Experiment
The different experiments we conducted
Milk Log
We wanted to understand the environment of microorganisms in milk to best design our biosensor. In our study of background literature, we found unsatisfactory information on a number of important elements of the system, namely the species found in milk, their rate of growth, and the impact of spoilage temperature and fat percentage on other variables.
In order to develop this knowledge, we ran the “milk log”. For this experiment, we purchased six quarts of milk from a major grocery store chain, two each of the skim, 2% and full fat varieties. We then split the milk into two sets, allowed one set of milks to spoil at room while the other expired in the fridge, at * and * degrees Celsius, respectively. Every 24 hours, samples from all quarts were sequentially diluted to several concentrations based on results from the previous day, plated on LB agar plates and incubated at 37 * Celsius. During the run of the trial, we documented the number of colonies for the most countable dilution of each sample for each day. We ceased sampling from quarts when the formation of solids made pipetting from the bottle difficult and concluded the experiment once all of the quarts had spoiled.
Additionally, PCR of the 16S rRNA of bacterial colonies which grew on the plates was performed and the product was sequenced to identify bacteria in the spoiled milk.
Site-directed mutagenesis
In order to save time, we had our construct synthesized in two unequally sized parts. The smaller of the two parts was synthesized and delivered to us with no complications. The larger part could not be synthesized correctly. The larger part that we received had a deleted adenine at the 509 position. To fix this we did a site directed mutagenesis.
We first designed primers with an adenine at the end such that they anneal to our incorrect part at the site of the missing base. We then ran a PCR with our primers and incorrect part, this added an adenine at the 509 position. We then sequence the resulting PCR product to confirm that the missing base had been added.
Well Diffusion Assay
A well diffusion assay was chosen as our AHL quantification method. Our well diffusion assay was conducted by creating agar plates with liquid bacterial culture evenly dispersed agar medium. A circular section of medium is removed from the center of the plate forming the “well”. A small volume of sample containing AHL is placed into the well, and the plate is left to incubate in at 30 degrees Celsius. During incubation, the sample diffuses through agar medium, where the bacteria in the agar detect presence of AHL as it diffuse. The chosen bacteria used in the method were CV026. This bacterial strain was chosen because are designed to produce colored pigment only in the presence of AHL. At a certain radius, the concentration of AHL is high enough to trigger a bacterial response, causing chromoproteins to be transcribed and color to be shown. Outside of this radius, the concentration is too low to be detected by the bacteria in the plate and no pigment is produced. Because the radius is indicative of the concentration of AHL, we used this method to quantify pure concentrations of AHL to compare with 2%, skim, and whole milk samples. We were able to demonstrate the effectiveness of well diffusion assays by testing them with CV026 monitor strain. CV026 well diffusion plates displayed a dark blue-violet color in the presence of various concentrations of reagent-grade AHL. Next, we wanted to see if CV026 could detect the concentration of AHL present in a sample taken directly from spoiled milk. The first round of tests did not yield any color change, indicating that the concentration of AHL in very spoiled milk is too low to be detected.
Liquid Liquid Extraction
We decided to see how easy it would be to purify the milk samples to increase the concentration of AHL within it. A broad literature review revealed that Liquid-Liquid Extractions (LLE) are often employed for this very purpose - to isolate AHL from a bacterial sample. AHL molecules are nonpolar and milk is primarily composed of water, so we figured that an LLE with a very nonpolar solvent should be able to remove the AHL from the milk. We acquired the necessary LLE equipment as well as solvents such as dichloromethane, ethyl acetate, and hexanes.
We were initially concerned that the milkfat present in the sample would interfere with the extraction of similarly-nonpolar AHLs. It turned out that separation between the water in the milk, the proteins, and the milkfat was very poor. The aqueous and organic fractions took very long to separate. Dichloromethane was particularly troublesome as a solvent because it caused an emulsion with the impure water and refused to separate completely, even after being left overnight. In the end, we concluded that the papers written on LLE for extracting AHLs from an aqueous medium did not address solubilities in solutions as complex as milk. We ultimately were unable to purify the AHL from milk with an LLE to a concentration detectable with a Well brDiffusion Assay.
Protocols
Our basic lab protocols are identical to our previous years. They can be found here. For the experiments specific to this year, we've attached the protocols below.
AHL Quantification by Well-Diffusion Assay
Rationale: C.violaceum wild type produces violacein, a blue/purple pigment; CV026 mutant is defective in producing violacein, but its synthesis can be induced by adding AHL. We can quantify the amount of AHL by a well-diffusion assay: add AHL or AHL-containing samples to plates with punched wells and measure the diameter of induced zones. By comparing the diameter of milk-induced zones to pure AHL-induced zones, we will be able to quantify the amount of AHL produced by spoilage bacteria.
Materials: monitor strain CV026, LB media, LB agar, Kanamycin.
Procedures:
*All media for CV026 should be supplemented with Kanamycin at 20 μg/ml; CV026 is also resistant to some other antibiotics—different antibiotics may be adopted.
1. Preculture: prepare LB media following the “LB Media Protocol”.
2. Inoculate CV026 to a liquid LB culture and incubate on shaker (140 rev/min of agitation) to ensure aeration for 24hrs at 30℃.
3. Calculate the amount of preculture needed: 1ml of the preculture is used to inoculate 50ml of LB media. Incubate the bacteria culture on shaker for 24hrs at 30℃.
4. Prepare LB agar and maintain at 46℃ to keep LB agar in liquid form.
5. Pour the bacteria culture to twice as much 1.2% LB agar (i.e. 50ml of bacteria culture is mixed with 100ml LB agar). Immediately pour the agar-culture solution as 20ml portions into petri dishes.
6. Let the culture-agar plates to solidify. Cut a well in the center of the agar using the bottom of a sterile P200 pipette tip (TBD); do not puncture all the way through the media. Make sure all cuts are equal in diameter and depth. (To do this, we can draw a line 4mm (TBD) from the bottom of the pipette tip so that all wells are cut 4mm deep)
7. Pipette up to 60μl (TBD) of sample to punched wells. Incubate plates for 48hrs at 25℃. DO NOT incubate the plates upside-down.
8. Measure the diameter of the induced zones and record data in lab notebook. Take pictures and upload to google drive.
References:
See page 243 for well-diffusion assay. See “bacterial strains and growth conditions”
LLE Protocol
1. Centrifuge 210ml of spoiled milk for 12,000 rcf for 3mins and capture supernatant solution. The AHL is extracellular and in solution, so discard the centrifuge pellet.
2. Make sure the separatory funnel is closed from the bottom. Transfer (aqueous) solution to separatory funnel. Add 90ml?(700:300 of supernatant: organic solvent, or try 50ml if 90ml is too much to evaporate with nitrogen later) amount of solvent to funnel.
3. Find a beaker to transfer your organic-AHL mixture to and pre-weight it.
4. Shake/stir/mix LLE vial at low-medium intensity so as to form emulsion. Remember to vent the built-up gas inside the funnel (due to volatile solvent going into gas phase). You may hear a small hiss due to escaping gas.
5. Put funnel in stand and let emulsion separate out into two fractions. The top fraction should be the less-dense, more volatile, organic fraction(DCM would be the bottom layer since DCM has a density of 1.33g/cm^3) containing the AHL and milk fat, and the lower fraction should be the denser aqueous fraction. Pour off the organic fraction into beaker.Repeat steps 2-4 twice more with the same amount 50/90ml of solvent each time.
6. Once you’ve performed the LLE thrice, discard of the aqueous fraction.
7. Put the organic fraction into the fume hood and apply a stream of nitrogen to it to accelerate evaporating off excess solvent
8. Once “dry” measure the weight of the beaker again to find weight of residual organics. a. Cows milk 3-4% fat b. Cows fat ballpark MW 650-900 g/mol c. AHL ballpark MW ~¼ of fat triglyceride
9. Reconstitute residue with minimal amount of desired solvent