Team:MIT/Results
Results
Our experimental approach to verifying transport of the ComCDE system to mammalian cells is outlined in the tables below. We broke the system verification down into two main parts.
Experiment 1: Validate ComD membrane localization and CSP binding
We ran a ComD titration experiment to see if ACTB:CD4-comD-mKO2 was expressing in HEK cells. We compared the fluorescent output from ACTB:CD4-comD-mKO2 to that of ACTB:mKO2 alone, with hEF1a:iRFP as a transfection marker. We saw a similar correlation between the iRFP expression and the mKO2 expression of each cell, indicating that CD4-comD-mKO2 was, in fact, being expressed in a similar manner as normal mKO2.
We are currently running further experiments to localize CD4-comD-mKO2 to the cell membrane of HEK cells and characterize CSP-AlexaFluor 405 binding.
Experiment 2: Validate that active ComE drives engineered promoter
We are currently in the process of running the above experiments.
Biofilm Characterization
In order to quantitatively measure the effect both of our system and selected output protein on the S. mutans biofilm, we had to first characterize biofilm accumulation without any added deterrents or inhibitors. By characterizing biofilm growth under our lab’s conditions and the resources available to us, we could better understand the effect of our synthetic system.
To actually get our hands on the bacteria S. mutans, as well as protocols and tips on how to best observe and treat the species, we met with Dr. Caroline Ribbeck from the MIT Ribbeck lab, which studies the mucus barrier and how it selectively allows and blocks different molecules and pathogens from passing through it.
Dr. Ribbeck gave us our S. mutans strain (UA159) and our protocol for tracking CFU (colony forming units) growth.
Initially, we wanted to optimize growing conditions to maximize the amount of biofilm formed. We learned from existing literature and Dr. Ribbeck that the way in which S. mutans grows on polystyrene (the material of our 96 well plates) is similar to the way they would grow on human teeth. While the shape and placement of S. mutans in our experiments were not exactly like that of the human mouth, we assumed our setup was a good model of the environment our bacteria naturally exist in.
For our experiments we varied sucrose concentration, growth media concentration, S. mutans volume, and incubation times.
From the data we collected in this experiment, we decided to use 75% TH broth and 2ul of 2% sucrose for our future S. mutans experiments. Figure 1A clearly shows that 2ul of 2% sucrose is the optimal concentration for biofilm growth, and while Figure 1B shows a similar about of growth in the 75%-100% range, we elected to use 75% in order to conserve our resources. (It should be noted that while pure water and no TH broth shows 100% of CFUs in biofilm (Figure 1B), this is an anomaly in our data, as TH broth is known to be absolutely required for cell growth and proliferation. Additionally, the protocols we received from Dr. Ribbeck recommended a range between 50% and 100% TH broth, so we were confident that the results for 0% TH broth could be safely disregarded.) We would have liked to run these experiments in a cleaner, more staffed environment to improve the data yield and our confidence in any collected data.
Unfortunately, due to long incubation times required for this experiment and its susceptibility to human error, we were not able to collect a large amount of reliable data for our sucrose concentration and logarithmic sucrose volume experiments. We ran many versions of each CFU experiment, but many became contaminated, as we were not allowed to transform S. mutans with antibiotic resistance genes, which forced us to use plates without any antibiotics. Even after adding a bunsen burner and disinfectant to upgrade our aseptic technique, we were unable to completely prevent contamination, thus invalidating much of our collected data.
Crystal Violet Assays
In order to perform our characterization in a more quantitative way, we found and modified an existing protocol that tracks biofilm growth through crystal violet staining. We can consider this method to be more quantitative as it doesn’t rely on counting colonies from dilutions done by hand but rather light being excited by our crystal violet stain.
Unfortunately, due to long incubation times required for this experiment and its susceptibility to human error, we were not able to collect a large amount of reliable data for our sucrose concentration and logarithmic sucrose volume experiments. We ran many versions of each CFU experiment, but many became contaminated, as we were not allowed to transform S. mutans with antibiotic resistance genes, which forced us to use plates without any antibiotics. Even after adding a bunsen burner and disinfectant to upgrade our aseptic technique, we were unable to completely prevent contamination, thus invalidating much of our collected data.
In order to perform our characterization in a more quantitative way, we found and modified an existing protocol that tracks biofilm growth through crystal violet staining. We opted to use this method because of its quantitative nature-it doesn’t rely on counting colonies from dilutions done by hand, but rather tracks the excitation of light by our crystal violet stain.
For this experiment, we also decided to put a solution of hydroxyapatite, a substance known to be an even closer simulation of enamel than polystyrene, into the bottom of our wells.
We then repeated the above experiment, this time adding a varying amount of kappa casein, our actuation protein of choice. All experiments were run with 2ul of S. mutans overnight culture, 2ul of 2% sucrose, and 100ul of 75% TH broth.
All of these Kappa casein experiments were run with 2ul of S. mutans overnight culture, 2ul of 2% sucrose, and 100ul of 75% TH broth. We observed that 1,000 ng of Kappa casein resulted in less biofilm accumulation than 10, 100, 10,000, and 50,000 ng of kappa casein (Figure 3). However, contrary to our expectations, all the wells with added kappa casein resulted in more biofilm growth than our negative control (0 ng kappa casein). This suggests some flaw in our experimental design, since previous literature showed very clearly that the opposite trend should have been observed.1 This difference may in part be due to the fact that we were not able to exactly replicate the experimental conditions used in (Vacca Smith 1994)--namely, they used small hydroxyapatite beads that were coated with saliva, which we were not able to procure. Additionally, since the data consistently showed less biofilm at 1,000 ng, we believe that the interaction between kappa casein and the S. mutans biofilm is more complex than we initially thought, and that there is some optimal concentration of kappa casein above and below which there is lowered efficacy.
Ideally, we would run this experiment a few more times with varied experimental conditions to try to identify the reason behind this inconsistency. In addition, our first crystal violet experiment (Figure 2A) showed that 6-9 ul of overnight culture produces the best biofilm results so we would want to use that amount for future experiments.