Part:BBa_K5425004
Contents
Short description
The population control module is a carefully engineered circuit that regulates cell density through quorum sensing. It detects when the population reaches a predetermined threshold and triggers a reversible cell cycle arrest. The arrest is lifted by incorporating negative feedback loops—using a repressor and an AHL-degrading enzyme—that halt the production of the arrest-inducing molecule once the population falls below the threshold. This allows the cell population to recover and maintain a stable density.
Fig:A pictorial analogy to depict how our module acts as a traffic light to regulate the cell population ,leading to smoother operations and lesser chaos.
General introduction to usage and biology
In a nutrient-rich medium, bacteria can proliferate rapidly, leading to uncontrolled population growth that overwhelms the medium's capacity and causes nutrient depletion. As bacterial numbers increase, they consume resources faster than they can be replenished, shortening the system's functional lifespan. This unchecked growth also results in the accumulation of metabolic byproducts, which, without regulation, can become toxic to the bacteria, ultimately reducing the system’s overall efficacy. Furthermore, overcrowded populations experience stress from competition for limited resources, leading to decreased productivity and failure to produce the desired compounds, such as linalool in the footbed application. These challenges underscore the key limitations of living microbial systems in long-term practical applications.
To explain the working principle of this circuit we divide it into three modules -
Sensing Module (LuxI and luxR)
The Sensing Module detects bacterial population size using quorum sensing, where LuxI produces AHL (N-acyl homoserine lactone) as the population grows. When AHL levels reach a critical threshold, it binds to the LuxR transcription factor, forming complexes that activate the plux promoter. This triggers gene expression in the Execution and Regulation modules which are placed under these plux promoters.
Execution module (cca gene)
In the Execution Module, the cell cycle arrest gene (cca) is expressed, halting cell division once the population hits a predefined threshold, preventing overgrowth and ensuring efficient use of resources like nutrients.The cca gene is designed by us and codes for an antisense RNA against dnaA protein(key initiator protein for replication).Expression of this modules arrest the cell cycle and the cells enter a quiescent state where they are metabolically active but can't divide exponentially. charecterisatiion of this part is reported on https://parts.igem.org/Part:BBa_K5425002
Regulation Module (AiiA,tetR)
The Regulation Module provides reversibility with two feedback loops: TetR controls AHL production by repressing the luxI gene, preventing continuous AHL synthesis, while AiiA degrades existing AHL to reduce its levels and allow population recovery. Both TetR and AiiA are regulated by the plux promoter and ensure that after the population is arrested, it can recover as AHL levels drop. This system maintains a controlled, stable bacterial population, optimizing conditions for linalool production while preventing toxic byproducts from accumulating.
The complete circuit
Modelling
Our initial target is to determine a general bacterial growth curve without any transformation. We thus generate a system of 2 ODEs governing the rate of change of substrate (S) with time and population (N) with time. For population dynamics determination, we have modified the Logistic Growth Equation to enhance the analysis of our model, by incorporating in a new death term which is shown and explained below eventually. For substrate consumption, we have employed the Monod Substrate Model.
We observe that our bacterial population oscillates (oscillation is due to the negative feedback loop in the QS circuit) at a cell concentration level of around 250, quite below the carrying capacity of the original population, which is 700. As a result, due to a lower population concentration, the substrate consumption, as well as the accumulation of toxic secondary metabolites in the medium, slows down in case of the bacterial population with quorum sensing and thus, the population life of the bacteria with the quorum sensing circuit gets extended upto a period of around 25 days, quite longer than population life of the original bacterial population, which is around 10 days. We are thus able to forecast the increase of bacterial population life in the medium by incorporating the quorum sensing circuit(QS circuit) inside the bacteria.
We accordingly fit the wetlab CFU data with the math model and plot the CFU vs t for the fitted math model for both the Control and the QS Circuit (Hifi) bacterial populations. The fitted curves are given below :-
All the equations used to model the circuit can be found on :- https://2024.igem.wiki/iiser-kolkata/
Characterization
We characterized this circuit by measuring optical density (OD at 600 nm,scattering) using a plate reader and spectrophotometer. For the characterization, we induced the bacteria harboring our plasmid with varying concentrations of AHL, aTc, and IPTG. Below are the OD vs. time curves corresponding to the different inducer concentrations
X axis=od Y axis=time(minutes)
We clearly see how the engineered population recovers after an initial decline in cell density.
X axis=od Y axis=time(minutes)
Arrest below the stationary phase of the control is Cleary evident here.
X axis=od Y axis=time(hours).
Sequence and Features
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal suffix found in sequence at 3784
Illegal EcoRI site found at 3368 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3368
Illegal SpeI site found at 3785
Illegal PstI site found at 3799
Illegal NotI site found at 3374
Illegal NotI site found at 3792 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3368
- 23INCOMPATIBLE WITH RFC[23]Illegal suffix found in sequence at 3785
Illegal EcoRI site found at 3368 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 3368
Illegal SpeI site found at 3785
Illegal PstI site found at 3799
Illegal AgeI site found at 2493 - 1000COMPATIBLE WITH RFC[1000]
Cloning and expression
All the sequences were sourced from NCBI and the Parts Registry, then assembled in silico using SnapGene. For cloning, we divided our complete construct into three fragments and had them synthesized by Twist Bioscience. To assemble the fragments, we used NEBuilder® HiFi DNA Assembly. This process required individual amplification of the fragments and linearization of the destination plasmid via PCR, followed by a four-fragment assembly.
Insert 1 (i1) = 1711 bp [transcriptional units of lux I and luxR]
Insert 2 (i2)= 972 bp [transcriptional unit having tetR and AiiA inintial fragment(split between i2 and i3)]
Insert 3(i3) = 1164 bp [residual part of AiiA and transcriptional unit of anti sense]
Linearised plasmid backbone = 2098 bp (pSB1C3A backbone)
Fig:Right to left,Gradient PCR of i1,i2,i3 and the anealing temp was found to be 62o for all the fragments.
Fig: Amplification insert 1 and 2.
Fig: Amplification insert 3.
Fig:Backbone plasmid linearisation
Fig: Single digest at BamH1 and linearisation of PCM.
Discussion
Overall, we observed a reduction in the stationary phase population upon the induction of our circuit, with the decline measured at approximately 0.2 OD (absorbance at 600 nm). We had initially incorporated a degradation tag into one component of the circuit (the AiiA enzyme), but through the course of our experiments, we found that to achieve more sensitive oscillations and a robust circuit, additional degradation tags should be added to other components, such as LuxI and TetR. For our control, we used an uninduced construct and a dummy plasmid containing the chloramphenicol resistance gene (Chl) M18 from kit plate 1, 2024.
To get insights over the experiments and design please visit :- https://2024.igem.wiki/iiser-kolkata/
References
1)Zhang, J., Hasty, J. & Zarrinpar, A. Live bacterial therapeutics for detection and treatment of colorectal cancer. Nat Rev Gastroenterol Hepatol 21, 295–296 (2024). https://doi.org/10.1038/s41575-024-00901-8
2)tricker, J., Cookson, S., Bennett, M. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519 (2008). https://doi.org/10.1038/nature07389
3)Danino, T., Mondragón-Palomino, O., Tsimring, L. et al. A synchronized quorum of genetic clocks. Nature 463, 326–330 (2010). https://doi.org/10.1038/nature08753
4)Gao, X., & Chen, L. (2003). "Antisense RNA-mediated gene regulation: a review." Journal of Molecular Biology, 332(4), 667-676. doi:10.1016/S0022-2836(03)00893-3.
5)Tay, S. K., & Zhang, H. (2007). "Production of long antisense RNA to modulate gene expression." Nucleic Acids Research, 35(18), e131. doi:10.1093/nar/gkm691.
6)Baker, K. E., & Loughran, G. (2015). "Long antisense RNA: a powerful tool for gene regulation." Current Opinion in Genetics & Development, 30, 50-55. doi:10.1016/j.gde.2015.03.002.
7)Liu, Y., & Schaffer, S. (2018). "Engineering long antisense RNAs for targeted gene regulation." Methods in Molecular Biology, 1630, 191-202. doi:10.1007/978-1-4939-7118-7_14.
8)Lee LF, Yeh SH, Chen CW. Construction and synchronization of dnaA temperature-sensitive mutants of Streptomyces. J Bacteriol. 2002;184(4):1214-1218. doi:10.1128/jb.184.4.1214-1218.2002
9)Menikpurage, I. P., Woo, K., & Mera, P. E. (2021). Transcriptional Activity of the Bacterial Replication Initiator DnaA. Frontiers in Microbiology, 12. https://doi.org/10.3389/fmicb.2021.662317
| None |