At a Glance
By using Peptidosomes we introduce a new powerful platform for co-culturing. This technique physically separates bacterial populations without limiting their ability to communicate with each other via signaling molecules. This part of EncaBcillus is focused on proving the concept of communication between encapsulated bacteria by making use of the native regulatory system for competence development in Bacillus subtilis which is based on quorum sensing.
In order to solve complex tasks, it is reasonable to break down a problem into small subunits that can be addressed by specialists individually. This concept is widely implemented in modern economy but can also be applied to synthetic biology . In nature, such strategies have been evolved over millions of years. A good example is the formation of biofilms by the gram-positive bacterium Bacillus subtilis. Here, cell-cell interactions lead to cellular differentiation processes, which results in an effective division of labour . However, development of synthetic consortia consisting of several different bacterial strains, with each executing distinct tasks remains challenging .
By encapsulating subpopulations within molecular cages, called Peptidosomes, we want to physically separate co-working bacteria while still assuring their cooperativity to fulfil one major task. In this context communication between the bacteria is indispensable, thus we had to make sure that the Peptidosome membrane is permeable for small molecules and that the bacteria are still able to interact with each other. In this part of EncaBcillus we will show that the encapsulation of one population does not limit its ability to communicate with cells in the surrounding medium.
The regulatory system for competence development in B. subtilis
Communication between encapsulated bacteria and bacteria in the surroundings
After extensive research, an iGEM project starts with designing genetic constructs and planning experiments. It takes some time until the strains are finished and the first assays are running. Anyway, it is no secret that the most important results come in the last few weeks before the Giant Jamboree. Figure 3 gives an overview of this subproject in chronological order.
Genetic design and cloning
Sender strain (SeSt)
Receiver strain (ReSt)
|PcomK mut||BBa_K2273013||In order to remove a SpeI restriction site we had to exchange one nucleotide (nt 32, A to T).|
All parts have been amplified via PCR from gDNA of Bacillus subtilis W168, verified by sequencing and cloned into the pSB1C3 for storage and submission to the iGEM registry. Cloning was done according to standard protocols. Plasmids have been multiplied with Escherichia coli DH10β. All assays have been conducted with B. subtilis W168 carrying one or more of the earlier-described constructs.
In order to investigate quorum sensing-dependent effects we determined the promoter activities of PsrfA, PrapA, PrapF, PcomG and PcomK mut in WT and in comX-deficient strains by monitoring the luminescence. In the knockout-strains comX has been replaced with either an erythromycin or a kanamycin resistance cassette (comX::ery or comX::kanR, for simplicity from now on referred to as ΔcomX).
Furthermore, we examined the influence of different media on promoter activity using Luria-Bertani (LB) broth as full medium as well as two minimal media: MCSE and MNGE medium. The latter is commonly used for the transformation of B. subtilis. All plate reader assays (PR-Assays) have been performed with the CLARIOstar® microplate reader from BMG Labtech.
For co-cultivation of ReSt and SeSt we used either ThinCerts™ - TC inserts (6 well, 0.4 µm pores, transparent) by Greiner or Peptidosomes. Peptidosomes have been prepared according to standard procedures.
For the co-culture assays with SeSt and ReSt using ThinCerts™ (Figure 6), separate day cultures were inoculated (1:1000) from overnight cultures and incubated at 37°C and 220 rpm until they reached an optical density at 600 nm (OD600) of 0.2 – 0.4. From this day cultures, fresh MNGE medium was inoculated to an OD600 of 0.05 and distributed to the well plate according to the pipette scheme (Table 2) in a final volume of 2 mL for each insert and well respectively. After one hour of incubation (37°C, 200 rpm) SeSt were induced with 1% xylose (if necessary). Luminescence was detected directly after induction (t0) and once every following hour (t1 – t5) using a chemiluminescence imaging system.
Co-culture assays using Peptidosomes were performed as well scan experiments according to "Plate reader well scan Peptidosome" protocol, with the following changes:
Fresh MNGE medium was inocculated with SeSt from overnights in a final volume of 500 µL. Peptidosomes were inocculated with ReSt from overnight to an OD600 of 10. After one hour of incubation strains were induced with 1% xylose (if necessary). Luminescence was detected directly after induction and once every following hour using the CLARIOstar® microplate reader from BMG Labtech. Plates were constantly incubated at 37°C under shaking conditions (100rpm).
Promoter activity screening
Although quorum sensing induces various changes in gene expression, the engineering of the ReSt initially required an extensive screening of several promoters regarding their ComX-dependent activity (Figure 7).
Influence of different media on the activity of PsrfA
Overproduction of ComX
Co-culture of SeSt and ReSt using ThinCerts™
Co-culture of SeSt and ReSt using Peptidosomes
The last step to an overall success of this subproject was the demonstration of microbial communication between bacteria encapsulated in Peptidosomes and bacteria in the surrounding medium. Therefore, we repeated the co-culture assay as described before but instead of separating the distinct strains using cell culture inserts we encapsulated the ReSt within a Peptidosome while we cultivated the SeSt in the surrounding medium. Luminescence was detected via well scan to precisely detect even small changes as well as the spatial extent. We could show that there is only a rise of luminescence if you cultivate the encapsulated ReSt together with the SeSt in the surrounding medium.
Our team has made substantial progress in the evaluation of Peptidosomes as a tool for co-cultivation and studies of microbial interactions. We could prove communication between encapsulated bacteria and bacteria in the surroundings by exchanging signaling molecules (=ComX). Further research about pore sizes of the Peptidosome membrane and diffusion rates needs to be done to tap the full potential of Peptidosomes as a powerful co-culture technique.
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