New Chassis Development

Background

Lactobacillus crispatus is a Gram-positive bacterium classified as a lactic acid bacterium (LAB) and is a part of the qualified presumption of safety (QPS) list of the European Food Safety Authority (EFSA) [1]. This microorganism is naturally present in various human microbiomes, including the urinary tract, where it fulfills protective functions.

Our motivation to work with L. crispatus is grounded in numerous compelling reasons, as elaborated in our project description. However, we encountered a prominent challenge—the relative scarcity of research involving this bacterium. Notably, several iGEM groups in the past expressed the desire to work with L. crispatus but were dissuaded by the difficulties associated with its utilization. Despite these challenges, we opted to embrace this endeavor and address this notable gap in the field.

Growth Conditions

The first challenge we confronted was the cultivation of L. crispatus. While it was observed that many researchers in the literature employed L. crispatus strains obtained from diverse sources, we were committed to ensuring the reproducibility of our procedures. Consequently, we acquired a specific L. crispatus strain from ATCC with the assigned reference number 33802.

L. crispatus exhibits robust growth in MRS (de Man, Rogosa, and Sharpe) medium, both in liquid broth and on agar plates, under anaerobic or microaerophilic conditions, at a temperature of 37 degrees Celsius. On agar plates, L. crispatus colonies present with distinctive rough surface irregularities, while in liquid culture, the bacterium accumulates as a discernible white sediment at the bottom of the tubes.

To facilitate the creation of a microaerophilic environment conducive to L. crispatus growth, we adopted a cultivation method involving 15ml Falcon tubes containing 10ml of medium.

During our initial efforts to revive lyophilized L. crispatus, we found that it required 48 hours to become viable. Subsequent growth cycles, however, demonstrated that the bacterium became visible within 24 hours, in accordance with existing literature, and exhibited sustained growth even after 48 hours.

MIC test

As elaborated in our integrate and results pages, we wished to use L. crispatus’ natural tetracycline resistance as our target site for genomic integration. To this end we needed to confirm growth in which concentrations indicated resistance. After a period of five days there was growth in our L. crispatus strain with tetracycline concentrations of up to and including 64 µg/ml. By the end of the testing period the bacteria grown in this concentration still exhibited exponential growth, indicating expression of the TetM gene and resistance within L. crispatus. This information can assist future designs of our integration system, transformation attempts and various other designs that rely on antibiotic selection.

An additional aspect to emphasize is the significance of allowing bacteria an acclimation period before conducting experimental procedures.

In our initial Minimum Inhibitory Concentration (MIC) experiment, a starter culture of Lactobacillus crispatus was thawed from frozen storage and supplemented with tetracycline at a concentration of 6 micrograms per microliter. Notably, it took approximately 144 hours (or 6 days) for visible colonies to emerge at the base of the starter test tube. In contrast, as detailed in the MIC test results, clearly distinguishable colonies were observed at the base of the test tube after 24 hours. In this case, the culture originated from a starter that had been incubated for 19 hours before transitioning to a medium that contains tetracycline.

Given that we observed unhindered bacterial growth at tetracycline concentrations of up to 64 micrograms per microliter, it is plausible that the absence of an acclimation period before exposure to an antibiotic-containing medium in the initial experiment accounted for this disparity. It is noteworthy that the MIC experiment protocol, on which we based our experiments, included period of 16 to 24 hours to allow acclimation to the to the bacterium's environment, before transitioning to a medium containing antibiotics.

In light of these findings and to ensure the integrity of experimental outcomes, we recommend initiating any procedures involving Lactobacillus crispatus by first culturing a liquid starter in a clean MRS medium overnight (for 16 to 24 hours) before commencing research experiments. This precautionary measure aims to mitigate potential disruptions to bacterial growth caused by conditions detrimental to their viability.

Transformation

L. crispatus is a Gram-positive bacteria and as such, the transformation process is challenging due to its considerable cell wall. Unlike the heat-shock method commonly employed for E. coli transformations, L. crispatus transformations are conducted using electroporation. In order to have a successful electroporation, it is necessary that the bacteria and DNA intended for electroporation will be as free of salts as possible.

To achieve successful electroporation, it is imperative to minimize the presence of salts in both the bacterial suspension and the DNA intended for electroporation. The electroporation process entails the passage of an electrical current through the bacterial cells, thereby creating pores in the cell wall for plasmid entry. However, electrical currents tend to follow the path of least resistance. In the presence of excessive salts, the current may bypass the bacterial cells, leaving the cell walls intact. The tau value, a parameter automatically measured by the electroporation device *, gauges the time required for current passage through the electroporation cuvette. Longer tau values indicate higher resistance and a greater chance of bacterial electrocution, with a maximum tau value of five milliseconds.

For these reasons, it is imperative to undertake transformation attempts with meticulously purified plasmids, characterized by a nanodrop value of 260/230 ratio closely approximating 1.8, indicative of optimal DNA-to-salt ratios. Furthermore, the transformation protocol outlined in our L. crispatus guidebook, accessible at the bottom of this page and in our protocols section, incorporates several steps involving centrifugation and bacterial washing to eliminate residual salts. Due to the time-consuming nature of these washing steps, it is imperative to work with chilled reagents and instruments.

L. crispatus guidebook

Comprehensive information and protocols relevant to our work with L. crispatus are compiled within our guidebook, including the protocols employed for plasmid assembly in E. coli.