Team:Caltech/Project/Experiments

Testing Export of Signaling Ligand

To test these quorum sensing systems’ viability in E. coli, we tried to construct plasmids containing our genes of interest and transformed them into E. coli. Due to technical issues, we were successful in cloning plasmids carrying the lamBDCA and fsrABC quorum systems' export genes. In addition to the genes of interest, each plasmid also contained the coding sequence for 3xFLAG at the 5' end of the sequence encoding the signaling ligand (resulting in a 3xFLAG at the N-terminus of the signaling ligand after translation). These FLAG tags were blotted against in the Western blots we eventually ran to detect the presence of the signaling ligands. These 3xFLAG tags were ultimately removed from the plasmids after their usefulness in Western blotting had expired.

Based on how the peptide precursors of the signaling ligands of the quorum sensing systems were translated and cleaved, we expected to see several discrete protein fragments in the Western blot. These fragments are detailed in Figure 1 & Figure 2.

Figure 1. Potential Western Blot Fragments for lam QS System. Based on the known domains of the lamB & lamD operon that we synthesized in our construct, we deduced that, depending on how the protein is translated and cleaved, any one of the three protein fragments presented above containing the 3xFLAG domain could have appeared on the Western blot.

Figure 2. Potential Western Blot Fragments for fsr QS System. Based on the known domains of the fsrB membrane protein, we deduced that, depending on how the membrane protein is cleaved, any one of the five protein fragments presented above containing the 3xFLAG domain could have appeared on the Western blot.

Liquid chromatography mass spectroscopy (LC/MS) analysis was also performed on the supernatants of cell cultures expressing the plasmids we had constructed. LC/MS is a process that starts by separating a liquid sample by size and composition via injection into a filtration column. After the sample passes through, its variously sized components are ionized, and their mass-to-charge ratios (m/z) are computed via mass spectroscopy by analyzing the effects of electromagnetic fields on the particles. The data obtained from these experiments were compared to existing characterization data for these signaling ligands to determine the presence or absence of the signaling ligand in the supernatant. According to the scientific literature, previous analysis of the lam signaling ligand had demonstrated that strong peaks were expected to be seen at m/z ratios of 260.1, 345.2, 373.2, and 577.3 after roughly 27 minutes flowing through the column (at a flow rate of 0.2 mL/min) [1].

Testing Scaffold Protein System

Before working on our two-component system, we tested to ensure that the scaffold is functional. We had two response regulators and a histidine kinase plasmid. The plasmid p1521 doesn't have the scaffold and plasmid p1523 does have the scaffold protein. We did this by transforming a two-component system that we know is functional, plating it with the appropriate antibiotic, growing a liquid culture with arabinose and using a plate reader to read GFP levels.

Testing Signaling Ligand Reception

To test the AgrC/AgrA two-component system in E. coli, we designed three plasmid constructs. The first plasmid construct contained the AgrC gene with 4 SH3 scaffold domains attached at the C-terminus. The second and third constructs both contained the AgrA gene along with a GFP reporter regulated by the P2 promoter, but the constructs differed in that only one of the constructs had an SH3 peptide attached to the AgrA gene. The SH3 scaffolds were used in order to allow us to induce signal transduction without the AIP inducer, by means of colocalizing the AgrC histidine kinase and AgrA response regulator. We were able to successfully clone two of the three test constructs, but unfortunately, induction of the two-component system via the scaffold induction could not be tested because we failed to successfully clone the plasmid containing the AgrA-SH3 gene.

To test the LamA/LamC and FsrA/FsrC two-component system in E. coli, we designed 6 different plasmid constructs. There was the histidine kinase, FsrC/LamC plasmids that contained 4 SH3 scaffold domains attached at the C-terminus. Then, for FsrA/LamA, we desgned two plasmids for each system. One contained an SH3 peptide, which would allow us to induce a signal without an AIP inducer and one did not have the SH3 peptide.

Testing Combinatorial Promoter Logic

As part of our original project design (see "Project Overview"), we sought to test the expression logic of several combinatorial promoter constructs. Promoter designs were picked from work done by Robert Sidney Cox et al [2]. Each test construct contained a combinatorial promoter, followed by a bicistronic RBS (BBa_J119024) and a super-folder GFP reporter gene. Using a different reporter gene than Cox et al, who used luciferase as their reporter, could have possibly changed the behavior of the promoters, as promoter activity has been shown to be dependent on the downstream sequence. Therefore, although Cox et al had already successfully characterized the expression logic of the promoters, it was still necessary for us to try and recapitulate their results.

We picked 3 combinatorial promoters to test. The first, abbreviated A90, (BBa_K1369000) consisted of a TetR operator site within the -35 and -10 RNA polymerase boxes and a LacI operator site downstream of the -10 RNA polymerase box. The second, abbreviated B83, (BBa_K1369001) consisted of a LacI operator site upstream of the -35 RNA polymerase binding site, and a TetR operator site within the -35 and -10 RNA polymerase boxes. The last, abbreviated D46, (BBa_K1369002) consisted of an AraC operator site upstream of the -35 RNA polymerase box, and two TetR operator sites, one within the -35 and -10 RNA polymerase boxes, and the other downstream of the -10 RNA polymerase box. After successfully cloning test constructs for each, DH5α-Z1 cells were transformed and were inoculated into liquid cultures. In order to then test the expression logic of each promoter, cells were grown for around 8 hours at 37&degC in MOPS media with different inducer concentrations. Then, GFP fluorescence was measured in a plate reader. Experiments were done in triplicates. For IPTG, we used concentrations of 0, 5, 50, and 500 μM. For aTc, we used concentrations of 0, 25, 50, and 100 ng/mL. For arabinose, we used concentrations of 0, 0.001, 0.01, and 0.1%.