Difference between revisions of "Part:BBa K218017"

Revision as of 22:30, 24 October 2009

LuxO D47E under constitutive expression of TetR repressible promoter

LuxO D47E from Vibrio harveyi with constitutive promoter and RBS. This circuit contains luxO D47E with constitutive promoter and RBS (J13002) and terminator (B0015). This circuit allows for the expression of the LuxO D47E protein, a mutant variant of the Vibrio harveyi LuxO protein. The LuxO D47E protein mimics the active (i.e.phosphorylated) form of the LuxO protein and is a transcriptional activator. Together with sigma factor 54, LuxO D47E binds to Vibrio harveyi's quorum regulatory small RNA promoters (Pqrr1, Pqrr2, Pqrr3, Pqrr4 and Pqrr5). We have also added the qrr4 promoter to the Registry as part of last year's project (BBa_K131017) and have created a reporter circuit containing the Pqrr4-gfp fusion (BBa_K218011) for the functional verification of this circuit. Furthermore, the KT1144 E. coli strain containing the Pqrr4-gfp fusion (obtained and used with permission from Dr. Bonnie Bassler of Princeton University) was used as an additional standard to test the functionality of the LuxO D47E protein. For the protocol and the results of this test, please refer to the main page of Part BBa_K131011.

Usage and Biology

Quorum-sensing bacteria produce and release chemical signal molecules termed autoinducers (AIs) whose external concentration increases as a function of increasing cell-population density. Bacteria detect the accumulation of a minimal threshold stimulatory concentration of these autoinducers and alter gene expression, and therefore their behavior. Using these signal-response systems, bacteria synchronize particular behaviors on a population-wide scale and thus function as multicellular organisms. The bioluminescent marine bacterium Vibrio harveyi uses three different AIs—AHL, CAI-1, and AI-2—to control the expression of genes responsible for bioluminescence and numerous other traits. We have designed our System 2 based on V. harveyi AI-2 signaling. V. harveyi AI-2 signal is a furanosyl borate diester, production of which requires the LuxS enzyme. Biosynthesis of AI-2 is dependent on the usage of S-adenosylmethionine (SAM) by the cell in various methylation reactions. For this reason, during periods of exponential growth, there is a very large production of AI-2, thus perhaps signaling to neighbors that a suitable environment for growth (i.e. rich in nutrients) has been found. LuxS catalyzes the formation of the (S)-4,5-dihydroxy-2,3-pentanedione (DPD) intermediate which spontaneously cyclizes and reacts with borate to give AI-2. AI-2 is bound in the periplasm by the protein LuxP, which is constitutively bound to LuxQ, a membrane bound histidine kinase sensor. The binding of AI-2 to LuxP is necessary in regulating the activity of the periplasm-bound LuxQ. At low cell density, in the absence of significant amounts of autoinducers, LuxQ acts as a kinase, autophosphorylates, and subsequently transfers the phosphate to the cytoplasmic protein LuxU. LuxU passes the phosphate to the DNA-binding response regulator protein LuxO. Phospho-LuxO, in conjunction with a transcription factor termed σ54, involved in nitrogen metabolism, activates transcription of the genes encoding five regulatory small RNAs (sRNAs) termed Qrr1–5 (for Quorum Regulatory RNA). The Qrr sRNAs interact with an RNA chaperone termed Hfq, involved in mRNA splicing. The sRNAs, together with Hfq, bind to and destabilize the mRNA encoding the transcriptional activator termed LuxR. LuxR is required to activate transcription of the luciferase operon: luxCDABE. Thus, at low cell density, because the luxR mRNA is degraded, the bacteria do not express the genes necessary for bioluminescence. At high cell density, when the autoinducers accumulate to the level required for detection, the kinase activity of LuxQ is overtaken by its phosphatase activity and thus drains phosphate from LuxO via LuxU. Unphosphorylated LuxO cannot induce expression of the sRNAs. This allows translation of luxR mRNA, production of LuxR, resulting in bioluminescence.

Reference: Waters C.M. and Bassler B.L. Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol. 2005;21:319-46.

Jeremy A. Freeman and Bonnie L. Bassler. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. 1999a. Molecular Microbiology. 31(2), 665-677.

Sequence and Features

Functional Parameters

Fluorescent Reading of the Preliminary trial

File:Fluorescent reading Calgary.jpg
Figure 1. Fluorescent readings when testing LuxO D47E mutants in KT1144 cells and testing the reporter circuit with functional LuxO D47E mutants.

This graph is divided into two lines of cells and a positive control. The left hand bars depict the KT1144 cells with and without LuxO D47E, and this test shows that the mutant is functional because there is an increase in fluorescence upon the addition of the mutant. See 'mutant circuits' on the side bar for more information on testing. The second line of cells is the reporter circuit with and without LuxO D47E, and the purpose here is to determine whether the reporter circuit is functional. Without the mutant circuit, fluorescence reads at 6699, whereas with the mutant, fluorescence reads at 12699. As there is an increase in fluorescence upon the addition of the LuxO D47E mutant, the reporter circuit is functional. The positive control is the TetR promoter followed by an RBS and GFP. TOP10 cells with pBluescript were used as a negative control and to blank the plate reader.

Characterization of the reporter (Pqrr4 + I13500) circuit

The functionality of this reporter circuit was tested by measuring the fluorescence of reporter circuit together with LuxO D47E Mutant Expression Circuit(BBa_K218017), and this fluorescence was compared to the fluorescence of our positive control (BBa_R0040+BBa_I13500). Top 10 E. coli containing the reporter circuit (BBa_K218011) were made chemically competent using standard CaCl2 treatment and then transformed with the LuxOD47E Mutant Expression Circuit (BBa_K218017). Furthermore, in order to test the activity of the LuxO D47E mutant protein (produced from the constructed expression circuit, part BBa_K218017), a standard was obtained from Dr. Bonnie Bassler. Part BBa_K218017 was transformed into the KT1144 E. coli strain containing the Pqrr4-gfp fusion on a cosmid (Bonnie Bassler, Princeton University). Liquid cultures of successful transformants (containing both BBa_K218011 and BBa_K218017) and KT1144 cells transformed with BBa_K21817 were grown overnight (16 hours) along with cultures of BBa_K218011 and BBa_R0040+Bba_I13500 (Positive control; constitutive GFP expression) and pBluescript (Negative control; culture lacking gfp). Overnight cultures as well as 1:10 and 1:100 dilutions and Luria Bertani Media were then aliquoted into a 96 well-plate and readings were taken using the Bio-tec Synergy HT plate reader at 37C. What follows are the detailed instructions for using the Bio-tec Synergy HT plate reader.

GFP fluorescent reading protocol
1. Grow overnight cultures of each sample
2. Power on the Bio-tec Synergy HT plate reader, or another plate reader, and KC4 application.
3. On a black 96 well plate, aliquot samples in required wells.
4. Go to wizard, and change the reading parameters to the following settings:
Reader: absorbance
Reading type: Endpoint
Wavelength: 570nm (it is as close as it gets to OD600)
5. Click ok.
6. Again, go to wizard, then in layout, mark the wells that contain samples and blank. Click ok.
7. Press the read button
8. Match the OD600 levels by diluting with corresponding Luria-Bertani (LB) broth.
9. Measure OD600 again.
10. Once OD600 are matching for all samples, serial dilute them (1 in 10, 1 in 100). To serial dilute, aliquot 100uL of original culture into a new tube containing 900uL of corresponding LB broth (1 in 10). To make 1 in 100, aliquot 100uL of 1 in 10 dilution into a new tube containing 900uL of corresponding LB broth (1 in 100).
11. Go back to wizard, change the reading parameters to the following settings*:
Reader: Fluorescence
Reading type: Endpoint
Excitation: 485/20
Emission: 528/20
Optics position: Top
Sensitivity: automatic adjustment, scale to high or low well.
Top probe vertical offset: 3mm
12. Click ok.
13. Again, go to wizard, change the layout of the cells.
14. Read.
*GFP reading protocol was obtained from Minenesota State University
http://www.mnstate.edu/provost/GFPPlateReaderAssayProtocol.pdf, date accessed: August 10th, 2009