GROWTH REGULATION
After incorporating the CcaS/R system in E.coli and the Opto-T7 system in Bacillus subtilis, we had to decide a growth-regulating gene to be expressed in the two genetic systems in order to maintain the desired bacterial ratios. For this purpose, we had two evident options :
Promoting the growth of the bacteria with a smaller population
Inhibiting the growth of the bacteria with a higher population
The former approach has certain complications, like promoting the growth of a particular bacterial species in a co-culture might lead to saturation of its population, beyond which the growth-promoting is practically redundant. Thus, we go by the latter approach.
Having decided to use a growth-inhibitory system we further had a choice of selecting a regulatory protein from either a bacteriostatic or a bactericidal class of proteins. Our natural instinct was to proceed with a bacteriostatic protein owing to its ease of operation, without increasing the concentration of dead bacteria in the culture.
We listed out the following methods that can be adopted to design a bacteriostatic growth regulatory system:
Auxotrophic Approach
In this technique, we exploit the properties of auxotrophic bacterial strains. The gene of interest has to be one which is essential for the biosynthesis of the particular nutrient for which the bacteria is auxotrophic
Initially, when the medium lacks the aforesaid nutrient, the gene is expressed naturally. Once we shine the light of specific frequency the gene inhibition occurs which interferes with the biosynthesis of the particular nutrient and the bacteria being an auxotroph is unable to grow. (Eg. LeuB system which is involved in the biosynthesis of leucine is a common system used in co-cultures)
The reason behind not going for the auxotrophic approach is that it requires us to use auxotrophic strains which need not end up creating a bacteriostatic effect. Moreover, using auxotrophic strains of bacteria will restrict the application of our project.
Using auxotrophic strains is impractical at later stages of development as it will pose as an additional burden to the cell along with the engineered system it might be carrying.
Antibiotic Approach
For this method, we select a bacteriostatic antibiotic (like chloramphenicol, tetracycline, erythromycin) which is initially present in the media. The bacterial strain will have the resistance genes for a particular antibiotic (like Cat system for chloramphenicol, TetR for tetracycline). When we shine the light of a particular frequency, the antibiotic resistance gene is inhibited and the bacteria is acted upon by the bacteriostatic antibiotic, freezing its growth while the other species continues to grow.
Antibiotic mediated growth inhibition requires quite some time and since we are aiming for a quicker system it is not best suited for us. Moreover, this approach can lead to antibacterial resistance in the microbe strains which is really harmful to the healthcare industry and the environment in the long term. Further, this antibiotic resistance that is inevitable would hamper with the feasibility of the project.
Bacteriophage Proteins
Certain bacteriophage proteins act as inhibitors for essential growth proteins of bacteria. The expression of these genes restricts the growth of the bacteria by inhibiting the functioning of their constitutive growth proteins.
This approach was best suited for our project due to the availability of a certain phage protein which had all the desired attributes of being a perfect bacteriostatic growth-inhibitory protein.
Gp2 is a protein obtained from the T7 bacteriophage which naturally infects Escherichia coli which is a potent inhibitor of bacterial RNA Polymerase.
Being independent of any auxotrophic system, it can be directly introduced into a wide variety of species without requirements of highly specific strains, increasing the applications of our project.
Mechanism of action of Gp2 protein
Bacteriophage T7 encodes a potent inhibitor of the Escherichia coli host RNA polymerase (RNAP). The product of gene 2 (Gp2), which forms a protein-protein interaction with σ70 subunit of RNA Polymerase prevents the normal egress of σ70 subunit from the RNAP active site channel. Gp2 thus misappropriates a domain of the RNAP holoenzyme (σ70) to inhibit the function of the enzyme.
Moreover in case of Gp2 protein:
It has a small coding sequence (slightly more than 200 bp) and expressing it with the larger optogenetic systems in our project seems more feasible than other systems like LeuB which are pretty huge in comparison to Gp2.
It binds to the RNA polymerase reversibly and its effect (inhibition of bacterial growth) is visible within almost 200 seconds, implying that the effect of this protein is almost instantaneous in the biological time scale of our system.
Considering the aforementioned factors using gp2 as the gene of interest in our optogenetic systems seemed to be the most optimum solution to the problem of growth regulation.
