How to work with Lactobacillus jensenii?

This toolbox contains all kind of information that can be useful for working with Lactobacillus jensenii. First, we provide culture conditions that we used and were proven to be working. Second, it contains a transformation protocol that we optimized and found to be the most efficient of all those we tested. Finally, the toolbox contains all DNA sequences (vectors and biobricks parts including L. jensenii specific promoters) that we used in L. jensenii for our project. We hope that this toolbox will facilitate the use of L. jensenii by other iGEM and research teams. We plan to extend the toolbox in the future by providing the community with a physical distribution of well-characterized vectors and regulatory elements.

How to grow Lactobacillus jensenii?

Strain and medium

For our project we used the following strain and culture medium :

Bacteria

Lactobacillus jensenii Gasser et al. (ATCC® 25258™)

Culture

BD Difco™ Lactobacilli MRS Broth

We used this broth for both our liquid and solid culture media. The liquid medium was used to do the solid medium by adding agar to it. We tested different concentrations of Agar to measure which one was the best.

5g/L

7g/L

10g/L

12g/L

15g/L

Figure 1: The different concentrations of Agar used for solid MRS culture medium and L. jensenii grown on the plates for 24 hours.

The plates with 5g/L and 7 g/L of Agar were not showing any colonies. For the three others there were more colonies for 12g/L. From this experiment we decided to use 12g/L of Agar for our experiments.

Cell culture

Growth on solid medium

L. jensenii is a facultative anaerobic bacterium, it does not absolutely need CO₂ to survive but grows way better in anaerobic conditions. For our project we did not have a CO₂ incubator available for growing bacteria. We thus decided to grow our bacteria using an “old school” microbiology method. The goal of the method is to get rid of the Oxygen present in the environment of the bacteria. To do so, we put the plates in a hermetic container and placed a burning candle inside it before closing the lid. The candle consumes all the oxygen and turns it into in CO₂, until all O₂ is consumed and the candle extinguish.

For our culture we used two kinds of plates and thus two kinds of containers.

As L. jensenii grows in small colonies we usually used small plates (60mm diameter) and put them in a jar (Figure 2) as follows:

Figure 2: Small plates (60mm diameter) are placed in an hermetically closed jar with a burning candle. Using this method we were able to produce the anaerobic environment required for *L. Jensenii* growth.

For transformant selection, we needed more colonies so we used normal culture plates (100mm diameter). We put them in an airtight-closing bowl with three candles (Figure 3).

Figure 3: 100mm plates incubated in a airtight-closing bowl.

Growth in liquid medium

How to transform L. jensenii?

To transform L. jensenii, we found several protocols. All of these protocols were specific to Lactobacillus plantarum or Lactobacilli in general, but not to Lactobacillus jensenii.
We selected different protocols that used or not magnesium in the electroporation buffer. Indeed, our electroporation machine could not stand magnesium, as it can causes electric arcs and damage the machine. We had to find another electroporator at the University of Montpellier. These electroporator had advanced settings such as the resistance.

The three protocols that we chose were one from Berthier in 1996, one from Speer in 2012, and the last one from Chatel [1].

We tried to transform our bacteria several times with these three different protocols. Finally, we decided to focus on Berthier 1996 as we had better results. A reason why Speer 2012 did not work well was the addition of glycine to grow bacteria. It weaken the bacterial wall too much, and inhibited the grow. We did not focus on Chatel because the use of magnesium in the electroporation buffer gave us bad time constants, that means that bacteria were not well transformed.

Once we chose the most promising protocol, we had to optimize it. We tried different conditions for the following parameters:

Amount of DNA per reaction: 10ng, 100ng, 1000ng, 5000ng (of pLEM415) ⇒ did not change anything. We decided to add about 1000ng of plasmid per reaction.
Voltage: 9kV/cm, 10kV/cm, 12.5kV/cm ⇒ we obtained better time constant (closer to 11ms to 13ms as explained in Berthier 1996) with 12.5kV/cm for the electroporation.
Resistance (ohm Ω): 300Ω, 500Ω, 600Ω ⇒ to reach a time constant between 11ms and 13ms, we decided to follow the protocol and to use 600Ω.
Antibiotic concentration for transformant selection: erythromycin 0.5µg/mL, 5µg/mL ⇒ we chose 0.5µg/mL as it was enough to select the transformants. 0.5µg/mL can kill non-resistant L. jensenii.

You can find the exact protocol for transforming L. jensenii on our protocol page: protocol.

L. jensenii plasmid

The choice of the plasmid has been complicated because the strain is little used in synthetic biology. Thus, it has been difficult to find plasmids "free" of intellectual property. Indeed, the only existing plasmids (pOSELp23 and pOSEL144) were created by the company Osel Inc (USA). Unfortunately, we could not obtain the constructs from the company.

We therefore had to find a vector responding to the following specifications:

An E. coli specific origin of replication.
n antibiotic resistance gene for selection in E. coli ⇒ These parts are required to support cloning and propagation of our vectors into E. coli before transforming them into L. jensenii.
A L. jensenii specific origin of replication.
An antibiotic resistance gene for selection in L. jensenii ⇒ These parts will allow propagation and maintenance of the plasmids in L. jensenii.

We chose not to create a vector from scratch but to start by trying to reuse different already ones existing. Our choice turned toward specific vector with different origins of replications operating into Lactobacillus species available on the Addgene website. While none of them was previously used for L. jensenii, we hoped that at least one of these vectors could be functional in L. jensenii:

We were able to transform pLEM415. If we had more time, we would have tested all the plasmids in L. jensenii. Some of them might be functional.

Bao, S., Zhu, L., Zhuang, Q., Wang, L., Xu, P. X., Itoh, K., ... & Lin, J. 2013. Distribution dynamics of recombinant Lactobacillus in the gastrointestinal tract of neonatal rats. PloS one, 8(3), e60007.

Team:Montpellier/L jensenii