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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.

Applications of BBa_R0062

User Reviews

UNIQ514426d5802acd8f-partinfo-00000000-QINU

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ZJU-China 2016

For better description of single oscillation system, we measured the response intensity of pluxR to AHL. We found that the response threshold of pluxR to AHL isn’t exactly as what shown in BBa_R0062. The minimum response concentration was about 10^-9 M instead of 10^-11M, the maximum response concentration was about 10^-6M instead of 10^-7M. and the relation curve was different with ETH_Zurich 2014 too. Since our samples were much more than ETH_Zurich 2014, we believe that our result are more precise and our model are more in accordance with the reality.

ETH_Zurich 2014

ZJU China 2016

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ETH Zurich 2014

Background information

We used an E. coli TOP10 strain transformed with two medium copy plasmids (about 15 to 20 copies per plasmid and cell). The first plasmid contained the commonly used p15A origin of replication, a kanamycin resistance gene, and promoter pLux (BBa_R0062) followed by RBS (BBa_B0034) and superfolder green fluorescent protein (sfGFP). In general, for spacer and terminator sequences the parts BBa_B0040 and BBa_B0015 were used, respectively. The second plasmid contained the pBR322 origin (pMB1), which yields a stable two-plasmid system together with p15A, an ampicillin resistance gene, and one of three promoters chosen from the Anderson promoter collection followed by luxR (BBa_C0062). The detailed regulator construct design and full sequences (piG0041, piG0046, piG0047) are [http://2014.igem.org/Team:ETH_Zurich/lab/sequences available here].

Experimental Set-Up

The above described E. coli TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (50 μg/mL) and ampicillin (200 μg/mL) to an OD₆₀₀ of about 1.5 (37 °C, 220 rpm). As a reference, a preculture of the same strain lacking the sfGFP gene was included for each assay. The cultures were then diluted 1:40 in fresh LB containing the appropriate antibiotics and measured in triplicates in microtiter plate format on 96-well plates (200 μL culture volume) for 10 h at 37 °C with a Tecan infinite M200 PRO plate reader (optical density measured at 600 nm; fluorescence with an excitation wavelength of 488 nm and an emission wavelength of 530 nm). After 200 min we added the following concentrations of inducers (3OC6-HSL, 3OC12-HSL, and C4-HSL): 10^-4 nM and 10⁴ nM (from 100 mM stocks in DMSO). Attention: All the dilutions of 3OC12-HSL should be made in DMSO in order to avoid precipitation. In addition, in one triplicate only H₂O was added as a control. From the the obtained kinetic data, we calculated mean values and plotted the dose-response-curves for 200 min past induction.

Characterization of the promoter's basal leakiness with LuxR in the absence of 3OC6-HSL

The amount of regulator LuxR (BBa_C0062) in the system was shown to influence the pLux promoter's basal expression or leakiness. By using the three different constitutive promoters BBa_J23100, BBa_J23109, and BBa_J23111 for the production of LuxR we have measured this effect in terms of fluorescence (see Figure 1, left-hand side).

Results

The measurements of the uninduced system (H₂O only) showed an increase of the pLux (BBa_R0062) activity in terms of fluorescence per OD₆₀₀ for increasing strength of the promoter controlling LuxR (BBa_C0062) expression. For BBa_J23100 (strongest promoter chosen) the basal GFP expression was more than 2.5-times higher as compared to BBa_J23109 (weakest one chosen), with BBa_J23111 (medium strength) falling between the two values but close to the strong promoter. Overall, this is in line with the promoter strength given in the Anderson collection and suggests that the basal leakiness of the pLux (BBa_R0062) promoter increases with an increase of LuxR (BBa_C0062) expression.

Characterization of the promoter's sensitivity to 3OC6-HSL depending on LuxR concentration

The amount of regulator LuxR (BBa_C0062) in the system was shown to influence the pLux promoter's response to the inducer concentration (3OC6-HSL). By using the three different constitutive promoters BBa_J23100, BBa_J23109, and BBa_J23111 for the production of LuxR we have measured this effect in terms of fluorescence (see figure 1).

Results

The measurements of the induced system with 3OC6-HSL concentrations of 10^-13 M to 10^-5 M showed an increasing sensitivity of the pLux (BBa_R0062) promoter (in terms of fluorescence per OD₆₀₀) for increasing strength of the promoter controlling LuxR (BBa_C0062) expression. For BBa_J23100 (strongest promoter chosen) the sensitivity is highest (half maximal effective concentration EC₅₀ approximately 20 pM), for BBa_J23109 (weakest one chosen) the sensitivity is lowest (EC₅₀ approximately 100 pM), with BBa_J23111 (medium) falling between these two but closer to the strong promoter (EC₅₀ approximately 10 nM). Overall, this is in line with the promoter strength given in the Anderson collection and suggests that the sensitivity of the pLux (BBa_R0062) promoter increases with an increase of LuxR (BBa_C0062) expression. Also, the sensitivity and the approximate EC₅₀ values are in the range of previous experience.

Figure 1 Effect of varied promoter strength for the expression of LuxR (BBa_C0062) and the resulting change of pLux (BBa_R0062) sensitivity. The fluorescence per OD₆₀₀ is shown over an inducer-range of 10^-13 M to 10^-5 M. The promoters used are: BBa_J23100 (high LuxR expression, red), BBa_J23111 (medium LuxR expression, green), and BBa_J23109 (low LuxR expression, blue). All three promoters are part of the Anderson collection. Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction ± standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.

Characterization of crosstalk

Background information

The E. coli strain used and the experimental set-up are described above. However, here we focus on the characterization of crosstalk and as a result we used only one, strong promoter (BBa_J23100) controlling the three different regulators (LuxR, LasR, and RhlR) used in the experiments in order to quantify crosstalk with pLux. In the following, we describe all the different levels of crosstalk we have assessed.

First-order crosstalk

In the first order crosstalk section we describe activation of pLux due to LuxR binding to inducers different from 3OC6-HSL or pLux itself binding a regulator-inducer pair different from LuxR-3OC6-HSL.

First Level crosstalk: LuxR binds to different AHLs and activates the pLux promoter

In the conventional system 3OC6-HSL binds to its corresponding regulator, LuxR, and activates the pLux promoter (figure 2, light blue). However, LuxR can potentially bind also other AHLs and then activate pLux (figure 2, 3OC12-HSL in red and C4-HSL in green). This leads then to unwanted gene expression (crosstalk).

Figure 2 Overview of possible crosstalk of the LuxR/pLux system with three different AHLs. Usually, 3OC6-HSL binds to its corresponding regulator, LuxR, and activates the pLux promoter (light blue). However, LuxR may also bind 3OC12-HSL (red) or C4-HSL (green) and then unintentionally activate pLux.

Second Level crosstalk: other regulatory proteins, like LasR and RhlR, bind to their natural AHL substrate and activate the pLux promoter

In the conventional system 3OC6-HSL binds to its corresponding regulator, LuxR, and activates the pLux promoter (figure 2, light blue). However, pLux can potentially be activate by other regulators (LasR, RhlR), binding their corresponding regulator (figure 3, 3OC12-HSL in red, C4-HSL in green). This leads then to unwanted gene expression (crosstalk).

Figure 3 Overview of possible crosstalk of the LuxR/pLux system with two additional regulators (LasR and RhlR). Usually, LuxR together with inducer 3OC6-HSL activate their corresponding promoter pLux (light blue). However, pLux may also be activated by the LasR regulator together with 3OC12-AHL (red) or by the RhlR regulator together with C4-AHL (green).

Second order crosstalk: Combination of both cross-talk levels

The second order crosstalk describes unintended activation of pLux by a mixture of both the levels described above. The regulator and inducer are being different from LuxR and 3OC6-HSL, respectively, and at the same time they do not belong to the same module. For example, the inducer C4-HSL (green), usually binding to the regulator RhlR, could potentially interact with LasR regulator (red) and together activate pLux (light blue). This kind of crosstalk is explained in figure 4.

Figure 4 Overview of possible crosstalk of the pLux promoter with both the regulator and inducer being unrelated to the promoter and each other. Usually, LuxR together with inducer 3OC6-HSL activate their corresponding promoter pLux (light blue). However, pLux may also be activated by another regulator together with an unrelated inducer. For example, the inducer C4-HSL (green) may interact with the LasR regulator (red) and together activate pLux (light blue).

Results

**Table 1** Crosstalk matrix for the promoter pLux (BBa_R0062)
In all the measurements conducted to create this matrix the promoter pLux was the basis and was induced in the six different variations shown. The dark blue points in the graph top left show the activation of gene expression when pLux is induced by 3OC6-HSL (Lux-AHL) binding to the corresponding LuxR regulator. The observed transition occurs at a concentration of approximately 1 nM of 3OC6-HSL. The light-blue curve plotted shows modeling data of pLux induced by 3OC6-HSL (Lux-AHL) binding to the corresponding LuxR regulator. This curve from the model and the dark blue data points obtained from experiments were plotted as a reference in all the other graphs describing pLux. Crosstalk can be observed for the cases where the 3OC12-HSL (Las-AHL) binds the LuxR regulator. Additionally for 3OC12-HSL binding to its corresponding regulator LasR and then binding to the pLux as seen in the middle of the top row and center of the matrix. For the case of Las-AHL binding the regulator LasR and subsequently the promoter pLux, the transition occurs at 1 nM and reaches half the fluorescence as comared to pLux induced by 3OC6-HSL binding LuxR. In the case of 3OC12-HSL binding LuxR and inducing the promoter pLux, the transition is observed at approximately 100 nM and severe crosstalk is observed, showing that the ON-OFF-ratio is not significantly different from the reference curve. The experiments with C4-HSL have shown that there is no significant crosstalk with the LuxR regulator and LasR regulator binding C4-HSL and subsequently to pLux. This is indicated on top right and middle right graphs. However, RhlR induced with its corresponding inducer (C4-HSL) binds to pLux and activates expression of GFP at about 100 nM.

Modelling Crosstalk

Each experimental data set was fitted to an Hill function using the Least Absolute Residual method. The results are shown in table 2.

The fitting of the graphs was performed using the following equation :

rFluo = the relative fluorescence (absolute measured fluorescence value over OD) [a.u.]
a = basal expression rate [a.u.] (“leakiness”)
b = maximum expression rate [a.u.] ("full induction")
n = Hill coefficient (“cooperativity”)
K_m = Half-maximal effective concentration (“sensitivity”)
[AHL] = AHL concentration [nM]

**Table 2** Parameters of Hill Function for crosstalk with Plux (with 95% confidence bounds)
	3OC6-HSL	3OC12-HSL	C4-HSL
LuxR	a = 1043 (966.8, 1139) [a.u.] n = 0.89 (0.79, 0.99) Km = 0.1892 (0.1392, 0.2391) [nM] b = 2.129e4 (2.065e4, 2.192e4) [a.u.]	a = 900.5 (882, 918.9) [a.u.] n = 1.049 (0.9976, 1.1) Km = 78.32 (73.18, 83.47) [nM] b = 1.866e4 (1.834e4, 1.899e4) [a.u.]	No crosstalk
LasR	No crosstalk	a = 362.4 (158.2,566.6) [a.u.] n = 0.7898 (0.3241,1.256) Km = 1.269 (0, 2.548) [nM] b = 1.253e4 (1.085e4, 1.422e4) [a.u.]	No crosstalk
RhlR	a = 313.2 (296.8, 329.5) [a.u.] n = 0.9427 (0.7565, 1.129) Km = 1210 (565, 1855) [nM] b = 8361 (7001, 9722) [a.u.]	No crosstalk	a = 309.6 (283.6, 335.6) [a.u.] n = 0.741 (0.6463, 0.8357) Km = 2738 (1411, 4064) [nM] b = 1.22e4 (1.045e4,1.45e4) [a.u.]

UNIQ514426d5802acd8f-partinfo-00000003-QINU

No review score entered. ETH Zurich 2013

Characterization of the pLuxR dose response to OHHL by plate reader analysis and single cell analysis

Fluorescence data analysis

The fitting of the following graphs was performed using this equation :

Output = eGFP levels [au]
Top = maximal eGFP level [au]("full induction")
Bottom = minimal eGFP level [au](“leakiness”)
n = Hill coefficient (“cooperativity”)
EC₅₀ = Half-maximal effective concentration (“sensitivity”)
[AHL]=OHHL concentration [nM]

Primary characterization of the dose reponse curve by microtiter plate analysis

Figure 1. Sensitivity curve of the wild type promoter analyzed on the Tecan Infinity M200 microtiter plate reader. The OHHL concentration range is plotted against the standarized fluorescence (au).
EC₅₀=5.86, R₂= 0.87,n=1.7
.All assays were carried out in triplicates, results are presented as mean ± standard deviation.

We cloned the promoter BBa_R0062 in the BBa_J09855 construct and cloned a eGFP gene as reporter. The eGFP part allows us to make fluorescence measurement in microtiter plate experiment as well as in single cell analysis to characterize the promoter depending on [OHHL], by recording the fluorescence [au]. According to literature [http://www.ncbi.nlm.nih.gov/pubmed/18760602 (Geske G.D. Evaluation of a focused library of N-aryl L-homoserine lactones reveals a new set of potent quorum sensing modulators)]we could define the first experimental OHHL ranges to induce the promoter. After some fine tunning and adjusting experiment we could narrow down the ranges to obtain a dose response curve in a reasonable resolution for the BBa_R0062 promoter to the OHHL (3-oxo-hexanoyl-l-homoserine-lactone). See Figure 1.

Single cell characterization of the dose response in liquid culture and on agar plate

To obtain high quality data we did single cell analysis over the range defined above. Obviously the EC₅₀ changed compared to the microtiter plate data.
See Figure 2.

Figure 2. Dose response comparison between [OHHL] induction in liquid culture and on agar plates, of the BBa_J09855 construct using the BBa_R0062 promoter and a eGFP gene as reporter</b> analyzed at different [OHHL] in the single cell analysis. The black curve shows the response of to OHHL in liquid culture, the orange curve shows the response to OHHL on agar plates. All assays were carried out in duplicates, results are presented as mean ± standard deviation.

Figure 3: Sensitivity curve of the wild type pluxR in agar plates and liquid culture For the liquid culture we got :EC₅₀=0.02nM, R²=0.84, n=1.7; For the agar plates we got :EC₅₀=4.45nM, R²=0.80, n=1.7
The black curve shows the response of to OHHL in liquid culture, the orange curve shows the response to OHHL on agar plates. All assays were carried out in duplicates, results are presented as mean ± standard deviation.

To obtain high quality data we did single cell analysis over the dose response range defined above. Obviously the EC₅₀ changed compared to the plate reader data. Figure 2. The plot shows the dose reponse curve of the promoter depending on AHL concentration. We performed this experiment in liquid culture as well as on agar plates (OHHL was added to the melted 1.5% agar), which was closer to our project. We observed a 220 time shift in the dose response between the liquid culture single cell analysis and the one in agar plates. See Figure 3

UNIQ514426d5802acd8f-partinfo-00000005-QINU

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SUN(Tsinghua)

Part was sequenced and functional.

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iGEM12_OUC-China

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In order to optimize the function of the lux promoter, We creat a hybrid promoter BBa_K737067 which combines plux with OmpR, and in this case, plux can be led to expression by HSL and obtain by phosphorylation of OmpR protein.. According to the research of it before, we camp up with a program . Keep the sequencing of Lux Box, -35box and -10box., Then put the sequencing of C3 point of OmpR promoter into -35box and -10box.

Figure 1 structural representation of Plux promoter

Figure 2 the analysis of conserved sequence of pLux by Weblogo

Plux promoter is organized from the promoter the shining gene of V. fischeri(R0062). Through analyzing common hybrid promoter of Lux by Weblogo , we found that the sequence of Lux Box is conserved. In benefit of LuxR Binding Site, -35box and -10box of the promoter the shining gene of V. fischeri(R0062).

Figure 3 : Our design of pLux-OmpR hybrid promoter

We link the hybrid promoter to the test circuit.

For control group experiment, we put strain A to LB medium adding 20% Sucrose solution and not adding any sucrose solution to cultivate in 37% shake flask , and detect the change of OD value and fluorescence value between 12h.

Though high concentration sucrose solution restrains the grain of E.coli, it doesn’t have effect on the work of circuit. In conclusion, we find that high osmotic pressure has no effect on quorum sensing circuit and the activated of plux promoter. Strain A could serve as control group of pLux-OmpR hybrid promoter.

Measurement of pLux/OmpR hybrid promoter's expression property

Place strain B in 100 mL LB medium with gradient osmotic pressure stress and culture in shake-flask in 37°C.Calculate RFU of each time point ,and we get a surprising result!

Figure 6 Experimental Group 1 Relative Fluorescence Unit--Time Scatter graph

From the figure 6 we can see that when stressed by 7% sucrose, GFP's expression is stronger than those cultured in normal condition. However,when stressed by 14% sucrose, GFP's RFU is 3 times as high as normal culture condition.This indicates that sucrose stress promotes GFP's expression via gene circuit.

UNIQ514426d5802acd8f-partinfo-00000009-QINU

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XMU-China 2011

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Site-directed mutagenesis at position 3,5 and 3/5 of BBa_R0062

On the basis of the nucleotide sequence of the lux pR promoter, the 20 base pair inverted repeat ACCTGTAGGA TCGTACAGGT might consititude a protein binding site. And we also learned that mutagenesis at position 3 and position 5 might cause dramatic change on the expression of downstream gene. Therefore, we generated 3 mutants (BBa_K658006 BBa_K658007 BBa_K658008) of the promoter lux pR by site-directed mutagenesis at position 3, 5 and 3/5.

By testing their strength in IR-GFP devices, we prove that mutagenesis at position 3 and position 5 can change the expression of downstream gene. Mutated promoters lux pR-3 (BBa_K658006) and lux pR-5 (BBa_K658007) dramatically enhanced the expression of the downstream gene compared with wild type promoter lux pR (R0062), while mutated promoter lux pR-3/5 (BBa_K658008) gave an even weaker expression of the downstream gene than promoter lux pR (R0062).

Based on the information about the mutated promoters, we constructed a series of population-control devices which can maintain the cell density of bacteria population at several certain values.

Lux pR strength testing device

To test the strength of promoters lux pR(BBa_R0062) and its 3 mutants lux pR-3 (BBa_K658006), lux pR-5(BBa_K658007), lux pR-3/5(BBa_K658008) , we constructed four devices( BBa_K658016 BBa_K658017 BBa_K658018 BBa_K658019).

If promoter lacl+pL(BBa_R0011) is induced by isopropyl-b-D-thiogalactopyranoside (IPTG), this device will be switched on. At sufficiently high cell density, this device produces greenish tint visible by naked eye. By measuring florescent intensities at steady state of the cell growth for these four IR-GFP devices, the strength of a promoter lux pR could be defined.

Four lux pR strength testing devices (BBa_K658016 BBa_K658017 BBa_K658018 BBa_K658019) were first cloned into plasmid pSB1A2 respectively, followed by transformation into E.coli strain BL21. Fluorescence was measured when cell growth reached a steady state (around 20h).

The results are shown in following figures:

Figure 1, figure 2 and figure 3 illustrate that mutated promoters lux pR-3 (BBa_K658006) and lux pR-5 (BBa_K658007) dramatically increased the fluorescence intensity at steady state compared with wild type promoter lux pR (R0062), while mutated promoter lux pR-3/5 (BBa_K658008) gave an even weaker expression of GFP than promoter lux pR (R0062). It might be explained that the mutagenesis at position 3 and position 5 of the sequence of lux pR (R0062) changed the binding strength between promoter lux pR and protein luxR.

Figure 1 Promoters strength relative to lux pR-3 (BBa_K658006).

Figure 5 Efficiency of promoter lux pR (BBa_R0062) and its three mutants Figure 2 Efficiency of promoter lux pR (BBa_R0062) and its three mutants.

Figure 3: Fluorescence of four IR-GFP devices at 20h. 5, 3, 3/5, N represent for IR-3-GFP (BBa_K658017), IR-5-GFP (BBa_K658018), IR-3/5-GFP (BBa_K658019) and IR-GFP (BBa_K658016) respectively.

Application----a series of population-control devices

The study of the mutated promoters lux pR-3 (BBa_K658006), lux pR-5(BBa_K658007) and lux pR-3/5(BBa_K658008) can be applied to construct a series of population-control devices based on iccdB0.6 (BBa_K658003). These devices—iccdB0.6(BBa_K658003), iccdB-3(BBa_K658009), iccdB-5(BBa_K658010) and iccdB-3/5(BBa_K658011) program the steady-state cell density maintaining at different levels.

With time limited, we only tested the performance of iccdB-3(BBa_K658009) compared to that of iccdB0.6(BBa_K658003) by measuring its steady-state cell density.

The results are shown in following figures:

Figure 4 Experimentally measured steady-state cell density of iccdB0.6(BBa_K658003) and iccdB-3 (BBa_K658009).

Figure 5 Average of experimentally measured cell densities of BL21’s cells with iccdB0.6 (BBa_K658003) and its mutant iccdB-3 (BBa_K658009).

Figure 4 and Figure 5 illustrate that the population-control device iccdB-3 programs a relatively lower steady-state cell density compared with iccdB0.6.

This matched the result of the test on four lux pR promoters’ strength in our IR-GFP device (BBa_K658016) mentioned above. As is shown in figure 2, promoter lux pR-3 has the highest strength of the four. It is probable that mutation at position 3 lowers the threshold for the binding reaction between LuxR/AHL protein complex and promoter lux pR, which starts the Quorum Sensing system at a relatively earlier period with a lower cell density compared with circuits regulated by wild type promoter lux pR (BBa_R0062).

Once the QS system is started, downstream killer protein expresses. The viable cell density reaches a steady state when cell growth rate equals to its death rate. Generally, steady-state cell density seems to fluctuate at the cell density when QS is started. Thus, the higher strength a promoter has, the earlier the population-control device is started, leading to a lower steady-state cell density.

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No review score entered. iGEM Tokyo_Tech 2010

In order to characterize R0062, Plux repression promoter, we constructed K395100 combining R0062 and K121013, which is a promoter-less gfp reporter (rbs-gfp-ter-ter) on pSB6A1 and used a fusion of PlacIq (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.

Overnight cultures of reporter strains grown at 37 °C containing appropriated antibiotics were diluted at least 1:100 and incubated at 37 °C as fresh cultures. After their OD₅₉₀ reached 0.6, added 100nM 3OC6HSL. After 3 hours of induction, fluorescence intensity was measured with flow cytometry. Also, we measured the fluorescence intensity under different concentration of 3OC6HSL (0nM, 1nM, 3nM, 5nM, 10nM, 30nM, 50nM, 100nM by flow cytometry 3 hours after 3OC6HSL induction.

After 3 hours of induction by 3OC6HSL, the expression of GFP with 3OC6HSL around 30 holds increased comparing with the expression without 3OC6HSL. Also, we found fluorescence intensity of luxR activation promoter is dependent on 3OC6HSL concentration. The threshold of fluorescence intensity of R0062, luxR activation promoter regulated by 3OC6HSL is around 5nM.

(→[http://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep more information])

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Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

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wmholtz

Using this part, I have successfully constructed and tested a quorum sensing circuit in E. coli.

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Aberdeen_Scotland 2009

Our miniprep, digest and gel gave expected results. However we did not use this part for our cloning.

No review score entered. NYMU-Taipei 2009

We have characterised the strength of the promoters pCI, p22, pLux, pLas relative to pCI. Has a noticeable basal expression level. More details are available at the [http://2009.igem.org/Team:NYMU-Taipei/Project/Promoter_Strength_Testing NYMU-Taipei iGEM09 wiki].

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Tsinghua-A 2017

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Crosstalk between Plux and Nine AHL-Receptor Combinations

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.

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

plux4: https://parts.igem.org/Part:BBa_K2267031

plux3: https://parts.igem.org/Part:BBa_K2267030

plux1: https://parts.igem.org/Part:BBa_K2267029

plux6: https://parts.igem.org/Part:BBa_K2267032

plux8: https://parts.igem.org/Part:BBa_K2267046

III 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

IV Results

The activated LuxR regulator binds to a LuxR-inducible promoter, pLux, upstream of a GFP reporter. As a result, GFP is expressed when the receiver device is induced with 3O-C6 AHL. We designed this part to characterize the activation range of the Lux receiver device. https://parts.igem.org/Part:BBa_K2267040 is Control， we did point mutation on https://parts.igem.org/Part:BBa_R0062 and the exact spots are

plux1: https://parts.igem.org/Part:BBa_K2267029

plux3: https://parts.igem.org/Part:BBa_K2267030

plux4: https://parts.igem.org/Part:BBa_K2267031

plux6: https://parts.igem.org/Part:BBa_K2267032

plux8: https://parts.igem.org/Part:BBa_K2267046

listed below. We linked gfp reporter genes luxr-plux1-gfp: https://parts.igem.org/Part:BBa_K2267043

luxr-plux3-gfp: https://parts.igem.org/Part:BBa_K2267041

luxr-plux4-gfp: https://parts.igem.org/Part:BBa_K2267042

luxr-plux6-gfp: https://parts.igem.org/Part:BBa_K2267044

luxr-plux8-gfp: https://parts.igem.org/Part:BBa_K2267045

on them respectively to test the function of modified promoters.

We characterised the activation range of this device using a GFP reporter https://parts.igem.org/Part:BBa_K319039. The results of our characterisation experiments can be found here

;

UNIQ514426d5802acd8f-partinfo-00000016-QINU

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-<I>Username</I>
+<I>username 2017</I>
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 Enter the review inofrmation here.
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+<!-- DON'T DELETE -->
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_C0062 AddReview 5</partinfo>
+<I>ZJU-China 2016</I>
+|width='60%' valign='top'|
+For better description of single oscillation system, we measured the response intensity of pluxR to AHL.
+We found that the response threshold of pluxR to AHL isn’t exactly as what shown in [https://parts.igem.org/Part:BBa_R0062:Experience BBa_R0062].
+The minimum response concentration was about 10^-9 M instead of 10^-11M, the maximum response concentration was about 10^-6M instead of 10^-7M. and the relation curve was different with ETH_Zurich 2014 too. Since our samples were much more than ETH_Zurich 2014, we believe that our result are more precise and our model are more in accordance with the reality.
+[[File:ETH_improvement.png|400px|thumb|center|ETH_Zurich 2014]]
+[[File:ZJU_ChinaParts.jpg|400px|thumb|center|ZJU China 2016]]
+|};
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_R0062 AddReview 5</partinfo>
+<I>ETH Zurich 2014</I>
+|width='60%' valign='top'|
+= Background information =
+We used an ''E. coli'' TOP10 strain transformed with two medium copy plasmids (about 15 to 20 copies per plasmid and cell). The first plasmid contained the commonly used p15A origin of replication, a kanamycin resistance gene, and promoter [https://parts.igem.org/Part:BBa_R0062 pLux (BBa_R0062)] followed by [https://parts.igem.org/Part:BBa_B0034 RBS (BBa_B0034)] and superfolder green fluorescent protein (sfGFP). In general, for spacer and terminator sequences the parts [https://parts.igem.org/Part:BBa_B0040 BBa_B0040] and [https://parts.igem.org/Part:BBa_B0015 BBa_B0015] were used, respectively. The second plasmid contained the pBR322 origin (pMB1), which yields a stable two-plasmid system together with p15A, an ampicillin resistance gene, and one of three promoters chosen from the [https://parts.igem.org/Promoters/Catalog/Anderson Anderson promoter collection] followed by [https://parts.igem.org/Part:BBa_C0062 luxR (BBa_C0062)]. The detailed regulator construct design and full sequences (piG0041, piG0046, piG0047) are [http://2014.igem.org/Team:ETH_Zurich/lab/sequences available here].
+= Experimental Set-Up =
+The above described ''E. coli'' TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (50 μg/mL) and ampicillin (200 μg/mL) to an OD<sub>600</sub> of about 1.5 (37 °C, 220 rpm). As a reference, a preculture of the same strain lacking the sfGFP gene was included for each assay. The cultures were then diluted 1:40 in fresh LB containing the appropriate antibiotics and measured in triplicates in microtiter plate format on 96-well plates (200 μL culture volume) for 10 h at 37 °C with a Tecan infinite M200 PRO plate reader (optical density measured at 600 nm; fluorescence with an excitation wavelength of 488 nm and an emission wavelength of 530 nm). After 200 min we added the following concentrations of inducers ([[3OC6HSL|3OC6-HSL]], [[AHL|3OC12-HSL]], and [[AHL|C4-HSL]]): 10<sup>-4</sup> nM and 10<sup>4</sup> nM (from 100 mM stocks in DMSO). Attention: All the dilutions of [[AHL|3OC12-HSL]] should be made in DMSO in order to avoid precipitation. In addition, in one triplicate only H<sub>2</sub>O was added as a control. From the the obtained kinetic data, we calculated mean values and plotted the dose-response-curves for 200 min past induction.
+== Characterization of the promoter's basal leakiness with LuxR in the absence of 3OC6-HSL ==
+The amount of regulator [https://parts.igem.org/Part:BBa_C0062:Experience LuxR (BBa_C0062)] in the system was shown to influence the pLux promoter's basal expression or leakiness. By using the three different constitutive promoters [https://parts.igem.org/Part:BBa_J23100:Experience BBa_J23100], [https://parts.igem.org/Part:BBa_J23109:Experience BBa_J23109], and [https://parts.igem.org/Part:BBa_J23111:Experience BBa_J23111] for the production of LuxR we have measured this effect in terms of fluorescence (see Figure 1, left-hand side).
+== Results ==
+The measurements of the uninduced system (H<sub>2</sub>O only) showed an increase of the [https://parts.igem.org/Part:BBa_R0062 pLux (BBa_R0062)] activity in terms of fluorescence per OD<sub>600</sub> for increasing strength of the promoter controlling [https://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)] expression. For [https://parts.igem.org/Part:BBa_J23100 BBa_J23100] (strongest promoter chosen) the basal GFP expression was more than 2.5-times higher as compared to [https://parts.igem.org/Part:BBa_J23109 BBa_J23109] (weakest one chosen), with [https://parts.igem.org/Part:BBa_J23111 BBa_J23111] (medium strength) falling between the two values but close to the strong promoter. Overall, this is in line with the promoter strength given in the [https://parts.igem.org/Promoters/Catalog/Anderson Anderson collection] and suggests that the basal leakiness of the [https://parts.igem.org/Part:BBa_R0062 pLux (BBa_R0062)] promoter increases with an increase of [https://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)] expression.
+== Characterization of the promoter's sensitivity to 3OC6-HSL depending on LuxR concentration ==
+The amount of regulator [https://parts.igem.org/Part:BBa_C0062:Experience LuxR (BBa_C0062)] in the system was shown to influence the pLux promoter's response to the inducer concentration ([[3OC6HSL|3OC6-HSL]]). By using the three different constitutive promoters [https://parts.igem.org/Part:BBa_J23100:Experience BBa_J23100], [https://parts.igem.org/Part:BBa_J23109:Experience BBa_J23109], and [https://parts.igem.org/Part:BBa_J23111:Experience BBa_J23111] for the production of LuxR we have measured this effect in terms of fluorescence (see figure 1).
+== Results ==
+The measurements of the induced system with [[3OC6HSL|3OC6-HSL]] concentrations of 10<sup>-13</sup> M to 10<sup>-5</sup> M showed an increasing sensitivity of the [https://parts.igem.org/Part:BBa_R0062 pLux (BBa_R0062)] promoter (in terms of fluorescence per OD<sub>600</sub>) for increasing strength of the promoter controlling [https://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)] expression. For [https://parts.igem.org/Part:BBa_J23100 BBa_J23100] (strongest promoter chosen) the sensitivity is highest (half maximal effective concentration EC<sub>50</sub> approximately 20 pM), for [https://parts.igem.org/Part:BBa_J23109 BBa_J23109] (weakest one chosen) the sensitivity is lowest (EC<sub>50</sub> approximately 100 pM), with [https://parts.igem.org/Part:BBa_J23111 BBa_J23111] (medium) falling between these two but closer to the strong promoter (EC<sub>50</sub> approximately 10 nM). Overall, this is in line with the promoter strength given in the [https://parts.igem.org/Promoters/Catalog/Anderson Anderson collection] and suggests that the sensitivity of the [https://parts.igem.org/Part:BBa_R0062 pLux (BBa_R0062)] promoter increases with an increase of [https://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)] expression. Also, the sensitivity and the approximate EC<sub>50</sub> values are in the range of previous experience.
+[[File:ETH_Zurich_2014_Different_LuxRConc.png|500px|thumb|center|'''Figure 1 Effect of varied promoter strength for the expression of [https://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)] and the resulting change of [https://parts.igem.org/Part:BBa_R0062 pLux (BBa_R0062)] sensitivity.''' The fluorescence per OD<sub>600</sub> is shown over an inducer-range of 10<sup>-13</sup> M to 10<sup>-5</sup> M. The promoters used are: [https://parts.igem.org/Part:BBa_J23100 BBa_J23100] (high LuxR expression, red), [https://parts.igem.org/Part:BBa_J23111 BBa_J23111] (medium LuxR expression, green), and [https://parts.igem.org/Part:BBa_J23109 BBa_J23109] (low LuxR expression, blue). All three promoters are part of the [https://parts.igem.org/Promoters/Catalog/Anderson Anderson collection]. Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction &plusmn; standard deviation. For the full data set and kinetics please [http://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [http://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.]]
+= Characterization of crosstalk =
+== Background information ==
+The ''E. coli'' strain used and the experimental set-up are described above. However, here we focus on the characterization of crosstalk and as a result we used only one, strong promoter [https://parts.igem.org/Part:BBa_J23100 (BBa_J23100)] controlling the three different regulators ([https://parts.igem.org/Part:BBa_C0062 LuxR], [https://parts.igem.org/Part:BBa_C0179 LasR], and [https://parts.igem.org/Part:BBa_C0171 RhlR]) used in the experiments in order to quantify crosstalk with [https://parts.igem.org/Part:BBa_R0062 pLux]. In the following, we describe all the different levels of crosstalk we have assessed.
+== First-order crosstalk ==
+In the first order crosstalk section we describe activation of [https://parts.igem.org/Part:BBa_R0062 pLux] due to [https://parts.igem.org/Part:BBa_C0062 LuxR] binding to inducers different from [[3OC6HSL|3OC6-HSL]] or [https://parts.igem.org/Part:BBa_R0062 pLux] itself binding a regulator-inducer pair different from [https://parts.igem.org/Part:BBa_C0062 LuxR]-[[3OC6HSL|3OC6-HSL]].
+==== First Level crosstalk: LuxR binds to different AHLs and activates the pLux promoter ====
+In the conventional system [[3OC6HSL|3OC6-HSL]] binds to its corresponding regulator, [https://parts.igem.org/Part:BBa_C0062 LuxR], and activates the [https://parts.igem.org/Part:BBa_R0062 pLux] promoter (figure 2, light blue). However, [https://parts.igem.org/Part:BBa_C0062 LuxR] can potentially bind also other [[AHL|AHLs]] and then activate [https://parts.igem.org/Part:BBa_R0062 pLux] (figure 2, [[AHL|3OC12-HSL]] in red and [[AHL|C4-HSL]] in green). This leads then to unwanted gene expression (crosstalk).
+[[File:ETH Zurich 1crosstalkPlux.png|400px|thumb|center| '''Figure 2 Overview of possible crosstalk of the [https://parts.igem.org/Part:BBa_C0062 LuxR]/[https://parts.igem.org/Part:BBa_R0062 pLux] system with three different [[AHL|AHLs]].''' Usually, [[3OC6HSL|3OC6-HSL]] binds to its corresponding regulator, [https://parts.igem.org/Part:BBa_C0062 LuxR], and activates the [https://parts.igem.org/Part:BBa_R0062 pLux] promoter (light blue). However, [https://parts.igem.org/Part:BBa_C0062 LuxR] may also bind [[AHL|3OC12-HSL]] (red) or [[AHL|C4-HSL]] (green) and then unintentionally activate [https://parts.igem.org/Part:BBa_R0062 pLux].]]
+==== Second Level crosstalk: other regulatory proteins, like LasR and RhlR, bind to their natural AHL substrate and activate the pLux promoter ====
+In the conventional system [[3OC6HSL|3OC6-HSL]] binds to its corresponding regulator, [https://parts.igem.org/Part:BBa_C0062 LuxR], and activates the [https://parts.igem.org/Part:BBa_R0062 pLux] promoter (figure 2, light blue). However, [https://parts.igem.org/Part:BBa_R0062 pLux] can potentially be activate by other regulators ([https://parts.igem.org/Part:BBa_R0062 LasR], [https://parts.igem.org/Part:BBa_R0062 RhlR]), binding their corresponding regulator (figure 3, [[AHL|3OC12-HSL]] in red, [[AHL|C4-HSL]] in green). This leads then to unwanted gene expression (crosstalk).
+[[File:ETH Zurich 2crosstalkPlux.png|thumb|400px|center|'''Figure 3 Overview of possible crosstalk of the [https://parts.igem.org/Part:BBa_C0062 LuxR]/[https://parts.igem.org/Part:BBa_R0062 pLux] system with two additional regulators ([https://parts.igem.org/Part:BBa_C0179 LasR] and [https://parts.igem.org/Part:BBa_C0171 RhlR]).''' Usually, [https://parts.igem.org/Part:BBa_C0062 LuxR] together with inducer [[3OC6HSL|3OC6-HSL]] activate their corresponding promoter [https://parts.igem.org/Part:BBa_R0062 pLux] (light blue). However, [https://parts.igem.org/Part:BBa_R0062 pLux] may also be activated by the [https://parts.igem.org/Part:BBa_C0179 LasR] regulator together with [[AHL|3OC12-AHL]] (red) or by the [https://parts.igem.org/Part:BBa_C0171 RhlR] regulator together with [[AHL|C4-AHL]] (green).]]
+== Second order crosstalk: Combination of both cross-talk levels  ==
+The second order crosstalk describes unintended activation of [https://parts.igem.org/Part:BBa_R0062 pLux] by a mixture of both the levels described above. The regulator and inducer are being different from [https://parts.igem.org/Part:BBa_C0062 LuxR] and [[3OC6HSL|3OC6-HSL]], respectively, and at the same time they do not belong to the same module. For example, the inducer [[AHL|C4-HSL]] (green), usually binding to the regulator [https://parts.igem.org/Part:BBa_C0171 RhlR], could potentially interact with [https://parts.igem.org/Part:BBa_C0179 LasR] regulator (red) and together activate [https://parts.igem.org/Part:BBa_R0062 pLux] (light blue). This kind of crosstalk is explained in figure 4.
+[[File:ETH Zurich 3crosstalkPlux.png|400px|thumb|center| '''Figure 4 Overview of possible crosstalk of the [https://parts.igem.org/Part:BBa_R0062 pLux] promoter with both the regulator and inducer being unrelated to the promoter and each other.''' Usually, [https://parts.igem.org/Part:BBa_C0062 LuxR] together with inducer [[3OC6HSL|3OC6-HSL]] activate their corresponding promoter [https://parts.igem.org/Part:BBa_R0062 pLux] (light blue). However, [https://parts.igem.org/Part:BBa_R0062 pLux] may also be activated by another regulator together with an unrelated inducer.  For example, the inducer [[AHL|C4-HSL]] (green) may interact with the [https://parts.igem.org/Part:BBa_C0179 LasR] regulator (red) and together activate [https://parts.igem.org/Part:BBa_R0062 pLux] (light blue).]]
+== Results ==
+{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"
+|+'''Table 1''' Crosstalk matrix for the promoter pLux ([https://parts.igem.org/Part:BBa_R0062:Experience BBa_R0062])
+|colspan="4" style='font-size:10pt';text-align:left|
+In all the measurements conducted to create this matrix the [https://parts.igem.org/Part:BBa_R0062 promoter pLux] was the basis and was induced in the six different variations shown.
+The dark blue points in the graph top left show the activation of gene expression when [https://parts.igem.org/Part:BBa_R0062 pLux] is induced by [[3OC6HSL|3OC6-HSL]] (Lux-AHL) binding to the corresponding [https://parts.igem.org/Part:BBa_C0062 LuxR regulator]. The observed transition occurs at a concentration of approximately 1 nM of [[3OC6HSL|3OC6-HSL]]. The light-blue curve plotted shows modeling data of [https://parts.igem.org/Part:BBa_R0062 pLux] induced by [[3OC6HSL|3OC6-HSL]] (Lux-AHL) binding to the corresponding [https://parts.igem.org/Part:BBa_C0062 LuxR regulator]. This curve from the model and the dark blue data points obtained from experiments were plotted as a reference in all the other graphs describing [https://parts.igem.org/Part:BBa_R0062 pLux]. Crosstalk can be observed for the cases where the [[AHL|3OC12-HSL]] (Las-AHL) binds the [https://parts.igem.org/Part:BBa_C0062 LuxR regulator].
+Additionally for [[AHL|3OC12-HSL]] binding to its corresponding [https://parts.igem.org/Part:BBa_C0179 regulator LasR] and then binding to the [https://parts.igem.org/Part:BBa_R0062 pLux] as seen in the middle of the top row and center of the matrix.
+For the case of Las-AHL binding the [https://parts.igem.org/Part:BBa_C0179 regulator LasR] and subsequently the [https://parts.igem.org/Part:BBa_R0062 promoter pLux], the transition occurs at 1 nM and reaches half the fluorescence as comared to [https://parts.igem.org/Part:BBa_R0062 pLux] induced by 3OC6-HSL binding [https://parts.igem.org/Part:BBa_C0062 LuxR]. In the case of [[AHL|3OC12-HSL]] binding [https://parts.igem.org/Part:BBa_C0062 LuxR] and inducing the promoter [https://parts.igem.org/Part:BBa_R0062 pLux], the transition is observed at approximately 100 nM and severe crosstalk is observed, showing that the ON-OFF-ratio is not significantly different from the reference curve.
+The experiments with [[AHL|C4-HSL]] have shown that there is no significant crosstalk with the [https://parts.igem.org/Part:BBa_C0062 LuxR regulator] and [https://parts.igem.org/Part:BBa_C0179 LasR regulator] binding C4-HSL and subsequently to [https://parts.igem.org/Part:BBa_R0062 pLux]. This is indicated on top right and middle right graphs. However, [https://parts.igem.org/Part:BBa_C0171 RhlR] induced with its corresponding inducer ([[AHL|C4-HSL]]) binds to [https://parts.igem.org/Part:BBa_R0062 pLux] and activates expression of GFP at about 100 nM.
+|-
+|style="width:200px"|[[File:ETH_Zurich_2014_qs-table_CornerLux.png|100px|link=https://parts.igem.org/Part:BBa_C0062:Experience]]
+|[[File:ETH_Zurich_2014_qs-table_3OC6-HSL.png|100px|link=https://parts.igem.org/Part:BBa_C0062:Experience]]
+|[[File:ETH_Zurich_2014_qs-table_3OC12-HSL.png|100px|link=https://parts.igem.org/Part:BBa_C0062:Experience]]
+|[[File:ETH_Zurich_2014_qs-table_C4-HSL.png|100px|link=https://parts.igem.org/Part:BBa_C0062:Experience]]
+|-
+|[[File:ETH_Zurich_2014_qs-table_LuxR.png|100px|link=https://parts.igem.org/Part:BBa_C0062:Experience]]
+|[[File:ETH_Zurich_2014_qs-table_PluxRef.png|200px]]
+|[[File:ETH_Zurich_2014_qs-table_PluxLuxRLasAHL.png|200px]]
+|[[File:ETH_Zurich_2014_qs-table_PluxLuxRRhlAHL.png|200px]]
+|-
+|[[File:ETH_Zurich_2014_qs-table_LasR.png|100px|link=https://parts.igem.org/Part:BBa_C0179:Experience]]
+|[[File:ETH_Zurich_2014_qs-table_PluxLasRLuxAHL.png|200px]]
+|[[File:ETH_Zurich_2014_qs-table_PluxLasRLasAHL.png|200px]]
+|[[File:ETH_Zurich_2014_qs-table_PluxLasRRhlAHL.png|200px]]
+|-
+|[[File:ETH_Zurich_2014_qs-table_RhlR.png|100px|link=https://parts.igem.org/Part:BBa_C0171:Experience]]
+|[[File:ETH_Zurich_2014_qs-table_PluxRhlRLuxAHL.png|200px]]
+|[[File:ETH_Zurich_2014_qs-table_PluxRhlRLasAHL.png|200px]]
+|[[File:ETH_Zurich_2014_qs-table_PluxRhlRRhlAHL.png|200px]]
+|}
+== Modelling Crosstalk==
+Each experimental data set was fitted to an Hill function using the Least Absolute Residual method. The results are shown in table 2.
+[[File:ETHZ_HillEq.png|center|200px]]
+<p>The fitting of the graphs was performed using the following equation :<br><br>
+rFluo = the relative fluorescence (absolute measured fluorescence value over OD) [a.u.]<br>
+a    = basal expression rate [a.u.] (“leakiness”)<br>
+b = maximum expression rate [a.u.] ("full induction")<br>
+n      = Hill coefficient (“cooperativity”)<br>
+K<sub>m</sub>  = Half-maximal effective concentration (“sensitivity”)<br>
+[AHL] = AHL concentration [nM]</p>
+<br clear="all"/>
+{| border="1" class="wikitable" style="margin: 1em auto 1em auto;text-align:center;"
+|+ '''Table 2''' Parameters of Hill Function for crosstalk with Plux (with 95% confidence bounds)
+!
+! [[3OC6HSL|3OC6-HSL]]
+! [[AHL|3OC12-HSL]]
+! [[AHL|C4-HSL]]
+|-
+![https://parts.igem.org/Part:BBa_C0062 LuxR]
+| a = 1043 (966.8, 1139) [a.u.]<br> n = 0.89 (0.79, 0.99)<br> Km = 0.1892 (0.1392, 0.2391) [nM]<br>b = 2.129e4 (2.065e4, 2.192e4) [a.u.]<br>
+| a = 900.5 (882, 918.9) [a.u.]<br>n = 1.049 (0.9976, 1.1)<br> Km = 78.32 (73.18, 83.47) [nM]<br> b = 1.866e4 (1.834e4, 1.899e4) [a.u.]<br>
+| No crosstalk
+|-
+! [https://parts.igem.org/Part:BBa_C0179 LasR]
+| No crosstalk
+| a = 362.4 (158.2,566.6) [a.u.]<br> n = 0.7898 (0.3241,1.256)<br>Km = 1.269 (0, 2.548) [nM]<br> b = 1.253e4 (1.085e4, 1.422e4) [a.u.]<br>
+| No crosstalk
+|-
+! [https://parts.igem.org/Part:BBa_C0171 RhlR]
+| a = 313.2 (296.8, 329.5) [a.u.]<br> n = 0.9427 (0.7565, 1.129)<br> Km = 1210 (565, 1855) [nM] <br> b = 8361 (7001, 9722) [a.u.] <br>
+| No crosstalk
+| a = 309.6 (283.6, 335.6) [a.u.]<br> n = 0.741 (0.6463, 0.8357) <br> Km = 2738 (1411, 4064) [nM] <br> b = 1.22e4 (1.045e4,1.45e4) [a.u.] <br>
+|}
+|}
+<!-- DON'T DELETE --><partinfo>BBa_R0062 EndReviews</partinfo>
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<!-- DON'T DELETE --><partinfo>BBa_R0062 AddReview number</partinfo>
+<I>ETH Zurich 2013</I>
+|width='60%' valign='top'|
+===Characterization of the pLuxR dose response to OHHL by plate reader analysis and single cell analysis===
+<b>Fluorescence data analysis</b>
+[[File:300-Formulapluxr1.png|left|300px]]
+<p>The fitting of the following graphs was performed using this equation :<br><br>
+Output = eGFP levels [au]<br>
+Top    = maximal eGFP level [au]("full induction")<br>
+Bottom = minimal eGFP level [au](“leakiness”)<br>
+n      = Hill coefficient (“cooperativity”)<br>
+EC<sub>50</sub>   = Half-maximal effective concentration (“sensitivity”)<br>
+[AHL]=OHHL concentration [nM]</p>
+<br clear="all"/>
+<b> Primary characterization of the dose reponse curve by microtiter plate analysis</b>
+[[File:Wtg1platereader.jpg|left|400px|thumb|<b>Figure 1. Sensitivity curve of the wild type promoter</b> analyzed on the Tecan Infinity M200 microtiter plate reader. The OHHL concentration range is plotted against the standarized fluorescence (au).<br> EC<sub>50</sub>=5.86, R<sub>2</sub>= 0.87,n=1.7<br>.All assays were carried out in triplicates, results are presented as mean &plusmn; standard deviation.]]
+<br><br>We cloned the promoter BBa_R0062 in the [https://parts.igem.org/Part:BBa_J09855 BBa_J09855] construct and cloned a eGFP gene as reporter. The eGFP part allows us to make fluorescence measurement in microtiter plate experiment as well as in single cell analysis to characterize the promoter depending on [OHHL], by recording the fluorescence [au]. According to literature
+[http://www.ncbi.nlm.nih.gov/pubmed/18760602 (Geske G.D. Evaluation of a focused library of N-aryl L-homoserine lactones reveals a new set of potent quorum sensing modulators)]we could define the first experimental OHHL ranges to induce the promoter. After some fine tunning and adjusting experiment we could narrow down the ranges to obtain a dose response curve in a reasonable resolution for the BBa_R0062 promoter to the OHHL (3-oxo-hexanoyl-l-homoserine-lactone). See Figure 1.<br>
+<br clear="all"/>
+<b>Single cell characterization of the dose response in liquid culture and on agar plate</b> <br><br>
+<p>To obtain high quality data we did single cell analysis over the  range defined above. Obviously the EC<sub>50</sub> changed compared to the microtiter plate data.<br> See Figure 2.</p>
+[[File:facs data.jpg|700px|center|thumb|<b>Figure 2. Dose response comparison between [OHHL] induction in liquid culture and on agar plates</b>, of the BBa_J09855 construct using the BBa_R0062 promoter and a eGFP gene as reporter</b> analyzed at different [OHHL] in the single cell analysis. The black curve shows the response of to OHHL in liquid culture, the orange curve shows the response to OHHL on agar plates. All assays were carried out in duplicates, results are presented as mean &plusmn; standard deviation.]]
+<br clear="all"/>
+[[File:AgaLiWT.jpg|400px|left|thumb|<b>Figure 3: Sensitivity curve of the wild type pluxR in agar plates and liquid culture</b> For the liquid culture we got :EC<sub>50</sub>=0.02nM, R<sup>2</sup>=0.84, n=1.7; For the agar plates we got :EC<sub>50</sub>=4.45nM, R<sup>2</sup>=0.80, n=1.7<br> The black curve shows the response of to OHHL in liquid culture, the orange curve shows the response to OHHL on agar plates. All assays were carried out in duplicates, results are presented as mean &plusmn; standard deviation.]]
+To obtain high quality data we did single cell analysis over the dose response range defined above. Obviously the EC<sub>50</sub> changed compared to the plate reader data. <i>Figure 2</i>. The plot shows the dose reponse curve of the promoter depending on AHL concentration. We performed this experiment in liquid culture as well as on agar plates (OHHL was added to the melted 1.5% agar), which was closer to our project. We observed a 220 time shift in the dose response between the liquid culture single cell analysis  and the one in agar plates. See <i>Figure 3</i>
+|}
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+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_C0062 AddReview 5</partinfo>
+<I>SUN(Tsinghua)</I>
+|width='60%' valign='top'|
+Part was sequenced and functional.
+|};
 {|width='80%' style='border:1px solid gray'
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-In order to optimize the function of the lux promoter, We creat a hybrid promoter ([BBa_K737067])which combines plux with OmpR, and in this case, plux can be led to expression by HSL and obtain by phosphorylation of OmpR protein..
+In order to optimize the function of the lux promoter, We creat a hybrid promoter [[Part:BBa_K737067|BBa_K737067]] which combines plux with OmpR, and in this case, plux can be led to expression by HSL and obtain by phosphorylation of OmpR protein..
 According to the research of it before, we camp up with a program . Keep the sequencing of Lux Box, -35box and -10box., Then put the sequencing of C3 point of OmpR promoter into -35box and -10box.
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 ==Site-directed mutagenesis at position 3,5 and 3/5 of BBa_R0062==
 On the basis of the nucleotide sequence of the lux pR promoter, the 20 base pair inverted repeat ACCTGTAGGA TCGTACAGGT might consititude a protein binding site. And we also learned that mutagenesis at position 3 and position 5 might cause dramatic change on the expression of downstream gene. Therefore, we generated 3 mutants (BBa_K658006 BBa_K658007 BBa_K658008) of the promoter lux pR by site-directed mutagenesis at position 3, 5 and 3/5.
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 |};
+<partinfo>BBa_R0062 AddReview 5</partinfo>
+<I>Tsinghua-A 2017</I>
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_R0062 AddReview 5</partinfo>
+|width='60%' valign='top'|
+=Crosstalk between Plux and Nine AHL-Receptor Combinations=
+==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
+https://static.igem.org/mediawiki/2017/thumb/e/ea/TsinghuaA-Project_1.png/320px-TsinghuaA-Project_1.png
+(Fig. 1: Results of our crosstalk experiment)
+OC6HSL-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:
+. Transform MG1655ΔsdiAΔlacI with 9 kinds of combinations of plasmids mentioned above.
+. 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.
+. Dilute the overnight culture to 1/50 in fresh LB medium (3ml) containing 50 microgram/ml Ampicillin and 30 microgram/ml Kanamycin.
+. Incubate the fresh cultures at 37℃ until OD600 reaches 0.2.
+. 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℃.
+. 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.
+===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===
+https://static.igem.org/mediawiki/2017/2/2a/T--TUST_China--part_Experiment_Design.png
+plux4:  https://parts.igem.org/Part:BBa_K2267031
+plux3:  https://parts.igem.org/Part:BBa_K2267030
+plux1:  https://parts.igem.org/Part:BBa_K2267029
+plux6:  https://parts.igem.org/Part:BBa_K2267032
+plux8:  https://parts.igem.org/Part:BBa_K2267046
+===III 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
+===IV Results===
+The activated LuxR regulator binds to a LuxR-inducible promoter, pLux, upstream of a GFP reporter. As a result, GFP is expressed when the receiver device is induced with 3O-C6 AHL. We designed this part to characterize the activation range of the Lux receiver device.
+https://parts.igem.org/Part:BBa_K2267040  is Control，
+we did point mutation on https://parts.igem.org/Part:BBa_R0062 and the exact spots are
+plux1: https://parts.igem.org/Part:BBa_K2267029
+plux3: https://parts.igem.org/Part:BBa_K2267030
+plux4: https://parts.igem.org/Part:BBa_K2267031
+plux6: https://parts.igem.org/Part:BBa_K2267032
+plux8: https://parts.igem.org/Part:BBa_K2267046
+listed below.
+We linked gfp reporter genes
+luxr-plux1-gfp: https://parts.igem.org/Part:BBa_K2267043
+luxr-plux3-gfp: https://parts.igem.org/Part:BBa_K2267041
+luxr-plux4-gfp: https://parts.igem.org/Part:BBa_K2267042
+luxr-plux6-gfp: https://parts.igem.org/Part:BBa_K2267044
+luxr-plux8-gfp: https://parts.igem.org/Part:BBa_K2267045
+on them respectively to test the function of modified promoters.
+We characterised the activation range of this device using a GFP reporter https://parts.igem.org/Part:BBa_K319039. The results of our characterisation experiments can be found here
+https://static.igem.org/mediawiki/2017/e/e6/T--TUST_China--part_part_results.png
+|};
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Difference between revisions of "Part:BBa R0062:Experience"

Latest revision as of 03:38, 2 November 2017

Applications of BBa_R0062

User Reviews

Background information

Experimental Set-Up

Characterization of the promoter's basal leakiness with LuxR in the absence of 3OC6-HSL

Results

Characterization of the promoter's sensitivity to 3OC6-HSL depending on LuxR concentration

Results

Characterization of crosstalk

Background information

First-order crosstalk

First Level crosstalk: LuxR binds to different AHLs and activates the pLux promoter

Second Level crosstalk: other regulatory proteins, like LasR and RhlR, bind to their natural AHL substrate and activate the pLux promoter

Second order crosstalk: Combination of both cross-talk levels

Results

Modelling Crosstalk

Characterization of the pLuxR dose response to OHHL by plate reader analysis and single cell analysis

Measurement of pLux/OmpR hybrid promoter's expression property

Site-directed mutagenesis at position 3,5 and 3/5 of BBa_R0062

Lux pR strength testing device

Application----a series of population-control devices

Crosstalk between Plux and Nine AHL-Receptor Combinations

I Background

II Results

III Experiment Design and Measurement Methods

Background information

Experiment Design

III Methods

IV Results