Part:BBa_K2357028
TraR Receiver
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 937
Illegal NheI site found at 960 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 9
Illegal BamHI site found at 1513 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 809
Illegal AgeI site found at 1094
Illegal AgeI site found at 1393 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 725
Short Description
This 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 receiver system of TraR is an Agrobacterium transcriptional regulator whose activity requires the pheromone N-3-oxooctanoyl-l-homoserine lactone (Zhu). An Agrobacterium is a Gram-negative bacteria that causes tumors in plants. It uses horizontal gene translation. It has a unique ability to transfer its DNA to plants, which is useful for genetic engineering. In addition, TraR is a Lux-R type transcription factor. It regulates genes for replication and conjucation for tumor-inducing plasmids in plants (Costa).
The TraR binds to the pheromone 3-oxo-octanoylhomoserine lactone (OOHL) and requires this molecule for folding into a protease-resistant, soluble conformation. This TraR protein is used within our receiver systems to transcribe those proteins. Although, we are not testing the protein itself to see if it does promote tumor production in plants, it is used within our system to determine if there are any orthogonal AHL signals. We a looking a designing genetic circuits for quorum sensing for genetic engineers and synthetic biologist to use for the production of proteins.
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 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 as displayed below:
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.
Tra 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 TraR; 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 TraR.
GFP Expression of TraR 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.
This test showed some notable results. As seen in the last graph, the AubI and the EsaI expressed much higher than the LuxI. This is a result that does not match other tests performed by the team. This was also not a result that was able to be replicated and it is thought that there may have been an issue with the LuxI that caused it to not induce the TraR.
The TraR receiver is orthogonal with most senders, even the TraI synthetic AHL barely induces its own receiver. The only two senders that induced the Tra receiver (aside from the TraR synthetic AHL) within a noticeable range, was the AubI and the EsaI.
GFP Expression of TraR with Synthetic AHLs
In the graph displayed below, it shows the GFP expression of various concentration of synthetic AHL Rhl with the receiver system TraR.
An interesting comment on this particular experiment was that after the first dilution with a GFP expression in arbitrary units of around 20 the expression of GFP is negligible. This could be an indication of orthogonality between the receiver TraR and RhlI sender. Within our experiments, we implied that orthogonality is defined as not expression or having no communication within the systems. This might be an important find for synthetic biologist and genetic engineering to use this orthogonal quorum sensing circuit within their genetic circuit to be able to, with confidence, produce results of a protein or phenotypic expression.
Another interesting result was the with the synthetic Rpa AHL. As depicted in the graph, it shows an almost steady state of GFP production, independent of the concentration of the AHL signal. This might be a useful finding if researchers wanted to use a smaller amount of signal due to limiting resources of RpaI sender. However, it could be an orthogonal pathway as the baseline is very close to zero and had overall minimal GFP expression.This graph depicts the relationship between Tra receiver and synthetic Sin AHL. A notable comment that can be made from this graph is that overall highest GFP production occurred at the 1E-4M concentration. While it was the highest, it only reached 3 arbitrary units, indicating that induction was very low for this system. However, it does not indicate orthogonality, as there was some induction that occurred no matter how small.An interesting comment on this induction, was the minimal overall expression of GFP. As seen with LasR and LuxR and they're corresponding senders, they had an overall high GFP expression of more than 5 arbitrary units. Here, it is not the case as the highest concentration of 1E-4M only reaches 6 arbitrary units.In summary, the Tra receiver appeared to be low inducing as the highest concentration only reached 1E-4M. As seen in the above graphs, there was little to no GFP expression with the AHLs tested in the lower concentrations. This could be a clear indicator to researchers that Tra receiver could be useful when striving for a low expression of GFP in their system and paired with any AHL tested.These results are showed within the graphs below
Induction Tests on TraR
Velocity (cm/hr) versus Time (Hrs) displaying how fast the senders AubI,BjaI, BraI, CerI, EsaI, 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, 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 above shows the 3D analysis of TraR and the various senders that were tested. This image is at 16 hrs and was done with the genesys imaging system and the analysis of distance was done in Adobe Illustrator. Each image shows both the strength of induction (vertical direction not quantified) and the distance that each sender was able to induce the Tra receiver.
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.
In the following two graphs, it shows the two treatment methods were used for the disposal of our materials.This graph shows the 70% EtOH treatment for the Tra 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
References
- Costa, E. D., Y. Chai, and S. C. Winans. "The Quorum-Sensing Protein Trar of Agrobacterium Tumefaciens Is Susceptible to Intrinsic and Tram-Mediated Proteolytic Instability." Mol Microbiol 84.5 (2012): 807-15. Print.
- Zhu, Jun, and Stephen C. Winans. “Autoinducer Binding by the Quorum-Sensing Regulator TraR Increases Affinity for Target Promoters in Vitro and Decreases TraR Turnover Rates in Whole Cells.” Proceedings of the National Academy of Sciences of the United States of America 96.9 (1999): 4832–4837. Print.
| None |