Overview

For science to be dependable and reproducible, good measurement is essential. Characterization and standardization of systems are crucial, particularly in the context of synthetic biology's "Design - Build - Test - Learn" paradigm, which facilitates simpler debugging and strengthens data-driven predictions.

Bradford Assay

In preparation for our Western Blot, we needed to quantify the amount of protein in the cell lysate, so we could normalise the amount that we loaded into SDS-PAGE gel. To achieve this, we used a Braford assay to measure the protein content in a sample of cell lysate.

Materials:

• BSA standard solution (0.1 µg/µl)
• Bradford solution - Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol. Add 100 ml of 85% phosphoric acid while stirring continuously. When the dye has dissolved, dilute to 1L in distilled water. Filter to remove residual precipitate (Whatman filter paper) and store at 4°C in a dark bottle.

Procedure:

1. Warm up the spectrophotometer before use.
2. Add 20uL of BSA standards in increasing concentration of protein to successive wells in a 96 well plate.
3. Add 1uL of your protein samples in the wells after the standard wells.
4. Add 200uL Bradford reagent and incubate in the dark for 5 minutes.
5. Measure the absorbance at 595 nm and plot the absorbance to find concentration of the unknown protein samples in µg.

Protein conc of standards (ug/mL)	Absorbance measured for standards
0	0.397
156	0387
312	0.444
625	0.48
1250	0.51
2500	0.68
5000	0.78

Equation of the line of best fit: y = 0.00008x + 0.412

Samples	Absorbance measured for samples	Protein conc calculated for samples from the equation of the line of best fit (ug/mL)
scFv (16 degrees)	0.803	4881.25
scFv (25 degrees)	1.128	8943.75
scFv (30 degrees)	0.706	3668.75
scFv+linker (16 degrees)	1.2	9843.75

BSA Calibration for DoE

We wanted to determine the optimal growth and expression conditions of SHuffle B for the best yield of NeoFv protein.
We began with a BSA calibration SDS-PAGE triplicate gel where we ran varying concentrations of BSA protein, along with two concentrations of an arbitrary 25kDa protein, to assess concentrations based on the intensity of banding. We ran a triplicate of this gel as a check for loading errors.
Using SDS-PAGE band intensity provides a quicker and cheaper way of analyzing protein concentrations. It can be done by running gels of cell lysate (optionally seperated into supernatant and pellet). Specialised image analysis softwares like ImageJ are required to plot the band intensities SDS-PAGE bands.

Sample Preparation: We prepared a stock solution of BSA protein of 10 g/ml and made serial dilutions to get the other concentrations.

One limitation of this method, as we found out while conducting our design of experiments (DoE) studies is in predicting concentrations below 100 μg/ml and above 0.7 μg/ml, due to going behind the positive y-intercept of the graph. We ignored the 1mg/ml data point while plotting the BSA standard curve because it was not preserving the linear concentration-intensity relationship found between 0.1 and 0.7 μg/ml.

Growth Curves:

Growth curves track the division of an organism in culture, and plotting them gives us vital information regarding the replication of the organism. They allow us to identify and predict the rough time markers where an organism reaches a particular phase of growth, which is vital for protocols like competency and protein expression. Additionally, when a particular experiment does not conform to the values that a growth curve predicts, it is an easily visible marker that something has gone wrong.

We wanted to compare the growth rates of the two different E.coli SHuffle strains we were using. We took Optical Density measurements at 600 nm and compared the same. The results for both the strains were then plotted on a graph

Materials required:

Bacterial culture (E. coli), Broth (Luria Bertani (LB) Broth, Nutrient Broth)
Glass wares: Conical flasks, Measuring cylinder, Sterile test tubes, Sterile Petri plates
Reagents: Distilled water
Other requirements: Incubator, Shaker, Spectrophotometer, Micropipettes, Tips, Sterile Loop

Protocol:

1. Streak a loopful of bacterial culture onto the agar plate using a sterile loop.
2. Incubate for 18–24 hours at 37 °C
3. Select one colony of each strain off the agar plate, and then place it in a test tube with 10 ml of autoclaved broth as an inoculum
4. Overnight, incubate the test tube at 37 °C
5. Take 500 ml of sterile conical flask and fill it with 250 ml of autoclaved broth.
6. Inoculate the aforementioned flask with 5 cc of the overnight-grown culture.
7. OD at the hour of zero. At 37 °C, incubate the flask.
8. Aliquot 1 ml of the culture suspension should be added at intervals of 30 minutes, and optical density should be measured using a spectrophotometer at a wavelength of 600 nm until the reading becomes stable.

At the conclusion of the experiment, the optical densities of each aliquot can be measured.
Plot a graph of time in minutes on the X axis versus optical density at 600 nm on the Y axis at the conclusion of the experiment to produce a bacteria growth curve.

Time post induction	Control	BL21	Shuffle K12	Shuffle B
00:00	0	0	0
00:35	0.002	0.0857	0.0672	0.0687
01:12	0.0	0.2447	0.0482	0.0194
01:52	0.008	0.5978	0.0567	0.0171
02:22	-0.009	1.0377	0.0521	0.0566
02:53	-0.003	1.4611	0.0888	0.0717
03:28	-0.003	1.9466	0.1053	0.0857
04:08	0:00	2.4176	0.1847	0.1065
04:51	0.006	2.677	0.2647	0.1622
05:32	0:00	2.7753	0.4333	0.2775
06:02	0:00	2.8465	0.61115	0.43575
06:32	0:00	2.8465	0.789	0.594
07:12	0.00	2.8465	0.9914	0.8478
07:52	0.00	2.8465	1.4103	1.3189
08:30	0.0542	2.8465	1.8441	1.7109
09:09	0.0292	2.8465	2.1414	2.0013
09:47	0.0185	2.8465	2.4997	2.3381
10:26	-0.004	2.8465	2.5181	2.7211
12:12	0.008	2.8465	2.7176	2.764

Growth Curves: