Difference between revisions of "Part:BBa K575010"

Latest revision as of 20:53, 21 October 2019

LasR/PAI1 Inducible Promoter + RBS (B0030) + RFP

This part consists of the LasR/PAI1 (3-oxo-C12-HSL) inducible promoter, RBS (Part B0030), and a RFP reporter.

Team OhioState Characterization

Introduction

Quorum sensing is a process of bacterial cell-cell communication that uses acyl-homoserine lactones (AHLs), as signaling molecules. Pseudomonas aeruginosa produces AHLs endogenously using an autoinducer synthase called LasI that makes an AHL called oxoC12. LasR complexed with oxoC12 is able to interact with LasR-dependent promoters and activate transcription (Rust 1996).

Part BBa_K575010 is a plasmid that encodes a red fluorescent protein (RFP) and ribosome binding site (RBS) driven by the lasB promoter, which is a LasR-dependent promoter. Upon activation of the LasR-dependent promoter, RFP will be expressed and red fluorescence should be observed. RFP expression is thus dependent on the presence of LasR and AHLs. Hereafter we refer to this part as plasB-rfp. E. coli does not produce AHLs endogenously and its LasR homolog, SdiA, is unable to interact with the lasB promoter (Lindsay 2005). As a result, RFP should not be expressed in E. coli + plasB-rfp. In order to use this part in E. coli, a source of LasR would have to be introduced and AHLs added to the medium. We hypothesize that RFP expression in E. coli + plasB-rfp’’ requires the addition of LasR protein and the presence of exogenous AHLs.

This E. coli system is optimal for characterization because plasmids expressing LasR, or not, were available in our lab (pAL105 and pAL106, respectively (Lindsay 2005)). pAL105 and pAL106 are identical in that each encodes a LasR-dependent promoter (plasI) fused to luxCDABE from Photorhabdus luminescens. However, pAL105 encodes LasR while pAL106 does not. Therefore, strains containing pAL105 will show increased luminesce in response to oxoC12 (detection limit of 10 nM), while strains containing pAL106 will not. Additionally, the LasR expressed from pAL105 should activate the lasB-rfp fusion on the biobrick part that we are characterizing, plasB-rfp.

Methods

Bacterial strains and media

Bacteria were grown in Luria-Bertani (LB) broth. Tetracycline and chloramphenicol were used at a final concentration of 10 and 30 μg/mL, respectively. AHLs were dissolved in acidified ethyl acetate (EA) and used at the concentrations specified.

Resuspension and Transformation of Biobrick DNA into Mach1

As per the instructions on the iGEM DNA Distribution Help Page, 10 μL of distilled water was used to resuspend the dry DNA of our required part, Bba_K575010. 1 microliter of this resuspension was then used in a chemical transformation with Mach1 OneShot Cells (refer to Experiments page for more protocol details). Cells were immersed in SOC media, given a 1 hour outgrowth, and plated on LB+cam plates (chloramphenicol resistance conferred by plasmid). Transformants were obtained the following day and were set up in liquid LB+cam. The Biobrick plasmid, plasB-rfp, was miniprepped using the Qiagen Miniprep Kit and nanodropped to determine concentration.

Transformation

WM54 + pAL105 and WM54 + pAL106 were obtained from the Ahmer Lab Strain Collection and grown on LB+Tet.

These strains were made electrocompetent using a mannitol-glycerol (MG) rinse to eliminate salts. Cells were pelleted and resuspended in water, then washed with MG solution and resuspended in MG (refer to Experiments page for protocol).

Biobrick Plasmid Bba_575010 (plasB-rfp) was electroporated into each of these strains using a BTX ECM630 Electroporator at 2500 V, 200 Ω, 25 μF (refer to Experiments page for protocol). 100 μl of each E. coli strain in MG solution was electroporated with 2μl of Biobrick plasmid at 71.2 ng/μl, and subsequently incubated for 1 hour in 1 mL of SOC media. These cells were plated on LB-Cam+Tet media to select for transformants that have chloramphenicol resistance from the BioBrick part (plasB-rfp) and tetracycline resistance from the pAL plasmids.

WM54 with pAL105 or WM54 with pAL106 were both separately electroporated with the Biobrick plasmid containing the lasB promoter fused to RFP. Transformants were selected on LB supplemented with chloramphenicol and tetracycline. Transformants were picked and grown in liquid culture containing LB+Cam+Tet at 37°C. These overnight cultures were used to inoculate a 96-well black, clear bottom plate.

The scheme of the assay is summarized in Figure 1A. In wells A1-A6, 200 microliters LB+Cam+Tet was added, with A1 and A4 containing the AHL oxoC12, A2 and A5 containing another AHL called C12, and A3 and A6 containing the AHL oxoC8. In this first row, all AHLs were at 1 uM concentration. In wells A7-A9, LB+Cam was used instead, and with oxoC12, C12, and oxoC8 in those wells in order. This was identical to A10-A12, except LB+Tet was used. In wells B1-B6, 180 microliters LB+CamTet was added. In B7-B9, LB+Cam was added, and in B10-B12, it was LB+Tet. 20 microliters of A1 was diluted into B1, and likewise for A2 into B2, and so on. Taking from B2, 20 microliters were transferred to 180 microliters of media in C2, and this was repeated for each well down to F6. Following this, our strains were added: WM54 + pAL105 +plasB-rfp, WM54 + pAL106 +plasB-rfp, WM54 +plasB-rfp, and WM54 + pAL105 in the 1.8 microliter amounts to each well.

This 96-well black/clear bottom plate was sealed with a BreathEasy film and incubated at 37°C for 16 hours. After incubation the plate was placed into the Spectromax with the following settings: Growth was measured by OD at 600nm; luminescence was measured at all wavelengths; fluorescence was measured with excitation of 573 nm and emission at 607 nm reading from the bottom of the plate. To ensure that luminescence readings did not interfere with fluorescence measurements, a positive control was run, as shown in Figure 1B. It was shown that luminescence does not interfere with RFP fluorescence due to the lack of a peak at 607 nm, the wavelength that RFP was measured at. This wavelength to measure RFP was determined empirically, as shown in Figure 1C.

Figure 1A. Plate assay set up.

Figure 1B. Scanning emission wavelength for Luminescence in the SpectraMax i3x plate reader.</b> Scanning analysis of a positive control for luminescence to establish if the luminescence would interfere with the emission wavelength of RFP at 607 nm.

Figure 1C(1). Establishing optimal excitation and emission for RFP in the SpectraMax i3x plate reader.</b> Scanning analysis of a positive control for optimal RFP excitation and emission. Started scanning every 10 nm from 460 nm to 610 nm (A), then narrowed the window scanning every 1 nm from 600 nm to 620 nm (B). Optimal excitation is 573 nm and emission is 607 nm.

Figure 1C(2). Establishing optimal excitation and emission for RFP in the SpectraMax i3x plate reader.</b> Scanning analysis of a positive control for optimal RFP excitation and emission. Started scanning every 10 nm from 460 nm to 610 nm (A), then narrowed the window scanning every 1 nm from 600 nm to 620 nm (B). Optimal excitation is 573 nm and emission is 607 nm.

Results

Experimental design

Strains and Plasmids used in this study are listed in Table 1.

Plasmid pAL105 provides the LasR protein (AHL responsive) and a LasR-responsive lasI-luxCDABE to serve as a reporter. Additionally, the LasR from pAL105 should activate the lasB-rfp fusion on the biobrick plasmid. pAL106 is identical to pAL105 except that it lacks the lasR gene. Therefore, pAL106 will not be able to activate the lasI-luxCDABE on pAL106 or the lasB-rfp fusion on the biobrick plasmid. The controls, which include WM54 with only pAL105, WM54 with only the plasB-rfp, and the combination of pAL106 and plasB-rfp should yield no discernible phenotype. This experiment was performed on four separate occasions with the mean and standard deviation graphed in Figure 2. Statistical significance was determined by comparing groups with a Mann-Whitney test assuming non-parametric data.

We were able to determine conclusively that the plasB-rfp fusion on the biobrick plasmid (previously listed as LasR-dependent) is NOT responding to LasR. Our data indicates that the LasR ligand, oxoC12, was indeed capable of stimulating luminescence from the lasI-luxCDABE fusion on the pAL105 plasmid (40-fold, p < 0.05). However, the plasB-rfp of the biobrick plasmid shows no activation in response to oxoC12 (1.3 to 1.9-fold, not significant). The lasI-luxCDABE fusion is an internal control that demonstrates that pAL105 is indeed providing LasR that is responding to AHLs. However, the biobrick part encoding the plasB-rfp is failing to respond.

Figure 2. LasR is specific to oxoC12 but the lasB promoter in Part Bba_K575010 is LasR-independent.

Usage and Biology

Sequence and Features