D imaging is needed: * click edit on the lower right side of the screen and click 3D on following screen (upper left) 16. If annotations are needed * proceed to the edit screen * click on annotate option in upper left hand corner * then place text over parts of the image you want to be annotated * save image in preferred format "as displayed" to save annotations.

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# Arizona\_State

## Arizona\_State\_University\_iGEM\_2017\_Results

# Engineering Variable Regulators for a Quorum Sensing Toolbox - EVR-QST

### \*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

There are 4 types of experiments used to characterize new receivers: Sender Quest: Battle of the AHLs, Synthetic AHLs: Quest for GFP Domination, Side Quest: Lost in Translation for the Missing mCherry, and Diffusion Quest.

The first experiment, Sender Quest: Battle of the AHLs, used new Las and Tra receiver cells being induced by a variety of combinations of senders in supernatant form. The maximum GFP expression was analyzed to understand the effect of different percentages of sender supernatant had on the receiver expressing GFP. These results help in determining what circuits will produce maximum or minimum GFP, and what systems are orthogonal.

In the Synthetic AHLs: Quest for GFP Domination experiments, synthetic AHL chemicals under different concentrations induce the receivers of LasR and LuxR. The maximum GFP expression was analyzed using Transfer Function curves to understand the effect a broad range of AHL concentration had on the receiver expressing GFP.

In the Sender Quest: Battle of the AHLs experiments, Las and Tra receiver cells were induced by a variety of combinations of senders in supernatant form. The maximum GFP expression was analyzed to understand the effect different percentages of sender supernatants had on the receiver when expressing GFP.

In the Side Quest: Lost in Translation for the Missing mCherry, the mCherry gene, or red color, in sender cells was not expressing as expected. To investigate this problem, experiments were conducted to determine why the mCherry was not expressing properly. It was found that the presence of mCherry is not a good indicator of synthase expression or AHL production.

In the Diffusion Quest, included Las and Tra receiver cells that were spread on agar plates and induced by spread sender cells. Images are then taken at different time intervals of the agar plate to analyze the induction rate of GFP.

![]( https://static.igem.org/mediawiki/2017/0/0d/Results_Summ.png)

## Sender Quest: Battle of the AHLs

Some of the specific questions this research was aiming to answer were: How do combinations of senders affect gene output? Are there any combinations of senders that increase the overall GFP expression? Are there any combinations that do not affect the GFP expression?

The specific senders that were chosen for the induction tests were selected because previous research showed that they have either a very low or very high rate of GFP induction when used in a single sender/ receiver circuit. In other words, the chosen senders tend to either work very well or not very well at all. More data is needed on how well these senders express the gene when used in combination with another. By combining two senders at a time, sometimes with senders that have shown to induce a high GFP expression and sometimes with senders that have shown a weak induction, the goal is to see if there is any increase or decrease the GFP expression on demand. The controls used for the experiment were single sender inductions on the same plate as the combinations, the use of blank wells (LB AMP 100%), a positive GFP control, and a negative control with negative receiver cells and negative sender supernatant.

**This next section of results is for the tests done with the next receiver, LasR.**

Test #1 with LasR: Sender A: AubI, B: EsaI, C: CerI

The first set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.

![Design Flowchart]( https://static.igem.org/mediawiki/2017/e/e9/A12.png  )
![Design Flowchart](https://static.igem.org/mediawiki/2017/8/8a/A13.png   )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/9/93/A14.png  )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/8/8c/A15.png  )
![Design Flowchart](https://static.igem.org/mediawiki/2017/5/52/A16.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/f/f9/A17.png   )

### Results from test #1 with LasR:

This test showed some notable results. As seen clearly in the last graph, the AubI showed a higher expression when mixed with 10% of a second sender (even when that sender was a negative control sender). The 40% AubI mixed with 10% negative sender and the 40% AubI mixed with 10% EsaI or 10% CerI both expressed higher than the 50% AubI by itself.

### Test #2 with LasR: Sender A: BraI, B: LasI, C: AubI

The second set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.

![Design Flowchart](https://static.igem.org/mediawiki/2017/9/98/A18.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/2/23/A19.png   )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/a/a6/A20.png  )

### Results from test #2 with LasR:

This test showed some notable results. As seen clearly in the last graph, the BraI showed a higher expression when mixed with 10% of a second sender (even when that sender was a negative control sender). The 10% BraI + 40% AubI mixed expressed higher than the 50% AubI by itself. Close behind the 50% AubI was the 50% LasI. Interesting result because the AubI expresses higher than the matching sender LasI to its own LasR receiver.

### Test #3 with LasR: Sender A: AubI, B: RpaI, C: BjaI

The third set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.

![Design Flowchart]( https://static.igem.org/mediawiki/2017/c/ce/A21.png  )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/3/3c/A22.png  )
![Design Flowchart](https://static.igem.org/mediawiki/2017/2/29/A23.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/4/46/A24.png   )

### Results from test #3 with LasR:

This test showed some notable results. As seen clearly in the last graph, the 40% AubI showed a higher expression when mixed with 10% of a second sender (even when that sender was a negative control sender). The 10% BjaI + 40% AubI mixed expressed higher than the 50% AubI by itself. Interesting result because the AubI expresses higher when mixed with another sender, seemingly with both LuxR and LasR.

**All the below tests are with the TraR Receiver.**

### Test #1 with TraR: Sender A: AubI, B: EsaI, C: LuxI

The first set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.

![Design Flowchart](https://static.igem.org/mediawiki/2017/a/aa/A26.png   )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/0/06/A25.png  )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/e/e7/A27.png  )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/4/49/A28.png  )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/a/ac/A29.png  )
![Design Flowchart](https://static.igem.org/mediawiki/2017/9/9f/A30.png   )

### Results from test #1 with TraR:

This test showed some notable results. As seen in the last graph, the AubI and the EsaI expressed much higher than the LuxI. This is a result that does not match other tests performed by the team. This was also not a result that was able to be replicated and it is thought that there may have been an issue with the LuxI that caused it to not induce the TraR.

### Test #2 with TraR: Sender A: LasI, B: RhlI, C: BjaI and SinI

The second set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.

![Design Flowchart](https://static.igem.org/mediawiki/2017/6/63/A31.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/c/c4/A32.png   )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/1/1d/A33.png  )

### Results from test #2 with TraR:

There are not any notable results in this test, the sender's did not induce the Tra receiver. The overall GFP expression (in A.U.) showed no induction and it may be orthogonal pathways when using filtered supernatants from cultured cells.

### Test #3 with TraR: Sender A: CerI, B: BraI, C: RpaI

The third set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.

![Design Flowchart](https://static.igem.org/mediawiki/2017/3/38/A34.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/4/4c/A35.png   )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/7/74/A36.png  )

### Results from test #3 with TraR:

There are not any notable results in this test, the sender's did not induce the Tra receiver. The overall GFP expression (in A.U.) showed no induction and it may be orthogonal pathways when using filtered supernatants from cultured cells.

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### Noteworthy (successful) results summary.

Here you will find all supporting data for the conclusions described in the results section including the error calculations and all additional graphs. Each section is broken down by the receiver and labeled at the start of the section.

### Summary for Lux Receiver:

When testing the LuxR the AubI showed a higher expression when mixed with 10% of a second sender (even when that sender was a negative control sender). The 40% AubI mixed with 10% negative sender and the 40% AubI mixed with 10% RhlI both expressed higher than the 50% AubI by itself. This result was confirmed in another test where 40% Aub mixed with 10% EsaI and 40% AubI mixed with 10% CerI both expressed higher than the 50% AubI alone. (See graphs below).

![Design Flowchart]( https://2017.igem.org/File:A4.png  )
![Design Flowchart](https://static.igem.org/mediawiki/2017/c/ce/A38.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/d/d2/A40.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/e/e9/A2.png   )

### Summary for Las Receiver:

Below we see an experiment where the AubI expresses higher when mixed with 10% of any other sender, these results are with the Las receiver. This evidence further confirms that AubI simply works best when mixed versus being used alone. The 40% AubI mixed with 10% negative sender, 10% EsaI and 10% CerI all expressed higher than the 50% AubI alone.

![Design Flowchart](https://static.igem.org/mediawiki/2017/8/86/A42.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/e/e1/A43.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/c/c2/A44.png   )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/9/9d/A45.png  )
![Design Flowchart](https://static.igem.org/mediawiki/2017/5/52/A16.png   )

Below, another test showed some notable results. As seen clearly in the last graph, the BraI showed a higher expression when mixed with 10% of a second sender (even when that sender was a negative control sender). The 10% BraI + 40% AubI mixed expressed higher than the 50% AubI by itself. Close behind the 50% AubI was the 50% LasI. Interesting result because the AubI expresses higher than the matching sender LasI to its own LasR receiver.

![Design Flowchart](https://static.igem.org/mediawiki/2017/a/a6/A20.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/4/46/A48.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/a/a0/A49.png   )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/e/e1/A43.png  )

Below, another test showed some notable results. As seen clearly in the last graph, the 40% AubI showed a higher expression when mixed with 10% of a second sender (even when that sender was a negative control sender). The 10% BjaI + 40% AubI mixed expressed higher than the 50% AubI by itself. Interesting result because the AubI expresses higher when mixed with another sender, seemingly with both LuxR and LasR.

![Design Flowchart](https://static.igem.org/mediawiki/2017/4/46/A24.png   )
![Design Flowchart](https://static.igem.org/mediawiki/2017/b/bd/A52.png   )
![Design Flowchart](https://2017.igem.org/File:A43.png   )

barely induces the Las receiver. May be orthogonal (below).

![Design Flowchart](https://static.igem.org/mediawiki/2017/7/70/A54.png   )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/7/70/A54.png  )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/3/31/A55.png  )
![Design Flowchart]( https://2017.igem.org/File:A48.png  )

AubI expresses higher than the LasI with the LasR (below)

![Design Flowchart](https://static.igem.org/mediawiki/2017/3/37/A58.png  )
![Design Flowchart](https://static.igem.org/mediawiki/2017/e/e1/A43.png  )
![Design Flowchart](https://static.igem.org/mediawiki/2017/a/a0/A49.png  )

### Summary of the Tra Receiver:

The TraR receiver is orthogonal with most senders, even the TraI synthetic AHL barely induces its own receiver. The only two senders that induced the Tra receiver (aside from the TraR synthetic AHL) within a noticeable range, was the AubI and the EsaI.

![Design Flowchart]( https://static.igem.org/mediawiki/2017/c/c4/A32.png )
![Design Flowchart](https://static.igem.org/mediawiki/2017/b/b7/A61.png  )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/4/49/A28.png )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/e/e7/A27.png )
![Design Flowchart](https://static.igem.org/mediawiki/2017/a/ac/A29.png  )
![Design Flowchart]( https://static.igem.org/mediawiki/2017/9/9f/A30.png )

### Unsuccessful results:

In the below test with the LuxR, there were some results that were not able to be replicated, meaning that there may have been some contamination or other unaccounted for error. The 40% LuxI + 10% negative sender expressed higher than the 50% LuxI alone, this result was not able to be replicated. Again the 40% LuxI + 10% RpaI expressed higher than the 50% LuxI alone but the result was unable to be replicated and lastly the 40% LuxI + 10% BraI expressed higher than the 50% LuxI and again, the result was unable to be replicated.

![Design Flowchart](https://static.igem.org/mediawiki/2017/6/64/A8.png  )

## Future plans for the project:

In consideration for the foundational advance of this project in the future, it would be advisable to add additional concentrations of the sender combinations with each receiver. These experiments tested combinations of 50%, 40%, 25%, 12.5%, and 10% and the notable results from these experiments could be used as a base for creating new experiments with additional concentrations and combinations. As new receivers are developed replicating these tests with them would be wise as it would grow the library of currently characterized receivers with their senders.

## Considerations for replicating the experiments:

A few important details to take into consideration when replicating these experiments are, noting that you get the best results from freshly transformed bacteria versus using bacteria from existing agar plates and that it is best to filter the supernatant from the sender cells immediately prior to running your experiment. Highest GFP induction occurred when freshly transformed samples were gathered and grown in liquid culture, using the shaking incubator, on the day of the experiment.

## References

Fuqua, C, Winans, SC, Greenberg, EP, et al. Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu. Rev. Microbiol. 1996; 50: 727–751. doi:10.1146/annurev.micro.50.1.727

Miller, MB, and Bassler, BL. Quorum sensing in bacteria. Annu. Rev. Microbiol. 2001;55: 165–199. doi:10.1146/annurev.micro.55.1.165

Davis, RM, Muller, RY, Haynes, KA. Can the natural diversity of quorum sensing advance synthetic biology? Front. Bioeng. Biotechnol. 2015; 3.30.1-9. doi: 10.3389/fbioe.2015.00030

Lindemann, A, Pessi, G, Schaefer, A, Mattmann, M, Christensen, Q, Kessler, A, et al. Isovaleryl-homoserine lactone, an unusual branched-chain quorum-sensing signal from the soybean symbiont Bradyrhizobium japonicum. Proc. Natl. Acad. Sci. U.S.A. 2011;108: 16765–16770. doi:10.1073/pnas.1114125108

Nasuno, E, Kimura, N, Fujita, MJ, Nakatsu, CH, Kamagata, Y, Hanada, S, et al. Phylogenetically novel LuxI/LuxR-type quorum sensing systems iso- lated using a metagenomic approach. Appl. Environ. Microbiol. 2012;78: 8067–8074. doi:10.1128/AEM.01442- 12

Eberhard, A, Burlingame, AL, Eberhard, C, Kenyon, GL, Nealson, KH, Oppenheimer, NJ, et al. Structural Identification of Autoinducer of Photobacterium fischeri. Biochemistry. 1981;20: 2444–2449.

Marketon, MM, Gronquist, MR, Eberhard, A, González, JE et al. Characterization of the Sinorhizobium meliloti sinR / sinI Locus and the Production of Novel N-Acyl Homoserine Lactones Characterization of the Sinorhizobium meliloti sinR / sinI Locus and the Production of Novel N -Acyl Homoserine Lactones. J. Bacteriol. 2002;184: 5686–95. doi:10.1128/JB.184.20.5686.

## Synthetic AHL Concentration Inductions of LasR, LuxR, and TraR

Questions these induction tests answer was how GFP expression is affected by different synthetic AHLs over a range of concentration. Conducting these experiments will also provide insight on what the optimal AHL concentration a sender is able to communicate a signal to the receiver to better express GFP. It will also be seen which systems of AHL and receiver are orthogonal.

The synthetic AHLs and range of concentrations chosen for these inductions came from the paper "Quorum Sensing Communication Modules for Microbial Consortia" written by Scott Spencer and Jeff Hasty at the University of California, and from last year's iGEM team. This year, it was decided to expand the characterization of the new and improved receivers of this year to include a broader concentration range, and more synthetic AHLs. To see these relationships, the synthetic chemicals induced receiver cells in liquid culture and analyzed the growth curves in a 96-well plate done in triplicate. The controls for these inductions included blank LB wells, a positive GFP control, a negative receiver control, a negative sender supernatant control, and a positive sender control.

The experiments performed were to characterize the Las, Lux, and Tra receivers included the synthetic AHLs of 3-oxo-C12 (Las), 3-oxo-C6 (Lux), 3-oxo-C14 (Sin), Coumaroyl (Rpa), C4 (Rhl), and 3-oxo-C8 (Tra). The following graphs show the maximum (DGFP/OD)/DT across a concentration range of 1E-14M to 1E-4M. Each data point was done in triplicate then averaged. These points determine where the GFP is changing the fastest, and represents the maximum rate of change at a specific synthetic AHL concentration.

The graphs below depict the GFP production from the F2620 receiver to be higher at a higher concentration of the synthetic AHL of Las. The second graph, depicts the same nature however at a higher concentration of the synthetic AHL of Lux. It is interesting to note that the Las sender, at a lower concentration, promotes a higher GFP expression than the Lux. Our group hypothesized that it would be opposite considering the LuxI sender comes from the same system as F2620. These results show differently.

![](https://static.igem.org/mediawiki/2017/9/9a/First_1.png)
![](https://static.igem.org/mediawiki/2017/7/70/First_2.png)

As Las receiver and Tra receiver are also induced by the synthetic AHLs, a similar trend emerges. There is little to no GFP expression for the lower concentration, between 1E-14 through 1E-9, 1E-8, or 1E-7 in some cases. Then a dramatic increase in GFP expression as the AHL concentration increases. This indicates that the system shows significant GFP expression at higher concentrations.

![](https://static.igem.org/mediawiki/2017/9/9d/First_3.png)
![](https://static.igem.org/mediawiki/2017/5/5b/First_4.png)
![](https://static.igem.org/mediawiki/2017/d/d3/First_5.png)
![](https://static.igem.org/mediawiki/2017/9/95/First_6.png)
![](https://static.igem.org/mediawiki/2017/d/d6/First_7.png)
![](https://static.igem.org/mediawiki/2017/1/1c/First_8.png)

Another interesting result was the with the synthetic AHL Rpa. As depicted in the graph below, it shows an almost steady state of GFP production, independent of the concentration of the AHL signal. This trend can also be seen in multiple systems such as the induction of LasR with a Rhl AHL, LuxR with Rhl AHL, and LuxR with a Rpa AHL. This might be a useful finding if researchers wanted to use a smaller amount of signal due to limiting resources of RpaI or RhlI sender. However, it did not show an orthogonal pathway since it did in fact promote GFP expression.

![](https://static.igem.org/mediawiki/2017/8/8f/First_9.png)
![](https://static.igem.org/mediawiki/2017/8/88/First_10.png)
![](https://static.igem.org/mediawiki/2017/5/51/First_11.png)
![](https://static.igem.org/mediawiki/2017/a/a6/First_12.png)

The trend outlined above is also expressed in the below graphs. From concentrations 1E-14M through 1E-5M there is a steady production of GFP that is independent of the AHL signal concentration. However, as the receiver is induced by the 1E-4M signal, an outlier of GFP expression is produced. This is an unusual result as the 1E-4M concentration in the LasR and Lux AHL system is expected to be around 2-6 arbitrary units. This unexpected point is also present in the TraR and Rhl AHL system. The max GFP expression of 1E-4M is an outlier as the trend indicates orthogonality between the receiver TraR and RhlI sender. Within our experiments, we implied that orthogonality is defined as not expressing GFP or having no communication within the systems. This might be an important find for synthetic biologist and genetic engineering to use this orthogonal quorum sensing circuit within their genetic circuit to be able to, with confidence, produce results of a protein or phenotypic expression. This orthogonality does not apply to the system of LasR and Lux synthetic AHL, however, as there was still induction and GFP was shown to express. As stated above, this system could be beneficial to researchers who have limited resources of the Lux sender and wish to express the same amount of GFP that would occur in higher concentrations.

![](https://static.igem.org/mediawiki/2017/9/91/First_13.png)
![](https://static.igem.org/mediawiki/2017/8/8f/First_14.png)

The graph of LasR and Las AHL displayed below is a combination of different sender concentrations of LasI in respect to the GFP expression of LasR. It is interesting to note that the corresponding sender and receiver system, Las, does not express as highly in GFP expression as other experiments with different senders. Our group hypothesized that this sender/receiver system would still promote a higher GFP expression, even at a more diluted concentration of synthetic AHL. This trend can also be seen in the graph of LasR and Sin AHL. The 1E-7M expresses a higher GFP of around 20 arbitrary units than the 1E-4M, which is an unexpected result. This is beneficial to researchers looking for maximum GFP expression using the Las receiver and a small supply of sender.

![](https://static.igem.org/mediawiki/2017/a/a7/First_15.png)
![](https://static.igem.org/mediawiki/2017/3/37/First_16.png)

A notable conclusion can be drawn from the below graph of the Tra receiver and Lux AHL system. It is seen from 1E-14 to 1E-7 that there is little to no GFP expression, while the 1E-4M concentration reached 6 arbitrary units with high error. This type of fluctuation also occurs in the LuxR with Sin AHL, although it is more varied throughout the concentration range. Both graphs appear to have an upward trend despite the fluctuations as concentration increases.

![](https://static.igem.org/mediawiki/2017/b/bc/First_17.png)
![](https://static.igem.org/mediawiki/2017/c/c1/First_18.png)

## Future plans for the experiment:

For the foundational advance of this experiment in the future, it would be advisable to expand the concentrations used for inductions. Currently, the concentrations used include 1E-14M, 1E-13M, 1E-12M, 1E-11M, 1E-10M, 1E-9M, 1E-8M, 1E-7M, 1E-6M, 1E-5M, and 1E-4M. From the experiments, it is seen that the most GFP expression occurs in higher concentrations from 1E-8M to 1E-4M. If the range is narrowed between the values to include 1E-4.5M, 1E-5.5M, 1E-6.5M, and 1E-7.5M, a better idea of how GFP is expressed can be analyzed. Also if more synthetic AHLs are able to be tested with these receivers, the characterization can be expanded and these receivers can be further analyzed on how they work in multiple systems.

## Considerations for experimental replication:

When replicating these inductions, it is important to note that these AHLs are dissolved in ethyl acetate and serial dilutions were performed to get the desired concentrations. These dilutions were done the day of induction, and kept at -20 degrees Celsius for optimum results and lowest chance of degradation before testing. Also when plating, the final concentration of each AHL in the wells were the concentrations listed, with 1% ethyl acetate. Freshly transformed bacteria cells and grown cultures also gave optimum results for induction.

## Case of the missing mCherry, Lost in Translation:

![](https://static.igem.org/mediawiki/2017/b/b5/Mcherry_no.png)

Why are the senders not expressing mCherry? All senders carry mCherry part as a indicator that our synthases are present within a plasmid, although randomly some express it while others do not. Our sender are bicistronic not fusion transcripts, giving us no guarantee that our synthases is present although they do share vector and if mCherry is expressed should still have synthases. This is an indicator on if our plasmid carries all of our needed parts, including our sender insert. Leading to explanations on why cross talk or sender are not working. For all gel runs, all parts said in the plasmid have shown to be present. This could possibly be a random occurrence, fluorence of Mcherry may be select. Sequencing was completed, revealing that no matter the expression of the culture, our senders still induce GFP. Concluding that mcherry is not a indicator for our synthases in our sender bacteria. This also opens the door for future possibility on why mCherry is not being expressed, regardless the fluorescence is not a valid indicator.
![](https://static.igem.org/mediawiki/2017/c/ce/MCherryGel.png )

- 1% agarose gel run at 110 volts for 40 minutes. This is the gel shows psb1C3 plasmid DNA purified from red cells on the left and beige on the right an digested with the corresponding labeled restriction enzymes. Results indicate the presence of mCherrry in both samples.

![](https://static.igem.org/mediawiki/2017/9/94/SinSender.JPG )

Sequencing results from a red Sin sender (Sin old) and a non-red Sin sender(Sin new). Results showed the presence of mCherry in the bicistronic gene.

![](https://static.igem.org/mediawiki/2017/e/e4/AubmCherry.png)

Induction of 2610 with supernatant harvested form non-red AubI cells. Results indicate that AHLs are still being produced regardless of mCherry.

## Modeling for Results page:

These results outline the dynamics of the plated agar inductions for LasR and TraR.

![](https://static.igem.org/mediawiki/2017/8/88/Im-66.png)
![](https://static.igem.org/mediawiki/2017/e/e8/Im-77.png)
![](https://static.igem.org/mediawiki/2017/e/ef/Im-88.png)
![](https://static.igem.org/mediawiki/2017/b/b2/Mmm-9.png)
![](https://static.igem.org/mediawiki/2017/1/16/Mmm-10.png)
![](https://static.igem.org/mediawiki/2017/7/70/Mmm-11.png)
![](https://static.igem.org/mediawiki/2017/f/fb/Mmm-12.png)

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