Difference between revisions of "Part:BBa K2033006"
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The CerI part arises from the soil bacterium Rhodobacter sphaeroides. The designed part by Ryan Muller was cloned into competent DH5AT E. coli cells. These were ligated into the psB1C3 vector and plated, as well as sequenced with help from ASU's Core Laboratory. | The CerI part arises from the soil bacterium Rhodobacter sphaeroides. The designed part by Ryan Muller was cloned into competent DH5AT E. coli cells. These were ligated into the psB1C3 vector and plated, as well as sequenced with help from ASU's Core Laboratory. | ||
| − | <div style="text-align: center;">[[File:T--Arizona State-- | + | <div style="text-align: center;">[[File:T--Arizona State--cersequence1.png|600px]]</div> |
| − | <div style="text-align: center;">[[File:T--Arizona State-- | + | <div style="text-align: center;">[[File:T--Arizona State--cersequence2.png|600px]]</div> |
An optical density test was conducted on the produced CerI construct to determine if the AHL is produced. The plate reader ran an 8-hour read from 580-610nm, indicating the presence of the mCherry fluorescent molecule. The AHL gene lies upstream of the mCherry gene, so successful production of mCherry is a good indicator that the AHL molecule is being produced. A positive growth curve was found for the CerI construct over the 8-hour read. Overall, mCherry production increased over time, suggesting that the CerI Synthase had been produced in E. coli. | An optical density test was conducted on the produced CerI construct to determine if the AHL is produced. The plate reader ran an 8-hour read from 580-610nm, indicating the presence of the mCherry fluorescent molecule. The AHL gene lies upstream of the mCherry gene, so successful production of mCherry is a good indicator that the AHL molecule is being produced. A positive growth curve was found for the CerI construct over the 8-hour read. Overall, mCherry production increased over time, suggesting that the CerI Synthase had been produced in E. coli. | ||
<div style="text-align: center;">[[File:T--Arizona State--CERRFP.png]]</div> | <div style="text-align: center;">[[File:T--Arizona State--CERRFP.png]]</div> | ||
| + | |||
| + | Gas chromatography was also done on the E. coli cultures to confirm production of the AHL molecule by the E. coli chassis. These tests are still in progress and will be completed at a later date. | ||
| + | |||
| + | Finally, the ability of the Cer AHL to induce the well-characterized Receiver Device Bba_F2620 was tested. F2620 relies on the LuxR part to output PoPS and GFP production. First, a visual induction was performed by plating the AHL Sender in the center of the plate with a GFP positive control, negative receiver control and F2620. As shown below, Cer is able to induce F2620, as some colonies in the top right section began producing GFP. This indicates crosstalk occurred between the two systems. | ||
| + | <div style="text-align: center;">[[File:T--Arizona_State--cerplate.png]]</div> | ||
| + | <div style="text-align: center;">Plate with GFP+(top left), Sender(center), -Receiver(bottom) and F2620(top right)</div> | ||
| + | |||
| + | The figure below compares CerI at 10% and 50% concentrations compared with the natural AHL synthase, LuxI at 10% and 50% concentrations. CerI is shown to induce F2620 strongly. This suggests that the Cer system will crosstalk with Lux and F2620. | ||
| + | <div style="text-align: center;">[[File:T--Arizona_State--cerind.png]]</div> | ||
| + | <div style="text-align: center;">GFP absorbance from CerI over time</div> | ||
===Safety=== | ===Safety=== | ||
| Line 46: | Line 56: | ||
====Disposal==== | ====Disposal==== | ||
| − | In order to properly dispose of 3OH-7-cis-C14-HSL, the sample should be autoclaved. This AHL does not possess a beta-ketone group in the acyl tail, and so, bleach is not capable of effectively degrading it. Further details about proper AHL disposal can be found here: http://2016.igem.org/Team:Arizona_State/WhitePaper. | + | In order to properly dispose of 3OH-7-cis-C14-HSL, the sample should be autoclaved. This AHL does not possess a beta-ketone group in the acyl tail, and so, bleach is not capable of effectively degrading it. This was tested via a differential bleach treatment and F2620 inductions were measured over an 8-hour period. As shown below, bleach decreased F2620 inductions, but did not completely eliminate the ability of the AHL to induce F2620. This is consistent with the results found by SA Borchardt, which found that non-3-oxo AHLs would not be degraded by bleach. |
| + | <div style="text-align: center;">[[File:T--Arizona State--cerbleachgraph.png]]</div> | ||
| + | |||
| + | The effect of autoclaving was also tested on the Cer AHL, as this is a standard EH&S sanitation protocol. The ability of AHLs after autoclaving to induce F2620 was tested. The figure below supports the nearly complete deactivation of the 3OH-7-cis-C14-HSL. The extreme pressures and temperatures generated by the autoclave are more than enough to destroy the vast majority of AHLs in the solution. | ||
| + | <div style="text-align: center;">[[File:T--Arizona State--cerautoclavegraph1.png]]</div> | ||
| + | Further details about proper AHL disposal can be found here: http://2016.igem.org/Team:Arizona_State/WhitePaper. | ||
| + | |||
====Other Considerations==== | ====Other Considerations==== | ||
| Line 52: | Line 68: | ||
===References=== | ===References=== | ||
| − | (1) | + | (1) Hwang W. "Genes of Rhodobacter sphaeroides 2.4.1 regulated by innate quorum-sensing signal, 7,8-cis-N-(tetradecenoyl) homoserine lactone." J Microbiol Biotechnol. 18.2.(2008) 219-27. |
| + | (2) Borchardt, S.A., Allain, E.J., Michels, J.J. “Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials” Applied and Environmental Microbiology. 67.7. (2001); 3174-3179. | ||
Latest revision as of 08:10, 27 October 2016
3OH-7-cis-C14-HSL Sender Device-CerI
This synthase is produced by the bacteria Rhodobacter sphaeroides and interacts with the receiver molecule CerR.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Short Description
This part produces the AHL quorum sensing molecule 3OH-7-cis-C14-HSL ((Z)-3-hydroxy-N-[(3S)-2-oxooxolan-3-yl]tetradec-7-enamide). This AHL synthase is designed to be inserted into a modular sender vector BBa_K2033011 with a constitutive Tet promoter, 2 ribosome binding sites (RBSs), an RFC10 prefix and mCherry.
Cer System
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 the cell, which is then detected by the second module, the "Receiver." Once a certain threshold of AHLs is breached, 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.
The Cer system originates from the aquatic proteobacteria Rhodobacter sphaeroides. It produces an 3OH-7-cis-C14-HSL, also known as ((Z)-3-hydroxy-N-[(3S)-2-oxooxolan-3-yl]tetradec-7-enamide). The structure is shown below:
This AHL notably has an unsaturated acyl tail, forming a Z-orientation double bond, which will serve as a unique binding domain for the transcription factor.
The CerI part arises from the soil bacterium Rhodobacter sphaeroides. The designed part by Ryan Muller was cloned into competent DH5AT E. coli cells. These were ligated into the psB1C3 vector and plated, as well as sequenced with help from ASU's Core Laboratory.
An optical density test was conducted on the produced CerI construct to determine if the AHL is produced. The plate reader ran an 8-hour read from 580-610nm, indicating the presence of the mCherry fluorescent molecule. The AHL gene lies upstream of the mCherry gene, so successful production of mCherry is a good indicator that the AHL molecule is being produced. A positive growth curve was found for the CerI construct over the 8-hour read. Overall, mCherry production increased over time, suggesting that the CerI Synthase had been produced in E. coli.
Gas chromatography was also done on the E. coli cultures to confirm production of the AHL molecule by the E. coli chassis. These tests are still in progress and will be completed at a later date.
Finally, the ability of the Cer AHL to induce the well-characterized Receiver Device Bba_F2620 was tested. F2620 relies on the LuxR part to output PoPS and GFP production. First, a visual induction was performed by plating the AHL Sender in the center of the plate with a GFP positive control, negative receiver control and F2620. As shown below, Cer is able to induce F2620, as some colonies in the top right section began producing GFP. This indicates crosstalk occurred between the two systems.
The figure below compares CerI at 10% and 50% concentrations compared with the natural AHL synthase, LuxI at 10% and 50% concentrations. CerI is shown to induce F2620 strongly. This suggests that the Cer system will crosstalk with Lux and F2620.
Safety
This section aims to provide safety information and suggestions about the CerI part. The greatest concern from this part is the activation of pathogens via crosstalk. According to Integrated DNA 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 actual AHL molecules are the chief concern.
Crosstalk Partners
CerI produces 3OH-7-cis-C14-HSL, which is known to have 4013 Biosystem pathways in quorum sensing, as shown https://pubchem.ncbi.nlm.nih.gov/compound/5281979#section=InChI. With this level of characterization, potential crosstalk partners are well understood.
Disposal
In order to properly dispose of 3OH-7-cis-C14-HSL, the sample should be autoclaved. This AHL does not possess a beta-ketone group in the acyl tail, and so, bleach is not capable of effectively degrading it. This was tested via a differential bleach treatment and F2620 inductions were measured over an 8-hour period. As shown below, bleach decreased F2620 inductions, but did not completely eliminate the ability of the AHL to induce F2620. This is consistent with the results found by SA Borchardt, which found that non-3-oxo AHLs would not be degraded by bleach.
The effect of autoclaving was also tested on the Cer AHL, as this is a standard EH&S sanitation protocol. The ability of AHLs after autoclaving to induce F2620 was tested. The figure below supports the nearly complete deactivation of the 3OH-7-cis-C14-HSL. The extreme pressures and temperatures generated by the autoclave are more than enough to destroy the vast majority of AHLs in the solution.
Further details about proper AHL disposal can be found here: http://2016.igem.org/Team:Arizona_State/WhitePaper.
Other Considerations
No other safety information is available for 3OH-7-cis-C14-HSL.
References
(1) Hwang W. "Genes of Rhodobacter sphaeroides 2.4.1 regulated by innate quorum-sensing signal, 7,8-cis-N-(tetradecenoyl) homoserine lactone." J Microbiol Biotechnol. 18.2.(2008) 219-27. (2) Borchardt, S.A., Allain, E.J., Michels, J.J. “Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials” Applied and Environmental Microbiology. 67.7. (2001); 3174-3179.