Revision as of 08:45, 28 October 2017

Promoter (luxR & HSL regulated -- lux pR)

Promoter activated by LuxR in concert with HSL

The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.

This Plux promoter "pointing to the right" is the same sequence, but inverted, as part BBa_K199052.

Usage and Biology

Pretty good off in the absence of LuxR/HSL. [jb, 5/24/04]
The team from Davidson College and Missouri Western State University discovered that this part promotes "backwards transcription" when LuxR protein is present and AHL-3OC6 is absent. You can read [http://www.ibc7.org/article/journal_v.php?sid=265 the paper that documents this unexpected "backwards promoter activity"] in their open access paper.

Tsinghua-A 2017's characterization

Crosstalk between Plux and Nine AHL-Receptor Combinations

I Background information

The crosstalk between Plux and nine AHL-Receptor Combinations (Namely, 3OC6HSL-LuxR/3OC6HSL-LasR/3OC6HSL-RhlR/3OC12HSL-LuxR/3OC12HSL-LasR/3OC12HSL-RhlR/C4HSL-LuxR/C4HSL-LasR/C4HSL-RhlR/)is characterized by ETH_Zurich 2014. However, the way they characterize the crosstalk may cause inconvenience for us to use.

The response concentration of some receptor-promoter combinations to AHL molecule may be too high but the concentration range that ETH_Zurich considered is limited, however, in real situation, bacteria we used may be able to secrete higher concentration of AHL molecule. What is more, the reverse situation may also happen. In other words, bacteria we used may be unable to secrete so much AHL molecule to activate gene expression controlled by Plux. Because the synthesis rate of each AHL molecule by corresponding synthase is different, this can really happen, even other conditions of AHL receptor, AHL synthase are same. For example, in our project, we want to design two kinds of E.coli which can synthesize two kinds of AHL and respond to AHL secreted by another but not by itself. Therefore, according to results from ETH_Zurich 2014, shown below (Fig. 1), we may design gene circuit of our E.coli as follows. (Fig. 2)

(Fig.1 Results from ETH_Zurich 2014. From this results, we may think that gene circuit designed in Fig. 2 can satisfy our needs. However, actually, from our results, we can see since E.coli can actually synthesize much more C4HSL than maximal concentration considered above by ETH_Zurich 2014, gene circuit in Fig.2 cannot satisfy our needs.)

(Fig.2 Designed gene circuit by results from ETH_Zurich 2014. Here RFP is used to indicate intensity of response.) In conclusion, it is inconvenient for us to choose our gene circuit just according to results from ETH_Zurich 2014 since it does not consider the ability of E.coli synthesizing AHL. Therefore, we designed our experiment as follows, which consider E.coli’s ability to synthesize AHL.

II Experiment Design

First, we cloned three AHL synthases---luxI (BBa_C0061), lasI (BBa_C0078) and rhlI (BBa_C0070) to the low copy backbone---pSB3K3. At the same time, we cloned three receptor-promoter combinations to pSB6A1. The three receptors are luxR (BBa_C0062), lasR (BBa_C0179) and rhlR (BBa_C0171) while the promoter is Plux (BBa_R0062). RFP is attached to the promoters, which is used to detect the response of the promoters to AHL molecules via the receptors. Adding, together, there are nine AHL-Receptor-Plux combinations. After that, we cotransform the plasmids containing AHL synthases and plasmids containing receptor-Plux combinations. (Gene circuit of them is shown in Fig.3)

(Fig. 3 Gene circuit of 9 AHL-Receptor-Plux combinations)

Finally, we detect how RFP intensity per cell change with time, which can be used to indicate the intensity of the promoter response to AHL molecule via corresponding receptor.

III Methods

1. Transfect EPI300 with 9 kinds of combinations of plasmids mentioned above.

2. Pick bacterial clones from the petri plate, then shake it overnight in the LB medium (3ml) with 50 microgram/ml Ampicilin and 30 microgram/ml Kanamycin at 37℃. For each combination, 2 clones are picked.

3. Dilute the overnight culture to 1/50 in fresh LB medium (5ml) containing 50 microgram/ml Ampicilin and 30 microgram/ml Kanamycin.

4. Incubate the fresh cultures at 37℃ until OD600 reaches 0.2.

5. Add 0.5ml culture obtained from Procedure 4 to 5ml fresh LB medium containing 50 microgram /ml Ampicilin and 30 microgram/ml Kanamycin. Incubate the fresh cultures at 37℃.

6. Take out 200μl cultures obtained from Procedure 5 and measure the fluorescent intensity and OD600 after some time. (The cultures will not be put back again.) The excitation wavelength is 584nm, emission wavelength is 607nm.

IV Results

Crosstalk between Plux and Nine AHL-Receptor Combinations

(Fig. 4: Results of our crosstalk experiment: From this, we can see that if we design gene circuit as Fig.2, we will see that I cannot respond a lot to 3OC12HSL secreted by II, but can respond significantly to C4HSL secreted by itself when considering E.coli’s ability to synthesize AHL, this does not satisfy our needs at all.)

I Background

The crosstalk of Plux between 3 kinds of AHL molecules (3OC6HSL, 3OC12HSL and C4HSL) and 3 kinds of receptors (LuxR, LasR and RhlR) was characterized by ETH_Zurich 2014. However, they used AHL reagents to measure the dose-response of promoter activity, which may not be convenient in quorum sensing, because the concentration of AHL bacteria secrete may be out of the range they tested before. In other words, bacteria is unable to secrete so much AHL molecules to activate gene expression. Furthermore, the response time in the quorum sensing process is also important for designers to optimize their synthetic circuits with better precise and robustness. Therefore, we designed circuits to measure the crosstalk between receptors expressed in one kind of E.coli and AHL molecules secreted by another kind of E.coli and response time of these systems.

II Results

Crosstalk between Plux and Nine AHL-Receptor Combinations (Fig. 1: Results of our crosstalk experiment) 3OC6HSL-LuxR is the wild-type pair of Plux. However, from this result, we can see that there are many other pairs can activate Plux. For example, 3OC6HSL-LasR, 3OC6HSL-RhlR, 3OC12HSL-LuxR, C4HSL-LuxR, C4HSL-LasR, C4HSL-Rhl can all activate it. What is more, the quorum sensing needs more than 30 hours to establish a steady state, which may be important for designing dynamic functions in bacteria with quorum sensing.

III Experiment Design and Measurement Methods

First, we cloned three AHL synthases: luxI (BBa_C0061), lasI (BBa_C0078) and rhlI (BBa_C0070), each ligated with promoter (BBa_J23100), RBS (BBa_B0034) and terminator (BBa_B0015) on a low copy backbone (pSB3K3). At the same time, we cloned three receptor-promoter combinations to pSB6A1. The three receptors are luxR (BBa_C0062), lasR (BBa_C0179) and rhlR (BBa_C0171) (with the same promoter, RBS and terminator as AHL synthases). Sensor promoter Plux (BBa_R0062) drives RFP (BBa_E1010) expression (with the same RBS and terminator as above) to detect the response of the promoters to AHL molecules via the receptors. Adding together, there are nine AHL-Receptor-Plux combinations. After that, we co-transformed the plasmids containing AHL synthases and plasmids containing receptor-Plux combinations. (Gene circuit of them is shown in Fig. 2)

(Fig. 2 Gene circuit of 9 AHL-Receptor-Plux combinations) Finally, we detect how RFP intensity per cell change with time, which can be used to indicate the intensity of the promoter response to AHL molecule via corresponding receptor. Detailed measurement methods are as follows:

1. Transform MG1655ΔsdiAΔlacI with 9 kinds of combinations of plasmids mentioned above. 2. Pick bacterial clones from the petri plate, then shake it overnight in the LB medium (3ml) with 50 microgram/ml Ampicillin and 30 microgram/ml Kanamycin at 37℃. For each combination, 2 clones are picked. 3. Dilute the overnight culture to 1/50 in fresh LB medium (3ml) containing 50 microgram/ml Ampicillin and 30 microgram/ml Kanamycin. 4. Incubate the fresh cultures at 37℃ until OD600 reaches 0.2. 5. Add 1ml culture obtained from Procedure 4 to 2ml fresh LB medium containing 50 microgram /ml Ampicillin and 30 microgram/ml Kanamycin. Incubate the fresh cultures at 37℃. 6. Take out 200μl cultures obtained from Procedure 5 and measure the fluorescent intensity and OD600 after some time. (The cultures will not be put back again.) The excitation wavelength is 584nm, emission wavelength is 607nm.

TUST 2017's characterization

design of ultrasensitive responses basis of https://parts.igem.org/Part:BBa_R0062 use Point mutation

Background information

Quorum-sensing bacteria such as Vibrio fischeri, are able to detected their own population density and implement density-based decision-making，Using the luxI/luxR quorum-sensing system, synthetic biologists have designed a large number of devices in prokaryotic microorganisms.Inevitably, high performance is required from such devices, and this includes reliability, sensitivity, Hence, to improve the properties of population-density switches, we design of ultrasensitive responses base https://parts.igem.org/Part:BBa_R0062 use Point mutation

Experiment Design

We selected the successful strains of the experiment and sequenced part1: https://parts.igem.org/Part:BBa_K2267029 part3: https://parts.igem.org/Part:BBa_K2267030 part4: https://parts.igem.org/Part:BBa_K2267031 part6: https://parts.igem.org/Part:BBa_K2267032 part8: https://parts.igem.org/Part:BBa_K2267033

Methods

The experiments for the characterization of parts were performed as described previously For density-response testing, cells (E. coli strain BW25113) from single colonies on LB agar (BD, USA) plates were grown overnight in 1 ml nutrition-rich, acid-base equilibrium medium (REM) (15.2 g/l yeast extract (BD, USA), 0.5% (NH4)2SO4, 4 mM MgSO4, 2% glucose and 24 g/l K2HPO4.3H2O, and 9.6 g/l KH2PO4) in Falcon tubes overnight (8−12 h, 1000 rpm, 37 °C, mB100-40 Thermo Shaker, AOSHENG, China). The cultures were subsequently diluted 500-fold with REM in 96-well plates, which were further incubated at 37°C in a shaker at 1000 rpm. Once the diluted cultures reached an OD600 of 0.12–0.14 (~3 h), 10 μL aliquots were transferred into 1 mL REM in 24-well plates (Corning/Costar 3524). These plates were incubated at 37°C in a Varioskan Flash (Thermo Scientific, USA) under constant shaking at 1,000 rpm for 20 h to maintain exponential growth, during which the OD600 and fluorescence values were recorded

Results

Sequence and Features