Team:Oxford/Experiments
Experiments
This page is intended to document the experiments we have directly performed, describe how difficult we found that they were to perform and explain the reasoning behind the choices we made. Full protocols and buffer recipes can be found on our protocols page. A full list of results can be found on our wet lab overview page. For experiments relating specifically to the Interlab study see our Interlab page.
Plate Reader
We wanted to test our copper-sensitive promoter parts at a range of different copper concentrations over time. We decided that our ideal promoter system would show a large change in expression over the region 0.01-0.05mM because this is the range under which copper concentration would rise after a meal. Our ideal system would also induce quickly so the chelator could be produced and the free copper chelated before it could be absorbed.
However we decided to test the promoters to copper concentration outside this range (0mM to 2mM) and for longer period of time. This was to better understand how our promoter systems operated, provide a more complete data set for our dry lab team to parameter fit and to provide data for others wishing to use our parts for our specifications. In order to increase the chance of our data being applicable to a probiotic bacterial in the small intestine we set the plate reader to human body temperature of 37°C and gently shaking at 225 rpm to stimulate gastric movement. We also verified that our growth medium was close to pH7 which is around the pH small intestine.
Two different fluorescent proteins were used in our project. The fluorescence our each our samples is presumed to be proportional to the expression level our our promoters. Our CueR-linked systems use the GFP variant sfGFP allowing these to directly compared. (Excitation filter 485-12nm, emission filter 520nm). Our pCusC RFP sequence however was provided with the red fluorescent protein mKate and when we attempted to improve the part by adding a positive feedback loop with CusR we used the same protein so we could compare them on the same plate and see if there was any improvement. (Excitation 580-10nm, emission 620-10nm)
To account for the number of cells present at different copper concentrations and different times we measured the optical density (OD) as a proportional measure of the number of cells present. For our parts using GFP, the optical density at 600nm (orange light) was used as at this wavelength has less interference with the yellow broth. For our mKate parts however, measuring at cell density at 600nm wouldn’t be accurate because of the emission from the fluorescent protein at approximately this wavelength. Consequently we measured the optical density at 700nm for these parts.
When comparing our copper promoter systems at different copper concentrations we chose to compare the data at after they had been in the plate reader for four hours. We found that this was just before the optical density reached maximum so the cells were still about in the exponential growth phase.
Plate reader experiments were prepared by picking individual colonies off stored plates into 5ml of LB with 1 in 1000 chloramphenicol and grown overnight (at least 8 hours).
A range of copper concentrations were prepared from stock solutions. A large volume plate was then prepared with 10μl of copper solution, 10μl of overnight culture and 980μl of broth with antibiotic. This resulted in a 1 in 100 dilution of the copper solutions prepared:
This large-volume plate was then centrifuged to mix the solutions and then 200μl transferred to a small-volume plate with a clear lid and then placed in the plate reader. The delay between mixing the cells, broth and copper solution and the starting of the plate reader was found to be less than 30 minutes.
In each of our plates we had 8 rows of 12 columns (96 wells in total). In the first column we included the negative control part from the Interlab study inside our testing MG1655 strain at 0mM copper. This gave us the background growth curve of the cells and acted as baseline to compare the increase in fluoresce to and show how leaky our constructs were at 0mM copper. Two repeats from each of four separate picked colonies was used.
Similarly in the second column we used the positive control part from the Interlab study. Whilst using a different form of GFP it confirmed to us that the plate reader was performing correctly. Using these parts rather than untransformed, plasmid-free MG1655 strain allowed us to use antibiotic-containing broth to account for any effect the antibiotic had on measurement. As we lacked a positive control expressing red fluorescent protein for our CusSR-linked systems we still included the GFP positive control to see see if growth was different when expressing a large amount of protein with no benefit to the cell.
In the remaining wells of the plate we included four biological repeats of two parts we wished to compare (e.g. pCopA sfGFP and pCopA CueR sfGFP) across the range of copper concentrations. Four biological repeats was considered sufficient for our purposes especially when combined with our flow cytometry data. By putting the parts in the same plate we ensured that the conditions were identical for both.
The plate reader measured the fluorescence and OD every ten minutes for at least 12 hours, shaking between measurements.
Overall we found this experiment to be reasonably simple albeit time consuming to perform. With practice the procedure can be done in about two hours. Although the experiment was designed to use large volumes and the same volume in each well, pipetting exact volumes into was found to be quite monotonous and it was easy to loose track of which well you were working on. If a single volume was added incorrectly the entire plate would need to done from the start. Consequently we found it easiest to do prepare as much as possible (e.g. the copper solutions) the night before and perform the experiment early in the morning or just after lunch when relatively attentive.
The easiest method to keep track of position in the plate was to pipette against the sides of the wells to be more visible and then shaking the plate after each component was added. Audibly counting rows and columns also helped. The easiest method of filling the plate was to add the all the copper solutions first down the columns then add the positive and negative control cells into their respective columns followed by each of the parts from left to right across each row. Going from low to high copper concentrations meant that the same pipette tip could be used for the part overnight cultures with relatively tiny change in copper from contamination between wells. Using the same pipette tip reducing the amount of motions necessary for each well. Using a multi-pipette to go from the large volume plate to a small volume plate greatly increases the speed of the procedure.
Flow Cytometry
Subject 2
Microscopy
Subject 3
Alginate Bead Preparation
Subject 4
Cu Absorbance Assays
Subject 5
