(A-13) Building Genetic Circuits to Control Bacterial Growth Location

Preferred Subtrack: Theory and Practice of Synthetic Biology

When designing engineered bacteria for medicine, containment of microbial growth to disease sites to prevent off-target growth is an important consideration. Consequently, engineering genetic circuits to confine bacterial growth in specific physiological conditions has excellent potential to address the challenges of translating next-generation microbial therapeutics.

In this work, we engineered complementary bacterial strains to grow and perish in the presence of acyl-homoserine lactone (AHL) as a proof-of-concept system to be expanded to physiologically relevant inducers such as lactate and oxygen. To develop strains in which AHL induced cell death, AHL-sensitive bacterial promoters were engineered into plasmids with the bacteriophage 𝝓X174 lysis gene downstream. Conversely, strains were also engineered to grow exclusively in the presence of AHL by introducing complementary repressors to enable transcription of 𝝓X174 only in the absence of AHL. To test the performance of our strains, we constructed a microfluidic device to create a continuous concentration gradient of AHL-infused media across cell cultivation chambers, exposing cells to unique microenvironments across a single trap. Spatial control of each strain is validated via distinct bacterial populations of each strain growing in the presence and absence of AHL, according to their respective plasmids.

Genetic circuits require the tuning and proper functioning of several genetic components including promoters, coding regions, ribosome binding sites (RBS), and terminators. For our circuits, we used pLux and pTet promoters. The binding of LuxR to AHL forms a complex that activates the pLux promoter. pTet is a constitutive promoter repressed by TetR. An RBS enables ribosomes to bind to plasmids and start translation. Additionally, the strength of the bond between a ribosome and an RBS is directly correlated to the amount of protein translated downstream. We used RBS.1 (part BBa_B0030) which has a high affinity for ribosomes. Terminators are sections of DNA that mark the end of a gene circuit’s transcription. The strength of a terminator indicates its effectiveness at stopping transcription and two unique strong terminators were used in these circuits. 

Our strains were cultured in lysogeny broth (LB) media with 60ug/mL of spectinomycin along with 2% glucose, in a 37C shaking incubator. Plasmids were constructed by Gibson and Golden Gate assembly followed by transformation into MG1655 E.coli cells and 1301 Salmonella cells. 

After building our circuits and performing transformations, our strains were loaded onto polydimethylsiloxane (PDMS) based microfluidic devices bonded onto coverslips and placed on a microscope stage for imaging. Acquisition of images was performed with a Nikon TI-2 camera.

Our strains were tested in microfluidic chips where continuous growth rate dynamics can be studied under steady-state exponential growth phase conditions. Our microfluidic chip consists of a two-dimensional growth chamber with media channels. Two distinct media inlets one comprised of LB with 200uM AHL and one with standard LB to create a continuous gradient of AHL across the cell trap.  Initially, each strain was loaded individually into the chip to discover regions of colonization only dependent on environmental inducer influence.  For strains whose death was induced by AHL, we saw populations of cells in areas of the traps closest to the control LB channel where there was a low AHL concentration. For strains that grew in the presence of AHL, there were populations of cells closest to the 200uM AHL LB channel in areas of high concentration of the molecules. To discover the impact of co-culturing where nutrients and space become regions of competition, we then loaded both strains into the chip at the same time. When the complementary strains were tested in the chip in co-culture, we saw a distinct separation of their populations throughout the gradient concentration of molecules (see Figure 2b). This separation of populations throughout the gradient is a direct result of the balance between selection by the environmental inducer AHL and the competition between the two strains for nutrients and space. 

A future application of this work is to investigate bacterial-cell interactions in the tumor microenvironment (TME). Identifying where bacteria are likely to colonize in the TME is essential to understanding how they interact with tumor cells and how scientists can develop more targeted tumor therapeutics. We can induce selective bacterial growth in the presence of several molecules representative of conditions in the TME such as oxygen, lactate, and sodium, by introducing promoters sensitive to each of those molecules into our genetic circuits. We can use microfluidics to study the balance of selection by the inducer and interspecies competition as we did in our proof-of-concept. Additionally, we can study where our strains colonize in cancer spheroids, a model system better representing the TME.