Team:Peking/Description
Description and Inspiration
Bacterial physiology: the systems-level background for endogenous and synthetic bio-parts
Synthetic biology usually approaches tasks by breaking them down into small manageable modules, like engineering the cells for acquiring external signals, processing logical information, or delivery of ultimate effects. For these purposes, sensors, gene circuits and actuators are introduced into a host cell called 'chassis', which sounds like a rigid and immutable framework.
The rarely realized fact is that, even E. coli, the most ordinary host cell in a lab, can exhibit remarkable variance in its general physiology, which may be characterized by doubling time (ranging from 20 min to many hours), cell volume, metabolic activities, morphology, DNA content, etc. These parameters can affect the 'desired' function of synthetic bio-parts directly or indirectly, for example, by causing the cell to re-partition its carbon resources between genome maintenance and individual genetic circuit, adjust the number of cellular RNA polymerases and ribosomes[1], or change the intracellular concentration of signaling metabolites like cAMP[2]. Therefore, manageability of our chassis and control over its general physiological parameters has shown its necessity and value.
However, little is concerned about revealing and utilizing the impact of cellular global physiology on molecular elements and networks, despite the fact that basic properties of engineered cells are closely related to the function and safety of the designs (see below).
Based on that understanding, we are curious of the following questions:
- Can we develop a set of versatile experimental tools, allowing us to study bacterial growth state with unprecedented control?
- Is it possible to give some phenomenological and quantitative description of the relationship between the control we exert and the physiological state of the cell?
- Can these empirical relations facilitate our manipulation and prediction of simple yet typical biological behaviors?
- Is there any specific context where control over general growth parameters has strong relevance to the function of the synthetic bacteria?
After months of effort, we now give positive answers to all these questions.
Previous solutions
Although less attention is paid to bacterial physiology in synthetic biology, the topic has long attracted scientists who hope to reveal the general growth laws of cells. Two methods are widely used to modulate growth state: changing the nutrient content of culture medium, or adding antibiotics that take effect by blocking specific biological process (for example, sublethal dose of chloramphenicol inhibits translation) [1]. While these treatments helped reveal the coupling between general growth parameters, they have their disadvantages. Limiting culture medium can significantly slow down biomass accumulation and expression of protein of interest; preparing medium containing different levels of nutrients can also be too complex to operate. The use of antibiotics poses the risk of drug abuse and antibiotic resistance. Their limitations make them even more unpractical if we hope the engineered bacteria to function in human body.
Some methods are taken to specifically acquire slow growing bacteria. Slow growing bacteria may have special advantages in certain context. For example, we would like to be cautious about the growth of therapeutic bacteria to prevent severe infections or crowding out of normal colonies. Synthetic biologists may solve this problem by designing killer switch: instead of regulating cell growth, they eliminate the engineered cells under defined conditions, making them completely inviable. We ruled out this method for its lack of reversibility and tunability.
We know from experts that another method of obtaining slow growing therapeutic bacteria is site-directed random mutagenesis. The process can be extremely burdensome. See our Integrated Human Practice page for more detailed information.
In the next section, we introduce our method for tackling this problem.
Our solution: a growth control toolbox directly targeting genome replication
Since inheritance of the genome is the basic prerequisite of all biological behaviors, and that the maintenance, replication and segregation of genome DNA must be coordinated with cell growth, division, volume, and shape, we want to figure out if we can interrogate the dynamics of cell cycle and those related properties by controlling genome replication initiation.
In E. coli, genome replication initiates at one single locus, oriC. Formation of DnaA protein filaments on DnaA boxes within oriC accurately regulates replication-bubble opening and subsequent helicase loading [3]. Here, we managed to block DnaA binding, mainly based on competition of CRISPR/dCas9 to the arrays of DnaA boxes (Apart from this, we also explored the effect of blocking other regions) [4]. See our Design page for more detailed introduction.
Figure 1. Formation of DnaA filaments around oriC results in the unwinding of DUE and recruitment of the helicase. Accessory protein IHF (integration host factor) is also needed. This figure is adapted from the review of Costa, A. et al [5].
Our experiments reveal that multi-input control is achievable. The variables include expression and degradation rate of dCas9, expression of sgRNA, targeted box, and length of sgRNA. We observed corresponding changes in cell morphology (cell elongation in most cases), and production enhancement. See our Demonstration page for more detailed data and analysis.
Our control system provides a platform to interrogate the dynamics of cell cycle and the coupling between multiple physiological parameters. We highlight the following advantages of our control system:
- Tunability---this guarantees that control would occur when the optimum condition defined by the user is met, and that optimum intensity can be reached
- Compatible with multiple inputs
- Wide adjustable range
- Reversibility
- Keeping cells viable
- Able to switch between different states back and forth
A specific context: microbial therapies
Apart from its significance in laboratories, we also find controlling cell growth has real-world relevance in certain scenarios.
Microbial therapy is a typical one. Engineering microbiomes for eradication of cancer cells provides unique advantage: many types of bacteria show preference for hypoxic microenvironments and thus have tumor-targeting inclinations; their gene packaging capacity allows for expression of multiple therapeutic macromolecules as well as gene networks (for performing more sophisticated tasks in sensing cancerous signals, integrating disease-related information from multiple sources, and releasing drugs upon detection of tumor).
But real-world application is not just about 'what' our microbiomes do, but also about 'how' they perform well under strict control, defined by the designer and the actual situation of the patient. Again, we would like to be cautious about the growth of therapeutic bacteria to prevent severe infections, toxicities arising from overexpression of drugs, or crowding out of normal colonies. After conversation with experts, we further affirmed several existing safety challenges of microbial therapy. See our Integrated Human Practice page for more detailed information.
To be specific, we are facing challenges including but not limited to the following ones:
- Drug efficacy largely depends on the local concentration of the therapeutic molecules. Bacteria colonization and proliferation near the tumor may contribute to enhancing drug concentration [6]. But how to achieve better intratumoral colonization?
- Bacteria are proliferative, and therefore, there efficiency is less dependent on the administered dose [6]. How can we adjust their effective dose in situ?
- Engineered bacteria may be counterproductive if they escape or overwhelm their intended environment, leading to clinically serious infection and sepsis [7]. What can we do to restrict their overgrowth or confine their spatial distribution, preventing escape?
In short, an intrinsic trade-off lies between the toxicities associated with therapeutic microbiome infection and their clinical effect. We hope to fine tune their growth to achieve optimum result. See our Demonstration page for more detailed data and analysis, and our Future Plan page for planned characterizations.
Refences
1. Scott, M., Gunderson, C. W., Mateescu, E. M., Zhang, Z. & Hwa, T. Interdependence of Cell Growth and Gene Expression: Origins and Consequences. Science (80-. ). 330, 1099–1102 (2010).
2. Berthoumieux, S. et al. Shared control of gene expression in bacteria by transcription factors and global physiology of the cell. Mol. Syst. Biol. 9, 1–11 (2013).
3. Reyes-Lamothe, R. & Sherratt, D. J. The bacterial cell cycle, chromosome inheritance and cell growth. Nat. Rev. Microbiol. 17, (2019).
4. Wiktor, J., Lesterlin, C., Sherratt, D. J. & Dekker, C. CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res. 44, 3801–3810 (2016).
5. Costa, A., Hood, I. V., & Berger, J. M. Mechanisms for initiating cellular DNA replication. Annual review of biochemistry, 82, 25-54(2013).
6. Forbes, N. S. et al. White paper on microbial anti-cancer therapy and prevention. J. Immunother. Cancer 6, 1–24 (2018).
7. Wright, O., Stan, G. B. & Ellis, T. Building-in biosafety for synthetic biology. Microbiol. (United Kingdom) 159, 1221–1235 (2013).