Characterization

Overview

One of the key tenets of synthetic biology is reducing the chaos and unpredictability of biological systems by rigorous characterization of the parts used in each experiment. In keeping with this philosophy, we set out to further characterize the parts used in the 2017 Interlab Measurement Study. We looked at each of the 3 constitutively expressing GFP test devices and their responses to expression in different media. Each part was transformed into E. coli K-12 DH5α and grown in both an LB media and M9 minimal media. Colony forming units were compared between the devices grown in each media, as well as an empty DH5α strain. We hope this will aid future teams that may want to use these parts for other measurement studies or for their use in cell stress related applications.

Media Used

We used an LB media to simulate optimal growth conditions, and a M9 minimal media to represent cells grown under stress conditions. With there exact compositions as follows:

LB Media: 10g tryptone, 10g sodium chloride, 5g yeast extract, 1L volume; autoclaved and stored in fridge before use.

M9 Minimal Media: 20mL of M9 salts, 2ml 20% glucose, 200uL 1M MgSO4, 10uL 1M CaCl2, 78mL H20. Filter sterilized and stored at room temperature.

Parts Used

We decided to further characterize three of the common GFP producing test devices from the 2017 interlab study, commonly named test devices 1-3. All of the parts are stored in pSB1C3 plamids, and were transformed into E. coli K-12 DH5-alpha.

Construct	Biobrick	Kit Plate	Location
Test Device 1	BBa_J364000	Kit plate 7	Well 2F
Test Device 2	BBa_J364001	Kit plate 7	Well 2H
Test Device 3	BBa_J364002	Kit plate 7	Well 2J

Basic Protocol

In order to convert OD600 absorbance values to colony forming units, a standard curve using microspheres from the measurement kit was used. For exact protocol used click here.
Samples grown overnight in LB media + antibiotic were spun down and given fresh media. Samples were diluted in LB or minimal media to an OD600 of 0.1. Using a multichannel pipette, they were loaded onto a 96 well plate in triplicate. Three biological replicates and three technical replicates were used.
Samples in LB and minimal media control were run with a Spectramax i3X. Each of which were read for 16 hours at a run temperature of 37˚C. Pathlength correction was turned off and absorbance was read at 600nm.
Data analysis was done to account for LB absorbance, for the control, then the technical and biological replicate values were averaged. Standard deviation was determined, and the values were created as seen below.

Results

Figure 1: E.coli DH5α populations with measurement plasmids in LB as compared to DH5α cells without plasmid over time at 37˚C.

Figure 2: E.coli DH5α populations with measurement plasmids in Minimal media as compared to DH5α cells without plasmid in LB media over time at 37˚C.

Figure 3: Measurement part BBa_J364000 in LB and minimal media as compared to DH5α cells without a plasmid grown in LB.

Figure 4: Measurement part BBa_J364001 in LB and minimal media as compared to DH5α cells without a plasmid grown in LB.

Figure 5: Measurement part BBa_J364002 in LB and minimal media as compared to DH5α cells without a plasmid grown in LB.

Effect of Stress on Cell Growth

As evident from figure 1 there appears to be very little difference in colony forming units for all of the E. coli with plasmid inserts when grown on LB media. Under low stress conditions maintenance of the plasmid does not significantly impact the amount of colony forming units and therefore the growth rate observed in each population. However, this changes in response to growth in a minimal media. As seen in figure 2 the E. coli grown on LB media forms roughly 50% more colony forming units than any of the cells grown on minimal media regardless of whether or not a plasmid was inserted. Additionally from our experiments we concluded that BBa_J364001 appears to respond the best to cell stress conditions. Out of all 3 devices tested, J364001 grew the best in our minimal media, as observed in figure 4. This would make it a good candidate to use in other cell stress related applications.