Abstract

The ability of living organisms to form patterns is an untapped resource for synthetic biology. The HKU iGEM2008 team aims to generate unique patterns by rewiring the genetic circuitry controlling cell motility. Specifically, E. coli cells are programmed to autonomously regulate their movement by sensing local cell density. Interesting patterns are formed by two types of newly engineered cells. The low-density mover cells spread outwards and spontaneously form a distinctive ring of low cell density surrounded by rings of high cell density whilst the high-density mover cells form a Mt.Fuji-like structure. Moreover, we build a theoretical model that satisfactorily fits our current experimental data, and also predicts some parameters which may significantly affect the ring formation. The study of this self-organized spatial distribution of cells helps us to understand principles underlying the formation of natural biological patterns, and synthetic non-natural patterns have various potential applied uses.

Overview

The iGEM2008 iHKU team aims to deliver a brilliant project this year. We major in multiple disciplines including Biochemistry, Bioinformatics, Physics, and Chemistry. Using our different backgrounds and modalities of thought, we complement each other in developing new ideas, and in carrying out wet/dry lab work (Team).
Pattern formation is one of the most common yet fascinating biological phenomena happening in our daily lives, though for centuries, biologists, physicists and mathematicians have struggled to understand its nature. How do highly ordered patterns arise from a few living cells? How can our hands, our eyes, our bones form their shape with such low error rates? These questions are fascinating and crucial. The fundamental elements in biological pattern formation are cell growth, cell movement, cell-cell communication, and differential gene expression. In this project, we aim to form new patterns by controlling cell movement, using a single strain of engineered bacteria. Bacterium E. coli was chosen as our model system. E. coli cells use their flagella to move around. To generate a recognizable and stable pattern, bacterial motility must be controlled and coordinated. This can be accomplished by designing genetic circuits coupling bacterial quorum sensing system and genes controlling mobility. There are several key genes responsible for the movement of flagella, two of them are cheY and cheZ. CheY protein has two forms: its phosphorylated form makes flagella rotate clockwise and the cell will tumble; its dephosphorylated form makes flagella rotate counterclockwise and the cell will be driven straight in one direction (run). The CheZ protein is involved in dephosphorylation of protein CheY. It is known in the literature that cells are immobile in the absence of CheZ.
By rewiring the genetic circuitry that controls cell motility, we aim to generate unique patterns (Design). First, we applied the method of Recombineering to delete the cheZ gene in chromosome of wild type E. coli strain, MG1655 (Protocols). Then, a series of biobricks and strains were successfully constructed (Plasmids and strains), to (i) import the quorum sensing system of Vibrio fischeri and (2) control the expression of cheZ by quorum sensing, either positively or negatively. As expected, interesting patterns were observed (Results), such as Mt.Fuji-like and ring of troughs patterns, when cells are seeded on low density agar plates. Since the ring-like patterns are so counterintuitive -- they formed despite cell growth and diffusion, our remaining work mainly focused on the characterization and modeling of these patterns.
Considering E. coli movement as a random walk, a coarse-grained model was used to describe the basic cell motility in response to AHL (the quorum sensor) synthesized by the cells. Our model is based on time dependent partial differential equations including the effect of cell random walk, cell growth, AHL diffusion, AHL synthesis and degradation, and nutrient diffusion and consumption. (Modeling). Analysis of the model suggests a basic mechanism underlying the formation of the intriguing trough-like patterns: regions of low cell density are formed by cells being sucked to neighboring high density regions as the engineered cells move randomly at low density and become immobile at high density. Our simulation indicated some factors might significantly affect the development of ring-like patterns, such as the growth rate of the cell which was also observed in the experiments. By measuring these factors, we provide not only solid data to support our hypothesis for our model, but also the values of the parameters involved (Results). As a result, we were able to achieve a double-ring pattern by modifying the cooperativity of the response to AHL upon adding positive feedback to the genetic circuit. (Results).
During the experiments, we encountered numerous challenges and difficulties. To overcome them, we created several NOVEL protocols, software, and devices with the help of our knowledge from different fields, such as “growth curve on agar plate” (Protocols), “movie taker”, and “reflection spectrophotometer” (Novel devices). We believe more researchers will benefit from our inventions.
Last but not least, in this project, we created 15 biobricks and characterized one existing biobrick (Characterization), which are considered to be helpful to future iGEM competitions and the study of synthetic biology.

Acknowledgement

We thank Dr LingChong You, California Institute of Technology, for providing the plasmid pluxRI2, and thank Dr Ron Weiss, Princeton University, for providing the plasmid pLD.

Sponsors

Retrieved from "http://2008.igem.org/Team:iHKU" Recent changes What links here Related changes Special pages My preferences Printable version Permanent link Privacy policy Disclaimers