The following contains the experimental highlights of our project. The Protocols linked here are only improved version, which lead to an optimization of the experiment or the project plan.
We want to point out that this is a condensed version of our lab journal for simplicity and reproducibility for future iGEM teams. If any questions arise or additional information is needed, we can provide a more extensive list of experiments that we can share if required.
The core of our idea revolves around the apparent advantage that Lactobacilli exhibit in acidic surroundings and the preferred environment of our helper bacterium (E.coli at a higher pH) [1.].
To ascertain the bacteriological growth patterns at a pH 4.5 and pH 6.5 we had to:
To demonstrate the growth condition in natural surroundings:
At the very heart of our concept is the selective advantage Lactobacilli seem to have in acidic environments; while lowering the vaginal pH through production of lactic acid, our Lactobacilli maintain an environment in which it can thrive. While several studies have already proven the fact that some Lactobacillus strains do better at a lower pH, we wanted to test whether this is also true for the strains we used [1.].
We therefore wanted to compare the growth of Lactobacillus at different pH levels to the growth of Escheria coli and Bifidobacteria, as both can also be present in the vaginal microbiome [2.]. As microbial composition can vary greatly based on different factors, ethnicity included, we performed the experiments with two different Lactobacilli species: Lactobacillus crispatus and Lactobacillus gasseri, both were obtained via a probiotic pill sponsored by Yoni solutions. The bacteria present in the probiotic were obtained from a healthy vagina. Thus the behavior of the bacteria is comparable to the bacteria present in the native genital tract. While Lactobacillus crispatus is the most prevalent species in caucasian women, and makes up a big part of the microbiome in asian, black and hispanic women as well, Lactobacillus gasseri too is widespread throughout all ethnicities. By including both species in our experiments, we wanted to make sure our solution would be applicable to a wide range of patients and not be restricted to one ethnicity only.
We expected all bacteria to be able to grow at both pH values, however, the Lactobacilli strains should out-compete the other candidates at low pH environments [3.].
First we had to settle on a media in which all four species grow at low and high pH. After not succeeding with the LB medium we switched to LAPTg, which is preferred by Lactobacilli. All bacteria except Bifidobacteria grew, which is why we decided to try the experiment with E. coli and Lactobacillus crispatus.
To establish growth curves, we decided to perform colony forming unit (CFU) measurements. For this purpose, we plated a dilution series of a bacterial culture at several time points. We decided to use LB-agar plates for E.coli and LAPTg-agar plates for the Lactobacilli, because they showed optimal growth for colony counting.
At first, we wanted to perform our growth curves in a vaginal fluid simulant. Unfortunately, none of our tested vaginal fluid simulant media [4.][5.] were suitable to perform growth curves, measuring E. coli and L. crispatus each at high and low pH. We compromised and decided to measure the growth in VSF, where only the high pH (6.5) condition allowed growth for both bacteria.
Given the involvement of numerous self-designed plasmids in our concept, successful assembly and transformation of these plasmids was a prerequisite before we could proceed to characterize components and perform measurements.
For more background information on our assembly strategies visit the Engineering Page.