Part:BBa_K2357000
LasR Receiver
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 966
Illegal NheI site found at 989 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 838
Illegal NgoMIV site found at 1342
Illegal AgeI site found at 1539 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 754
Short Description
The receiver is designed to be inserted into a modular receiver vector pSB1C3 with an inducible promoter, a constitutive promoter, a green fluorescent protein (GFP) 2 ribosomal binding sites (RBS)- one for the Las regulator protein and one for a GFP.
System Introduction
The Las Receiver system comes from the bacterial strand Pseudomonas aeruginosa [3]. Pseudomonas aeruginosa is a common Gram-negative rod-shaped bacteria that is multidrug resistant pathogen recognized for its ubiquity and its intrinsically advanced antibiotic resistance mechanisms. According to Allesen, this bacterium is able to produce extracellular DNA to function in cell-to-cell communication in the creation of biofilms. In addition, it also employs two other interconnected quorum sensing systems aside from Las: rhIRI and PQS.
The LasR gene was characterized in a study done by the University of Rochester School of Medicine and Dentistry where they reported the LasR gene positively regulated the expression of elastase in Pseudomonas aeruginosa [2]. In addition, it is suspected to be an activator of E. coli. A ribosomal binding site was found 10 to 11 nucleotides upstream from the start of translation. In addition, in E. coli., the proteins were predicted to be hydrophilic [2]. The LasR receiver was used in a variety of studies through this iGEM experience. Studies included determining GFP expression with a variety of sender concentrations, determining GFP expression with a line up of synthetic AHLs, and plate inductions.
AHL quorum sensing functions within two modules. The first module, the "Sender," must be induced by certain environmental conditions, usually population density of surrounding organisms. This will begin production of the AHL by an AHL synthase, which is then detected by the second module, the "Receiver." The Receiver will cause the expression or silencing of certain genes to achieve the desired purpose of the communication, whether it is the production of GFP or to increase growth rate. For our system, the receiver LasR will cause expression of gene through the production of GFP.
A major difference between this year's receivers to ASU iGEM 2016 team's receivers is the orientation and the inducible promoter. The receiver system is divided into two subsections: the GFP protein and the regulator protein. In 2016, the regulator protein was placed before the GFP protein
In 2017, the ASU iGEM team switched the orientation to have the GFP protein components first, and the regulator components second, as displayed in a pictorial diagram below:
The second difference is the inducible promoter used within the Las receiver. A hybrid promoter with a replaced lux-box from the commonly used from PluxI promoter was replaced with the las-box that contained its respective receptor binding site.
Las System
There are three subsections within our data for this year's iGEM team. The first sub section included data on natural sender combinations with the receiver system Las; the second is synthetic AHL signals with our respective receiver system; and lastly, a subsection on induction plates for GFP production and determining how fast or slow these senders are communicating with the receiver system Las.
A sub section of our data was determining induction plates for the GFP production with various senders on the Las Receiver. The data allows us to have the 3D analysis of the Las receiver with these senders. In addition, it allowed our team to determine how fast the senders moved over a certain amount of time with this receiver system.
GFP Expression of LasR with Natural Sender Combinations
The first set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.
Below we see an experiment where the AubI expresses higher when mixed with 10% of any other sender, these results are with the Las receiver. This evidence further confirms that AubI simply works best when mixed versus being used alone. The 40% AubI mixed with 10% negative sender, 10% EsaI and 10% CerI all expressed higher than the 50% AubI alone.
Below, another test showed some notable results. As seen clearly in the last graph, the BraI showed a higher expression when mixed with 10% of a second sender (even when that sender was a negative control sender). The 10% BraI + 40% AubI mixed expressed higher than the 50% AubI by itself. Close behind the 50% AubI was the 50% LasI. Interesting result because the AubI expresses higher than the matching sender LasI to its own LasR receiver.
Below, another test showed some notable results. As seen clearly in the last graph, the 40% AubI showed a higher expression when mixed with 10% of a second sender (even when that sender was a negative control sender). The 10% BjaI + 40% AubI mixed expressed higher than the 50% AubI by itself. Interesting result because the AubI expresses higher when mixed with another sender, seemingly with both LuxR and LasR.
BraI barely induces the Las receiver. May be orthogonal (below).
AubI expresses higher than the LasI with the LasR (below)
GFP Expression of LasR with Synthetic AHLs
The following graphs show the maximum (DGFP/OD)/DT. These points determine where the GFP is changing the fastest, and represents the maximum rate of change at a specific synthetic AHL concentration.
The graph displayed below is a combination of different sender concentrations of LasI in respect to the GFP expression of LasR. It is interesting to note that the corresponding sender and receiver system, Las, does not express as highly in GFP expression as other experiments with different senders. Our group hypothesized that this sender/receiver system would still promote a higher GFP expression, even at a more diluted concentration of synthetic AHL
The following graphs show the maximum (DGFP/OD)/DT. These points determine where the GFP is changing the fastest, and represents the maximum rate of change at a specific synthetic AHL concentration. The first graph on the top left-hand side displayed below is a combination of different sender concentrations of LasI in respect to the GFP expression of LasR. It is interesting to note that the corresponding sender and receiver system, Las, does not express as highly in GFP expression as other experiments with different senders. Our group hypothesized that this sender/receiver system would still promote a higher GFP expression, even at a more diluted concentration of synthetic AHL.
The graph on the top right-hand side depicts the GFP expression Las receiver induced with the Lux sender AHL. A notable result that can be concluded from this graph is that from concentration 1E-14M to concentration 1E-5M, there is very minimal GFP expression then a sharp increase of expression for the 1E-4M concentration. This is very unusual, as it is expected that as concentration increases, so does the GFP expression. This is also serves as an interesting comparison with the Lux receiver and Las AHL induction, as there was an overall high GFP expression across all concentrations.
The graph on the lower left-hand side depicts the relationship between the Las receiver and the Rhl AHL. As shown, there is a steady state of induction in GFP expression as the AHL concentration increases. This data could be useful to researchers hoping to express GFP with a limited amount of Rhl sender, and get the same results as seen in a higher concentration.
The following graph on the bottom right-hand side shows the maximum GFP expression of LasR for each corresponding AHL concentration of Rpa. There is a steady state of GFP expression from concentrations 1E-14M through 1E-7M. Then as the concentrations increase, there is also an increase in GFP expression, indicating the 1E-4M concentration of Rpa AHL to be the highest inducer of LasR for maximum GFP expression. This would be a beneficial system for researchers looking to use the Las receiver and obtain the highest GFP expression.
In the graph on the left-hand side, the maximum GFP expression of LasR and Tra AHL is depicted. From concentrations 1E-14M to 1E-9M, GFP expression is very minimal it is as expected that there is some induction despite the AHL being at low concentrations. Then beginning at 1E-8M, GFP expression increases with increasing concentration furthering the hypothesis that GFP expression is greater at higher concentrations.
A notable result from this induction of Las receiver with Sin AHL is a steady state of GFP expression from 1E-14M concentration to 1E-10M concentration. Then the GFP expression begins to rise and peaks, indicating that at 1E-7M, the GFP expression as at an overall maximum. This is an interesting occurrence, as 1E-4M would be expected to have the highest GFP expression, and not a lower concentration. This could be especially useful for researchers looking for a lower concentration of Sin sender in their circuit, and need a high GFP expression at a single concentration.
Induction Tests on LasR
This data depicts the 3D analysis of the Las receiver with each the various senders tested. This data was used to calculate the modeling data (induction rates) that can be seen on the teams Modeling page. The imaging for the Las receiver was done from 0-40 hours
Velocity (cm/hr) versus Time (Hrs) displaying how fast the senders AubI, BjaI, BraI, CerI, EsaI, LuxI, LasI, -Sender, RhlI, RpaI, SinI moved over the course of 40hrs. Each dot represents when imaging was performed with the Gene Sys imaging system. The velocity is the rate between each individual imaging session for example the rate at 5 hours would be based on how fast the receiver was induced since the last imaging point at 3 hrs.
Distance (cm) versus Time (Hrs) displaying how far the senders AubI, BjaI, BraI, CerI, EsaI, LuxI, LasI, -Sender, RhlI, RpaI, SinI moved over the course of 40hrs. Each dot represents when imaging was performed with the Gene Sys imaging system.
Safety
This section aims to provide safety information and suggestions about the LasR and sender combination disposal. The greatest concern from this part is the activation of pathogens via crosstalk. According to Integrated Device Technologies, quorum sensing genes are not considered dangerous by themselves, as they do not directly cause the creation of a new pathogenic strain. They may contribute to pathogenicity, but so do synthetic promoters. So, the acurate disposal of these systems are our main concern to focus on.
This graph shows the 70% EtOH treatment for the Las AHL where the AHL was dissolved in Ethyl Acetate, treated with 70% EtOH (15 minutes) left to evaporate and resuspended in Ethyl Acetate and then imaged over the course of 10 hrs.
This graph shows the Ethyl Acetate control for the Las AHL where the AHL was dissolved in Ethyl Acetate, left to evaporate and resuspended in Ethyl Acetate and then imaged over the course of 10 hrs. This control was to serve as a procedural control to see if the process of allowing the AHL's to sit in a 37 degree Celsius environment till their solvent dissolved and then were resuspended would cause the degradation or not and how that related to the EtOH and 2-propanol treatments.
This graph shows the Freezer control for the Las AHL where the AHL was dissolved in Ethyl Acetate, left in the freezer and then imaged over the course of 10 hrs. This control was to serve as a procedural control to see if the process of allowing the AHL's to sit in a 4 degree Celsius environment till the induction plate was ready to be imaged how that related to the EtOH and 2-propanol treatments. These AHL's were not placed in the plate till just before the imaging started and all other treatments were added to the plates.
These graphs below shows the data for the autoclave treatment for the Las supernatant where the Las AHL was dissolved in ethyl acetate and subjected to the default setting (15 minutes) for the autoclave device and then placed in an overnight induction plate and imaged for 8 hours.
References
- [1] Allesen-Holm, M., et al. "A Characterization of DNA Release in Pseudomonas Aeruginosa Cultures and Biofilms." Mol Microbiol 59.4 (2006): 1114-28. Print.
- [2] Gambello, M J, and B H Iglewski. “Cloning and Characterization of the Pseudomonas Aeruginosa lasR Gene, a Transcriptional Activator of Elastase Expression.” Journal of Bacteriology 173.9 (1991): 3000–3009. Print.
- [3] Pesci, E C et al. “Regulation of Las and Rhl Quorum Sensing in Pseudomonas Aeruginosa.” Journal of Bacteriology 179.10 (1997): 3127–3132. Print.
| None |