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 to 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 https://parts.igem.org/Part:BBa_J23109 BBa_J23109] (weakest one chosen), with https://parts.igem.org/Part:BBa_J23111 BBa_J23111] (medium) 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 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 https://parts.igem.org/Part:BBa_J23109 BBa_J23109] (weakest one chosen) the sensitivity is lowest (EC₅₀ approximately 100 pM), with https://parts.igem.org/Part:BBa_J23111 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 Influence of varied promoter strength on the expression of LuxR (BBa_C0062) and the resulting effect on pLux (BBa_R0062). 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 two-level crosstalk on the promoter

Background information

System considered

Modeling crosstalk

First-order crosstalk

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

Second Level crosstalk: other regulatory proteins, like LasR, bind to their natural HSL substrate and activates the promoter

Second order crosstalk: Combination of both cross-talk levels

Other regulatory proteins, like LasR, bind to different HSL and activates the promoter

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 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 0.5 fold the fluorescence as 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, meaning that the ON-OFF-ratio is not significantly different from the reference curve. Observation of C4-HSL has 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.

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

SUN(Tsinghua)

Part was sequenced and functional.

iGEM12_OUC-China

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.

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 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.

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])

Antiquity

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

wmholtz

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

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