Part:BBa_K1799022
IPTG-Repressible RFP Generator
Device was designed and assembled using Synbiota's Rapid DNA Prototyping (RDP) system. The device consists of the LsrR ORF under the control of the Lac promoter and a medium-strength RBS, followed by a terminator, followed by an RFP ORF under the control of pLsrA and a medium-strength RBS.
When cultured in a LacI- strain or in the presence of IPTG, LsrR is produced, repressing pLsrA and preventing expression of RFP.
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 in a matter of hours (BBa_K1799020, BBa_K1799021, and BBa_K1799022) for testing our BBa_K1799000 (pLsrA-2) and BBa_K1799002 (LsrR) parts.
Experimental Data
In order to evaluate the LsrR regulatory protein and the pLsrA-2 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 BBa_K1799020: 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 BBa_K1799021: 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 BBa_K1799022. 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. This is followed by a pLsrA2 promoter followed by a medium-strength Ribosome Binding Site, in turn 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. 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 and is largely consistent across all samples, except for the Lsr-based system with 0mM IPTG (where each of the three fluorescent readings were similar according to the plate reader). While the absence of overlap in error bars seen above lends credence to the validity of these results, additional steps can be taken in the future to improve the statistical character of this experiment. These include the use of stronger Ribosome Binding Sites in plasmids p2 and p3 (to increase the degree of fluorescence), re-suspending the spun-down cells with fractional volumes of PBS to increase the effective cell concentration and increase the total fluorescence in a 100uL aliquot, and also to increase the statistical processing gain of the experiment by using more than three samples per test condition (e.g., ten or more samples).
The figure below demonstrates the results for the mid-log growth:
As before, the average values shown by the blue bars demonstrate the intended effect (i.e., increasing IPTG increases RFP for the control, while the opposite is true for the Lsr-based inverter). However, the error bars are now seen to overlap, which erodes confidence in this conclusion. The smaller number of cells in each aliquot when grown to only mid-log phase (compared to stationary phase) limits the ability of the fluorimeter to accurately measure fluorescence as it will be operating closer to its noise floor. It is important to note that the same steps noted above to improve accurate system measurements for overnights will also apply to mid-log growth (i.e., stronger Ribosome Binding Sites, greater cell concentrations in each aliquot, and increased sample processing gain).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1902
Illegal AgeI site found at 2014 - 1000COMPATIBLE WITH RFC[1000]
| None |