Describe the research, experiments, and protocols you used in your iGEM project.
The goal of completing a gene expression assay is to determine the best strain and circuit in regards to gene expression. For the validation, standardization, and development of the mathematical model associated with the gene expression system, we aim to identify and standardize the expression conditions using at least two variables. The first variable is based on the evaluation of a minimum of three expression strains that have been previously employed and validated in synthetic biology assays: E. coli BL21, DH5alpha, and K12. Each strain will undergo a minimum of 12 repetitions for each treatment. The second variable under investigation will encompass four types of genetic circuits. Each genetic circuit features a variant of the LuxR protein with characteristics that have been previously identified in the literature or have undergone prior validation of their functions.
Hence, the first circuit incorporates a system validated in E. coli by the iGEM team. The second and third circuits exhibit increasing affinity and stability, respectively. Meanwhile, the fourth circuit integrates characteristics through protein engineering, involving LuxR2 and LuxR3.
Consequently, the three E. coli strains will be subjected to the four circuits with LuxR variants to identify the optimal detection system based on the efficiency and stability of LuxR. The assays will involve cultivating the three studied strains for up to 12 hours, followed by induction with chitin to activate the biosensor. This will enable measurements of optical density (OD) to assess growth, as well as the measurement of fluorescence associated with reporter proteins within each circuit.
1.1. Prepare the bacterial strains: Obtain four strains - control Lux R, better specificity, better stability, and a combination of specificity with stability.
1.2. Prepare genetic circuits: Prepare Genetic Circuit 1 (with one variation) and Genetic Circuit 2 (with four variations).
1.3. Prepare petri dishes with LB medium.
The 1st Genetic Circuit is tested with one variation for each strain.
12 replicas x 3 strains = 36 tests
The 2nd genetic circuits are tested with one positive control and 3 other versions of LuxR:
Upon completion, we anticipate conducting 12 replicates for each of the 4 genetic circuits and across 3 strains, summing up to 144 tests. This will enable us to statistically evaluate the data by utilizing an analysis of variance (ANOVA) to determine the presence of any significant differences between the treatments.
There are four negative controls: 3 of which consist of the LB medium and the strain of the bacteria without a genetically engineered plasmid to observe how the bacteria grows without it; 1 consists of just the LB medium in order to have a baseline measurement of wavelength through a spectrophotometer, which provides a base value to compare the turbidity and therefore density of the samples. This comparison will be conducive to concluding which strain-circuit combination grew more. This will result in 12 negative controls.
144 + 36 + 12 = 192 replicates.
The other test we are conducting is fluorescence testing, which will involve electrophoresis in Polyacrylamide Gel.