Revision as of 16:26, 17 October 2016

Results

Testing with Exposure to Different Temperatures

All of our genetic device constructs were placed in to the same pSB1C3 vector backbone and transformed in to BL21 (DE3) E.coli cells. We chose BL21 cells as these are optimized for protein expression, since they lack the lon protease involved in protein degradation (Rosano and Ceccarelli, 2015). The cells were grown up in liquid culture of LB broth (with chloramphenicol in strains containing a pSB1C3 housed construct) overnight at 37 °C. The following day, the bacterial cells were diluted down to an appropriate optical density in the region of 0.05 at 600 nm using LB broth with [CONCENTRATION!!!] chloramphenicol. We then pipetted 100 µl of the diluted cells into the corresponding wells, Diagram 1. The plate was laid out in this manner, with a border of wells containing only LB broth, to allow for any inaccuracies that may occur as the heating of the plate occurs from the outer to central wells.

Diagram 1 shows the plate layout we used for these experiments.

The plate reader was then set at either 30°C, 37°C and 42°C and measured for growth using OD600 and fluorescence of GFP using 485nm excitation wavelength and 520nm emission wavelength with a gain of 250. The cells were left to grow for 18 hours with OD and fluorescence being measured every five minutes. In between measurements, the plate reader was programmed to shake, to ensure the cells didn’t clump together. The clumping of cells may have drastically effected the OD reading. The OD was also measured using a ‘well-scan’ function which takes 4 readings at different points within a well, resulting in a more accurate average reading.

Graph of plate at 30oC

Graph of plate at 37oC

Graph of plate at 42oC

Graph of Constructs at all temps???

Picture of plates from cultures grown at 37oC (and 30oC)???

Exposure to an Electrical Current

Here we replicated the method carried out by a team of undergraduates at Stanford.

Again, all BioBricks were placed in the same pSB1C3 backbone and BL21 E. coli cells. The cells were grown up in 10ml of liquid culture of LB broth with chloramphenicol overnight at 37°C.

The next day, 5ml of the concentrated cells along with 300ml of LB broth (with chloramphenicol) was poured into a gel electrophoresis tank. The tanks were then run for 40 minutes at 28V (400 mA). At each time point (0, 1, 3, 5, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37 and 40), the gel tanks were stopped for roughly 30 seconds to allow us to obtain 9 samples of 100μl 3 from the positive end of the tank, 3 from the negative end, and 3 from the middle of the tank in order to measure the OD600 and fluorescence (in triplicate) on a plate reader. We measured the GFP fluorescence using 485nm excitation wavelength and 538nm emission wavelength.

Figure ??. Experimental set-up for carrying out the Stanford Experiment

Figure??. Fluorescence divided by the OD600 value for our BBa_K1895000 construct over the 40 minutes it was electrically shocked.

Figure??. Fluorescence divided by the OD600 value for our BBa_K1895006 construct over the 40 minutes it was electrically shocked.

After carrying out the initial experiment, we came up with some improvements which we believed would yield more efficient and reliable results.

We grew up fresh cultures for four hours, to ensure that cells were still in the exponential phase. This means that the culture would not be under any form of stress from being in the stationary phase and we would hopefully see more of a fluctuation when the cells were exposed to an electrical current.

We also felt that the 5ml in 300ml was too dilute, so we increased the concentration by adding 10ml of culture to 250ml of LB broth with chloramphenicol in the gel electrophoresis tank.

The new, more concentrated solution was added to the gel tanks and this time we left the solution to run for an hour and a half, taking samples every five minutes instead of every 2/3 minutes as this gave us more time to accurately measure the samples.

This time, we took 1ml of sample from the positive end of the gel electrophoresis tank as we believed that the cells would migrate towards the positive end due to their negative charge and the overall flow of the tank. The OD600 was measured in a cuvette this time with an LB with chloramphenicol blank. When measuring fluorescence, 1ml of culture was spun down at 14,000rpm for 1 minute and 800μl was removed and the pellet was re-suspended. Using this method increases the cell concentration, therefore amplifying the GFP signal and also any fluctuations. The fluorescence was again measured on the plate reader using 485nm excitation wavelength and 538nm emission wavelength.

Figure??. Fluorescence divided by the OD600 value for our BBa_K1895000 construct over the 90 minutes it was electrically shocked with our new protocol.

Figure??. Fluorescence divided by the OD600 value for our BBa_K1895006 construct over the 90 minutes it was electrically shocked with our new protocol.

Figure?? Growth Curve for our BBa_K1895000 over the 90 minutes it was electrically shocked

Figure?? Growth Curve for our BBa_K1895006 over the 90 minutes it was electrically shocked