Team:Humboldt Berlin/Results

Results

Size distribution of produced minicells

We wanted to know whether there is a difference in size of minicells at time points during bacterial cell growth, and how filtration shifts this distribution.

Filtration was done using 0.45 µm filters. The protocol for the conducted measurements can be found here.

Comparing density distributions across optical densities for either filtered or not filtered minicells show no significant difference in mean size. Meaning, our measurements show that the time of extraction has no significant influence in the observed time frame. Therefore, we decided to compare all measured cells unfiltered and filtered (using 0.45 µm filter) in one (graphic all_raw and graphic raw_filtered).

Filtration or purification on the other hand influences the size distribution substantially, which is only logical. The mean diameter shifted from 529 nm to 394 nm after filtration. If it would be desirable to have minicells of increased size for applications (or not) is further investigated in Model C.

Please bear in mind that filtration took a significant hit on the minicell count compared to the non-filtered equivalent. We experienced a loss of up to 7/8 of total minicell count after filtration.

Figure 1: Violin Plots of the experimentally measured size distributions at different growth phases measured in nm. Minicell size was characterized as well in filtered and unfiltered samples (raw).

Proton Motive Force Measurements

The membrane potential plays a crucial role in maintaining the energy household of cells. Along with the chemical potential, it drives the proton motive force (PMF). The PMF is used in different essential physiological processes, e.g., to produce chemical energy sources such as ATP¹ and for the fueling of secretory machines, such as the type 3 secretion system of the injectisome². Thus, an important aspect of the minicells that must be characterized is their ability to maintain the membrane potential.

To quantify the time the minicells are able to execute their function we characterized the membrane potential as an indicator for minicell activity. For this, we used DiSC₃(5) (3,3'-Dipropylthiadicarbocyanine Iodide), a cationic dye able to accumulate within polarized membranes³. By this, the higher the membrane potential the higher the accumulation of DiSC₃(5) within the membrane. Therefore, this can be used as an indicator for the functioning of minicells.

We expect an exponential decay of the proton motive force as an indicator for metabolism and function executing minicells. An exponential fit onto the acquired data in the form of I(t) = a * exp( -c * t), where I is the intensity, c the reziproke of the lifetime and t the time. This resulted in a = 0.136572 and c = 0.03432747 and can be seen together with the recorded data in figure (PMF). Furthermore we use this acquired lifetime to calculate the lifetime of a minicell dependent on its size in our Model P.

Figure 2: Mean intensity of DiSC3(5) reporter of still gfp expressing minicells, extracted at different time points (black). Exponential fit (blue) with a = 0.136572 and c = 0.03432747.

Characterization of P_lldR (BBa_K1847008) in Salmonella

The lactate-inducible promoter (P_lldR) of E. coli is frequently used and well characterized by a variety of iGEM team, highlighted by the high frequency of part-characterization on the respective part web page. Since we are working with Salmonella Typhimurium and wanted to use a promoter that is induced by lactate, which is secreted in high amounts by cancer cells. For our project, we therefore decided to characterize the lldR promoter (BBa_K1847008) in the S. Typhimurium background.

For this, we fused P_lldR to the green fluorescent protein (gfp; BBa_E0840) reporter gene and cloned it into the pKF01 backbone we designed for our project. pKF01 expresses the lldR gene from a constitutive synthetic promoter (P_{syn_thyA}, BBa_K3861002), together with the thyA gene (BBa_K3861003).

We characterized the promoter by using lactic acid at different concentrations. If LldR is not present, P_lldR drives expression of gfp constitutively, which can be observed in the coloring of the colonies on LB-agar plates. In Figure 1A, the P_lldR-gfp construct is found in the pSB1C3 backbone, which lacks the repression of LldR. This results in constant expression of gfp and thus green colored colonies. On the other hand, when LldR is present in higher amounts, expression from P_lldR promoter is restricted/reduced. This can be observed Figure 2B, where P_lldR-gfp construct was cloned into the pKF01-backbone.

When LldR, the repressor of this promoter, is present in higher amounts, the promoter becomes inducible. Here, LldR binds to the promoter, thus blocking it from being targeted by the RNA-polymerase. Upon presence of an inducer LldR releafs DNA and P_lldR is free and transcription is possible. This promoter can be fine-tuned by varying different inducer concentrations (Fig. 3). The use of lactic acid for induction of the P_lldR was shown to be detrimental to bacterial growth at higher concentrations, as S. Typhimurium strains were able to grow at lactic acid concentration up to 12.5 mM. At higher concentrations (25 and 50 mM lactic acid) growth was inhibited (Fig. 4).

Fig. 3: Salmonella Typhimurium strains either transformed with pSB1C3-PlldR-gfp (on top) or with pKF01-_PlldR-gfp (at the bottom) and streaked on either an LB-chloramphenicol or an LB plate. (On Top) In this strain lldR is not additionally expressed from the vector, whereby GFP is constitutively expressed from P_lldR, resulting in green colored colonies. (At the bottom) lldR is constitutively expressed on pKF01, which allows for repression of gfp expression from P_lldR promoter, resulting in characteristic Salmonella colony color.

Fig. 4: Dose-dependent activity of P_lldR -gfp at low lactic acid concentrations (6h post induction). Mean relative fluorescence units (RFU) of GFP using lactic acid concentrations between 0 and 6.25 mM.

Fig. 5: Salmonella Typhimurium growth curve under different lactic acid concentrations. The optical density at a wavelength of 600 nm was measured for a period of 14 h. Growth was measured at different lactic acid concentrations (0-50 mM).

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List of Sources

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2. Diepold, A. & Armitage, J. P. Type III secretion systems: The bacterial flagellum and the injectisome. Philosophical Transactions of the Royal Society B: Biological Sciences (2015) doi:10.1098/rstb.2015.0020.
3. te Winkel, J. D., Gray, D. A., Seistrup, K. H., Hamoen, L. W. & Strahl, H. Analysis of antimicrobial-triggered membrane depolarization using voltage sensitive dyes. Front. Cell Dev. Biol. (2016) doi:10.3389/fcell.2016.00029.