Team:Cadets2Vets/InterLab

Cadets2Vets participated in the 2017 Interlab Measurement Study. The purpose of this study is to build upon past work and develop a method for standardizing the quantification of GFP expression using a plate reader or flow cytometer. Synthetic biology is challenging because of its overall complexity, as well as the difficulty in predicting the behavior of the genes when placed in a novel environment. iGEM is working towards reducing the difficulty of working with synthetic biology by creating a biobricks “interchangeable parts” model of plasmid and biological systems development, as well as creating a worldwide protocol for fluorescence measurement. During the interlab study we were able to test specific ribosome binding site and promoter combinations that could potentially become the new standard for protein production. The idea is that ribosome binding sites would affect the recruitment of ribosomes to the transcript, thus affecting the level of protein expression.

The Interlab protocol measured the Abs600 and fluorescence of bacterial cultures, and this gave us an idea of the cell density and the fluorescence of the culture over time. We had both technical and biological replicates to understand the variability that might occur in the experiment.

The Cadets2Vets found this study to be very straight forward and rewarding. Being a part of one of the largest studies in synthetic biology is a privilege, and understanding the complexity of the science gives us another level of appreciation for the competition and what it really means to be a sum of an important whole.

On August 2 and 8, 2017, Keshava Katti and Kian Croston ran two ticket assays to determine the effect of adjusting plasmid DNA proportionality. The past two tickets contained equal proportions of PcArsR and PARsGFP (1:1 ratio).

The Cadets2Vets team set up two tickets, positive and negative tests, on August 2. The positive ticket tested the dosage response at varied volumes of arsenic water; the negative ticket substituted all instances of arsenic water with molecular water.

Column 1: 2uL Master Mix + 2uL water (molec/arsenic)

Column 2: 2uL Master Mix + 4uL water (molec/arsenic)

Column 3: 2uL Master Mix + 6uL water (molec/arsenic)

Column 4: 2uL Master Mix

Positive Master Mix contained:

12 uL S30 Extract
16 uL S30 Promega
4 uL amino (-Cys)
0.8 uL amino (-Leu)
3.2 uL arsenic water
4 uL of PcArsR + Ars1.1 circuit plasmid

Negative Master Mix contained:

12 uL S30 Extract
16 uL S30 Promega
4 uL amino (-Cys)
0.8 uL amino (-Leu)
3.2 uL molecular water
4 uL of PcArsR + Ars1.1 circuit plasmid

The image (above) shows the results of our tickets. As expected, our control ticket with no arsenic displayed no GFP expression. On the other hand, our positive ticket showed the same lack of fluorescence, which was attributed to the excess regulatory protein. Unfortunately, the Cadets2Vets did not realize that Ars1.1 was actually causing an increased production in regulatory protein until after our fourth ticket assay on August 8.

Team Cadets2Vets created 8uL of PcArsR + Ars1.1 plasmid DNA by combining 5.33uL of Ars1.1 and 2.67uL of PcArsR (2:1 ratio, respectively). There was a fundamental misunderstanding behind this combination. Our team believed that Ars1.1 was our GFP-producing plasmid DNA; in reality, Ars1.1 was our constitutive promoter, producing our ArsR regulatory protein, and GFP plasmid DNA combined.

The original idea was to increase the amount of promoter binding sites for ArsR, which is how GFP expression is regulated in the arsenic circuit. The additional PArsGFP plasmid was supposed to prevent ArsR from being free in solution and binding to free arsenic ions, thereby unintentionally sequestering our arsenic ions and ArsR proteins away from the regulatory mechanism we designed. Instead, we accidentally ended up increasing the amount of constitutive promoter DNA (Ars1.1 added unwanted constitutive promoter DNA), thereby increasing the overall production of ArsR regulatory protein.The image (left) shows the results of our tickets. As expected, our control ticket with no arsenic displayed no GFP expression. On the other hand, our positive ticket showed the same lack of fluorescence, which was attributed to the excess regulatory protein. Unfortunately, the Cadets2Vets did not realize that Ars1.1 was actually causing an increased production in regulatory protein until after our fourth ticket assay on August 8.

Fluorescence Standard Curve Created

Note (Zoey): After speaking with Judy I learned that our original settings and interpretation of the interlab protocols settings for the plate reading where incorrect. We errored in not paying close attention to whether it required ABS (Absorption) or OD (Optical density) for the test. There is a specific tab in the plate reader program for Fluorescence (FL). Where we make our setting: Excitation (485 mn) / Emission (530/30). This error made the calibration for our culture/LB broth ratios to more than likely be off. It is expected we will need to redo this interlab experiment once more. In addition to the culture plate run, we should have used ABS setting of 595 nm allowed by the machine and not 450. This setting should have been the same for the Day 3 Culture hourly sequence plate read experiment.

After Fluorescence standard curve was created with plate reader we began the Day 3 section of interlab protocols. Using the ABS setting of 450nm (Read today’s note for details on our error here)

Entering this data into the excel spreadsheet contained on the team drive “Interlab 2017 Measurements.

We made up an LB broth + Chloramphenicol solution .25ml of CAT in 250 ml of LB Broth as our standard. To create a 25ug/ml stock.

Taking a sample from each of our incubation falcon tubes. Pos, neg, TD1, TD2, TD3, TD4, TD5, TD6 all from the “A” samples in cells A1 - A8 respectively and LB+Chloramphenicol in A9. We ran that 96 well plate through the plate reader again using the same ABS 450 nm setting as before. (See today’s note above for details on our error here)

Imputing the resulting numbers into the excel located on Team drive called, “2017 Interlab Dilution Calculation Sheet.” This was provided by IGEM and ran the required calculation to determine the ratio of LB + Chloramphenicol to how much of each culture was required.

Covering 50 mL falcon tubes in tin foil as per protocols following the ratios set down in the excel spreadsheet to equal 12 mL total. We labeled 16 total 1.5 mL microcentrifuge tubes 8 “A” & 8 “B”, a 500uL sample from each of the 16 new culture falcon tubes was placed into each respective microcentrifuge tube. Tape was wrapped over all 16 tubes at hour 0 and placed in ice. The Falcon tubes of culture were placed in the shake plate incubator and timer was set for 2 hours.

Hour 2. The process was repeated as per interlab protocols. 500 uL of culture solution were placed in the 16 microcentrifuge tube corresponding with the same numbered and lettered falcon tube. Labeled and placed into Ice.

Hour 4. The process was repeated as per interlab protocols. 500 uL of culture solution were placed in the 16 microcentrifuge tube corresponding with the same numbered and lettered falcon tube. Labeled and placed into ice.

Hour 6, repeated previous hours steps.

Once all were finished I then plated a sample of 100 uL from the microcentrifuge tube in a vertical pattern beginning with the negative control. A1, A2, A3, A4. For the B sample of the negative control, t went in the next four wells. A5, A6, A7, A8. This process was continued on down the rows. B row = Positive Control. C row = TD1, D row = TD2, E row = TD3, F row = TD3, G row = TD 4, H row = TD4, I row = TD 5, J row = TD 6, K row = LB broth+CAT. A single plate was prepared for each two-hour increment and sent through the plate reader.

All results were recorded manually into the team drive excel spreadsheet for later analysis.

Repetition of interlab measurements starts today.

LB broth made for overnight cultures. 500 mL broth made. 10g LB broth powder in 500 mL H2O.

Broth autoclaved on liquid cycle for ~1 hour.

Ludox

With black bottom/clear bottom Greiner 96 well plate. Prepared a column of 4 wells (A1-D1) with 100 µl 100% LUDOX and 4 wells containing 100 µl H 2O (A2-D2).

Method:

Added 100 µl LUDOX into wells A1, B1, C1, D1

Add 100 µl of H2O into wells A2, B2, C2, D2

Measured absorbance 595 nm of all samples in all standard measurement modes in instrument

	Ludox 100%	H20
Replicate 1	0.40	0.029
Replicate 2	0.40	0.030
Replicate 3	0.037	0.030
Replicate 4	0.041	0.030

Fluorescein standard curve repeat.

Fluorescein stock tube spun down. Prepared 2x fluorescein stock solution by adding 1 mL of PBS. Combined 500 µL of 2x fluorescein stock solution and 500 µL 1x PBS 1xPBS to make a 1x fluorescein solution.

iGEM Fluorescein standard curve method followed (dilution series over A-D, 1-12 on 96 well plate).

Plate read on FilterMax F5 Multi-Mode Microplate Reader:

1

2

3

4

5

6

7

8

9

10

11

12

A

1.1e9

8.8e8

6.0e8

3.2e8

2.0e8

1.0e8

5.0e7

2.5e7

1.3e7

6.4e6

3.1e6

8.7e4

B

1.2e9

7.8e8

6.1e8

3.4e8

1.7e8

9.2e7

4.5e7

2.2e7

1.1e7

5.5e6

2.7e6

7.6e4

C

1.2e9

8.8e8

5.8e8

3.4e8

1.8e8

9.4e7

4.7e7

2.4e7

1.2e7

5.7e6

2.9e6

7.6e4

D

1.2e9

8.6e8

5.4e8

3.8e8

1.9e8

9.6e7

4.9e7

2.4e7

1.2e7

5.9e6

3.0e6

7.6e4

Overnight cultures started:

Two colonies from positive, negative and TD1-6 inoculated in 10 mL of LB + CAT broth in 50 mL Falcon tube. Duplicates made. Falcon tubes placed in shaker overnight at 37C and 220 RPM.

Interlab day 3 attempt 2

100 uL taken from each Falcon tube and placed into 96 well plate. Abs600 read and placed into dilution calculation excel sheet for preloading volumes of culture and LB broth.

Zero, two, four and six hour time tables started. At each two hour interval, 500 uL of each culture is collected and placed on ice. These samples will be compiled at the end of the six hours in four 96 well plates and read based on fluorescence.

500 uL of each sample taken at hour zero and placed on ice.

500 uL of each sample taken at hour 2 and placed on ice.

Interlab Protocol Day 2 Attempt 3. Measured out 8 grams of LB in a 400ml solution of DI water. Ran solution through the autoclave liquid cycle.

0.4 mL of 25mg/mL chloramphenicol (CAT) solution was added to the 400ml solution to make a 25ug/mL concentration solution of LB+CAT.

Labeled 16x50 mL Falcon tubes, +, -, TD1, TD2, TD3, TD 4, TD 5, & TD6 and then labeled A & B to represent duplicates. Add in 5mL of LB+CAT solution into each tube. Then inoculated two separate colonies using the 90% plates from each corresponding Test device and controls into its respective 50mL tube. One colony went to the A while the other went to be the B.

Set the incubator to 37 degrees Celsius and the shake plate to 220 RPM for a 16-18 overnight incubation.

Interlab protocols for Day 3. Took a 100uL sample from each tube and placed them in a row on a 96 well plate, for initial dilution table. “A” samples deposited into respective rows 1-8 a ninth well used as a control with LB+CAT sample; this was repeated for the “B” row samples. Running the plate in the plate reader using ABS setting 595nm. The resulting measurement was placed in Interlab Dilution table to determine dilution into a 12mL Solution.

target Abs600		0.02
target volume (mL)		12
sample	Abs600 Reading	Volume of Preloading Culture	Volume of Preloading Media
positive control	0.523	0.490797546	11.50920245
negative control	0.469	0.5517241379	11.44827586
device 1	0.457	0.5673758865	11.43262411
device 2	0.484	0.5333333333	11.46666667
device 3	0.45	0.5769230769	11.42307692
device 4	0.461	0.5620608899	11.43793911
device 5	0.478	0.5405405405	11.45945946
device 6	0.46	0.5633802817	11.43661972
media+chl	0.034

We covered 16 new 50 mL falcon tubes in tin foil to block ambient light and labeled. Following the instructions of the interlab dilution table above, we added mostly LB broth+ CAT (media) solution and a sample from the overnight cultures of each culture in their respective counterpart. Total solution equaling 12 mL.

Took 500uL of each culture and placed them in their own 1.5mL microcentrifuge tube, storing them in ice. This is time 0hr time point. Then began the time sequence, repeating the same process taking samples at 2hrs, 4hrs, and 6hrs.

Plated each hour collection as shown in the diagram below, of Interlab Protocol into plates, one plate per hour time point for the plate reader. Ran each plate through first for ABS at setting 595nm. Then ran it through for fluorescence Excitation 485nm, and Emission 535 Recorded chart numbers down on team drive

Copied the resulting data points from the plate reader program into the table provided by Interlab project. Resulting example of the table below.

For the team there was a steep learning curve. Making sense of protocols and how to use them in a program and with equipment none of the team members had experience with was a chance to work through problem solving. It took our team 3 consecutive attempts to get all the data points with the correct settings and required filters. This provided the team with the ability to compare previous data points and confirm in triplicate our results in instance of Absorption rates.

In order to go beyond just the protocol requirements for interlab experiment, we discussed what are some hypothesis we could draw from the data we collected.

Team:Cadets2Vets/InterLab

InterLab

Overview

LAB NOTES

08/02/2017

09/06/2017

09/07/2017

09/08/2017

09/18/2017

09/19/2017

09/20/2017

09/24/2017

09/25/2017

What the Team Learned

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2304 Jefferson Ave, Tacoma, WA 98402

	10%	90%	Notes
Positive Control	2	77
Negative Control	10	131
TD1	6	38
TD2	4	52
TD3	6	37
TD4	3	7
TD5	3	3
TD6	3	49