Example Experiment

This project required many basic synthetic biology techniques. See below for the techniques we used, reagents required, and a brief protocol. Note that all techniques require basic microbiology tools (pipettes, tubes, plates, incubator, etc.)

1. Five constructs of plasmid were designed to be utilized as molecular biosensors for nutrient levels within soil. All constructs were synthesized in the medium copy number plasmid “pTwist-kan” These constructs include:
a. Copper-inducible construct
b. Nitrate construct
c. Nitrite construct
d. Phosphate construct with fully functioning Lac operon
e. Phosphate construct with LacI degradation tag

2. Transformation
Added to microcentrifuge tubes:
a. 50 uL DH5a electrocompetent E. coli cells
b. 5 uL Synthesized plasmid

i. Add solution to electroporation cuvette

c. Pulse
d. Quickly add 1 mL of LB broth to the cuvette
e. Transfer solution from cuvette to microcentrifuge tube
f. Incubate at 37 C for 30 min
g. Centrifuge at 13.7 x g for 1 min
h. Decant LB
i. Add 50 uL of fresh LB broth, resuspend pellet
j. Spread 100 uL on LB/ Kan agar
k. Incubate at 37 C overnight

3. Qualitative Color Change:
a. Multiple dilutions of Copper, nitrate, and phosphate buffers
b. Grew 5 ml overnight cultures of E. coli strains contain the 5 constructs
c. Five 1:10 dilutions of 0.1 M CuSO4, 1M NaNO3, and 0.1M NaNO2

i. NaNO2: 0.1M, 0.05M, 0.025M, 0.0125M, 0.00625M
ii. NaNO3: 1M, 0.5M, 0.25M, 0.125M, 0.0625M
iii. CuSO4: 0.1M, 0.05M, 0.025M, 0.0125M, 0.00625M
iv. NaH2PO4: 0.1M, 0.05M, 0.025M, 0.0125M, 0.00625M

d. 500 uL of each culture added to the corresponding 125 uL buffer
e. Incubated 5 minutes
f. 4 microliters of 10 mg/ml Nitrocefin were added to all tubes
g. Pictures were taken at 5 and 15 minutes after the addition of nitrocefin

i. Control: E. coli without plasmid
ii. Control: No nitrocefin added

h. Different ratios of the nitrocefin were then experimented with

4. Light Spectrophotometry:
a. Spectrophotometer ran at wavelength of 600nm

i. To measure concentration of bacteria

b. Absorbance readings were taken for different dilutions of copper sulfate and sodium phosphate

i. 4 dilutions – 1x10^-3,1X10^-4 M, 1X10^-5 M, 1X10^-6 M
ii. Control: No nitrocefin
iii. Control: No buffer

c. Later this was repeated with extracting nutrients out of dirt as well

i. 4 dilutions – 1x10^-3,1X10^-4 M, 1X10^-5 M, 1X10^-6 M
ii. Control: No nitrocefin
iii. Control: No buffer
iv. 10 minute dirt water samples
v. 25 minute dirt water samples

5. Soil Sample Preparation:
a. A 50 ml conical tube was used to acquire 40 ml of loosely packed soil
b. 25 ml of 10 mM Tris pH 7.5 was added to the soil sample

i. The sample was vortexed for 30 seconds

c. A funnel with filter paper was placed in a new 50 ml conical tube
d. The soil and buffer solution was poured into the funnel with filter paper to remove large soil particles

i. Liquid was allowed to collect until 5 ml was present

e. A 0.45 micron syringe filter was attached to a 10 ml syringe and placed in a 15 ml conical tube
f. The 5 ml soil solution was poured in the syringe and passed through the syringe filter to remove any remaining large soil particles
g. The solution that was obtained from the filtrations steps was used for testing with the E. coli strains containing the nutrient-inducible constructs

6. UV-Vis Spectrophotometry (for copper experiment)
E. coli was prepared similar to what is described in the Quantitative color change experiments above with some modifications

a. 50 ml cultures of E. coli was prepared the day before the experiments were to take place
b. The following day 4 ml of E. coli containing the copper-inducible construct were added to seven tubes
c. All seven tubes were centrifuged at 4500 rpm for 5 minutes
d. The supernatant was removed from the tubes
e. The E. coli cells were resuspended in 2 ml of 10 mM Tris pH 7.5 buffer
f. 0.5 ml of the 1x10-3,1X10-4 M, 1X10-5 M, 1X10-6 M copper sulfate solution were added to four tubes
g. To one tube, 0.5 ml of 1x10-3 M copper sulfate was added but no nitrocefin was added (“no nitrocefin” tube)
h. To one tube, 0.5 ml of 10 mM Tris buffer was added
i. To one tube, 0.5 ml of the soil sample solution was added
j. To all of the tubes except for the “no nitrocefin” tube, 4 microliters of 10 mg/ml nitrocefin was added
k. All tubes were allowed to incubate at room temperature for one hour
l. After incubation, 1.2 ml of each mixture was pipetted into a microcentrifuge tube
m. All of the tubes were centrifuged for 1 minute at 13,300 g in a microcentrifuge
n. One ml of liquid from each tube was transferred to a cuvette
o. Cuvettes containing these solutions were analyzed by the UV-Vis spectrophotometer at wavelengths from approximately 200 nm to 820 nm
p. Readings from the spectrophotometer were transferred to an excel file and the data was analyzed