Part:BBa_K1799000
pLsrA-2
Promoter for the E. coli LsrACDB operon. This promoter is repressed by LsrR, and derepressed by autoinducer 2 (AI-2) in the presence of LsrK.
Usage and Biology
The AI-2-sensitive Lsr pathway relies on a transmembrane protein (LsrACDB) to import AI-2, which is then phosphorylated by LsrK, allowing it to bind the repressor protein LsrR, which detaches from the pLsrA and pLsrR promoters, enabling transcription. This pathway is used in E. coli to regulate biofilm formation and is present in many other bacterial species as well. However, few components of this pathway were available through the Registry, and the ones that were available did not appear to function as intended. As a result, we had many of the relevant parts synthesized. We used Synbiota’s RDP system to quickly assemble several large constructs (BBa_K1799020, BBa_K1799021, and BBa_K1799022) in a matter of hours, which were then used to begin to characterize the Lsr quorum sensing pathway.
Experimental Data
In order to evaluate the LsrR regulatory protein and the pLsrA2 promoter, we devised a logical inverter system. In the absence of the Lsr-related components, a control system expressed a red fluorescent protein (RFP) in response to increasing levels of the inducer molecule IPTG. This is accomplished through the conformational effect of IPTG on the LacI regulatory protein which reduces binding to the Lac promoter and increases expression of the RFP. Hence increasing the amount of IPTG increases the amount of RFP.
In the Lsr-based inverter system, transcription of the LsrR regulatory protein is under control of the Lac promoter and transcription of the RFP is under control of the pLsrA2 promoter (which is inhibited by LsrR). In the absence of IPTG, LacI binds to the Lac promoter and inhibits LsrR transcription. This facilitates RNA polymerase binding to the LsrA2 promoter and ultimately increases expression of RFP. When IPTG is present, the binding of LacI to IPTG results in increased expression of LsrR, which inhibits the pLsrA2 promoter and in turn reduces the expression of RFP. Hence the inversion effect: increasing the amount of IPTG reduces the amount of RFP.
We constructed three plasmids using the Synbiota Rapid DNA Prototyping (RDP) assembly protocol. The first plasmid, p1, had an Ampicillin resistance and a medium-copy-number origin of replication. The p1 plasmid contained a single device: a constitutive promoter followed by a strong Ribosome Binding Site, followed by the coding sequence for the LacI regulatory protein. The p1 plasmid was used in both the control system as well as the Lsr-based inverter system under study.
The second plasmid, p2, had a Chloramphenicol resistance and a high-copy-number origin of replication. The p2 plasmid contained a single device: a Lac promoter followed by a medium strength Ribosome Binding Site, in turn followed by the coding sequence for a red fluorescent protein (RFP). The p2 plasmid was only used in the control system (i.e., p1+p2).
The third plasmid, p3, had a Chloramphenicol resistance and a high-copy-number origin of replication. The p3 plasmid contained two devices. The first device was a Lac promoter followed by a medium strength Ribosome Binding Site, in turn followed by the coding sequence for the LsrR regulatory protein and a terminator. The second device was a pLsrA2 promoter followed by a medium-strength Ribosome Binding Site, in turned followed by the coding sequence for a red fluorescent protein (RFP). The p3 plasmid was used only in the LSR-based inverter system under study (i.e., p1+p3).
The following steps were carried out for each individual plasmid: Synbiota RDP assembly, transformation into the DH5-alpha strain of E. coli, streaking onto LB-agar plates with selective antibiotics, colony PCR, overnight incubation using selective antiobiotics, glycerol stock preparation and plasmid min-prep. The DH5-alpha strain of E. coli was chosen because it was LuxS-negative. As such, the E. coli in the systems under study would not produce the AI-2 quorum sensing molecule and interfere with the intended experiment. The E. coli transformed with p1 were rendered competent and transformed with p2 and p3 to obtain p1+p2 (control) and p1+p3 (Lsr-based inverter) two-plasmid systems, respectively. The doubly-transformed cells were plated, colony-selected, and had glycerol stocks prepared. Then fresh 5mL overnights of the control and Lsr-test systems were commenced in LB plus Ampicillin and Chloramphenicol and either 0mM or 1mM IPTG (for a total of four overnights).
On the following morning (long after reaching stationary phase), 4mL aliquots of each overnight were spun down and double-washed with PBS and put on ice to serve as overnight specimens for fluorescent analysis. Each of the four overnights also underwent a 1:100 dilution into 5mL of fresh LB media with identical antibiotics and IPTG concentrations and grown to mid-log phase (OD600 between 0.3 and 0.4). These were also spun down and double-washed with PBS and put on ice to serve as mid-log specimens.
Fluorimetric analysis was conducted at Columbia University on a BioTek Synergy H1 Hybrid Reader (and the Genspace iGEM team gratefully acknowledges the generous help of the Columbia University iGEM team in this regard). The excitation and emission wavelengths for the fluorescent analysis were 503nm and 607nm, respectively (see http://link.springer.com/article/10.3103%2FS0096392508030036#page-1). The eight samples were transferred in triplicate to a 96 well plate in 100uL aliquots per well. A 100uL aliquot of PBS served as the blank reference against which OD600 and fluorescent measurements were comparatively made (i.e., by subtracting the OD600 and fluorescent readings of the blank from each aliquot under study). The comparative fluorescent reading of each aliquot was divided by the comparative OD600 reading of the same aliquot in order to obtain a measure of “per cell” fluorescence. The triplet samples were then averaged and the standard deviation calculated to obtain the results shown below for the overnights:
The average values of the fluorescence measurements shown above by the blue bars demonstrate the intended effect. As expected, the control system exhibits increased levels of fluorescence as the IPTG is increased from 0mM (“IPTG-“) to 1mM (“IPTG+”). Conversely, the Lsr-based system (“LSR”) exhibits reduced levels of fluorescence as the IPTG is increased. The error bars denote the range of +/- one standard deviation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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