Team:Newcastle/Results/EicosanolDetection
Eicosane detection
Summary
New Parts:BBa_K3206010, BBa_K3206011
Research has shown that eicosane is a volatile biomarker found on the skin associated with Parkinson's disease. The muninn project attempted to design a method of detecting eicosane levels by degrading eicosane into 1-eicosanol and detecting this product using a 1-eicosanol biosensor.
A 1-eicosanol biosensor was designed using an adhEp2 and an RFP reporter gene, which was thought to become active in the presence of 1-eicosanol. This part was successfully designed to be compatible with type IIs restriction and built into a DVK-AF destination vector. Characterisation of this part was done by growing the Escherichia coli DH5-alpha transformed with the biosensor at different concentrations of 1-eicosanol to determine if this could RFP transcription relative to these concentrations. It was found that 1-eicosanol was not able to cause the biosensor to become active by producing RFP. A variety of fatty alcohols were used in an attempt to activate the biosensor at different concentration: however, no RFP response occurred.
Upon designing the model for this part, research suggested that 1-eicosanol would not interact with the adhEp2 promoter as expected. Therefore, the proposed biosensor would not be able to detect varying levels of 1-eicosanol. Future research should focus on designing a construct that is able to detect eicosane or 1-eicosanol more effectively.
Introduction
Eicosane has been found to increase on the sebum of people living with Parkinson’s disease; this long chain fatty alcohol is produced by species of Malassezia yeast, which lives on the sebum found on the skin [ 1, 2 ]. This acts as an indicator of Parkinson’s disease as the yeast grows on the sebum, an oily layer found on the skin which increases in production for people living with Parkinson’s. The colonization of this yeast leads to the skin condition seborrheic dermatitis, a disease affecting the scalp and other oily parts of the skin. So far, eicosane has only be found to increase in the sebum of people living with Parkinson’s disease.
We have designed a biosensor to detect 1-eicosanol, which will be produced from eicosane using a long-chain alkane monooxygenase (LadA) We will use part BBa_K398017 from the iGEM registry as our LadA. 1-eicosanol will then bind to an adhEp2 promoter, activating the transcription of the reporter gene RFP.
In order to characterize this part, we decided to carry out a dose dependent response for different concentrations of 1-eicosanol and observe the effect on fluorescent output in relative fluorescent units.
Assembly and Transformations
We designed the g-block using the online tool benchling. The construct was made to have A-F ends compatible with Type IIs restriction, with sites specific to the restriction enzyme BsaI (Figure 1).
Figure 1. Eicosanol biosensor assembled in benchling. Construct contains an adhEp2 promoter which is active in the presence of 1-eicosanol.
The DNA was diluted in TE buffer to a concentration of 10 ng/uL as our stock concentration. We assembled the g-Block into a Dvk-AF plasmid backbone using MoClo procedure. A 1:1 ratio of g-Block DNA to Dvk-AF plasmid DNA was used to give a total of 20 fmol DNA was used in the reaction.
The products from the MoClo were used to transform our chassis Escherichia coli strain DH5-alpha using heat shock. A gel electrophoresis confirmed that the g-block had assembled into the Dvk-AF plasmid, as indicated by a higher size band (Figure 2).
Figure 2. 1% agarose gel electrophoresis of assembled DNA constructs after MoClo experimental procedure. GcvA, Eicosanol-RFP and nitroxoline have a higher band compared to Dvk-AF plasmid DNA (the positive control).
The ligated DNA was used to transform E. coli DH5-alpha. 5 uL of assembled plasmid was added to 50 uL of chemically competent E. coli DH5-alpha. 2 uL of sequenced verified Dvk-AF was used as a positive control and the same amount of TE buffer was used as a negative. Once added, the bacteria were incubated on ice for 30 min, heat shocked at 42 degrees celsius for 1 min and then transferred back into ice for a further 2 min. After this, 200 uL of LB was added and incubated for 1 hour at 200 rpm at 37 degrees celsius.
A successful transformation was confirmed from the growth of a white colony on LB agar plate containing x-gal and kanamycin (Figure 3). This suggests that the MoClo assembly has inserted the eicosanol biosensor construct into the LacZ gene, preventing the bacteria to produce beta-galactosidase and metabolise the x-gal which would produce a distinct blue colony.
Transformation with Dvk-AF showed that the E. coli DH5-alpha cells had successfully been made competent as they were resistant to kanamycin contained in the LB agar growth media. Additionally, untransformed E. coli DH5-alpha were unable to grow on LB plates containing kanamycin, suggesting that these cells are not resistant tot the antibiotic and no contamination occurred (Figure 3).
Figure 3. 1) Successful Transformation of E. coli DH5-alpha with 1-Eicosanol biosensor assembled into Dvk-AF plasmid using MoClo level 1 assembly method. 2) Positive control- E. coli DH5-alpha transformed with Dvk-AF plasmid giving blue colonies. 3) Negative control- E. coli DH5-alpha not transformed.
An overnight culture of the transformed bacteria was made, and the plasmid was isolated using the Miniprep procedure. Once the plasmid was isolated, the DNA was sequenced by Eurofins Scientific and sequence data was aligned to the construct made in Benchling (Figure 4).
Figure 4. Sequence alignment of Eicosanol-RFP reporter construct gBlock assembled into Dvk-AF reporter construct. Sequence alignment was performed using Benchling.
Solubilizing 1-eicosanol
The solubility of 1-eicosanol in water is 0.001 g/L, making it a very insoluble chemical [ 3 ]. We tried a range of organic solvents in an attempt to dissolve the chemical and found that diethyl-ether was the most effective. A literature search was performed to try and find what percentage of diethyl ether E. coli growth was not inhibited or effected: however, no data was found. To solve this problem, a plate reader assay was performed at a range of diethyl-ether percentages to determine when it was inhibited. Two assays were performed; a plate reader assay over 16 hours where OD600 was measured every 20 minutes and another assay after 20 hours incubation.
Figure 5 showed that 10% diethyl ether would be too high of a percentage to use as it had a negative effect on overall cell growth. The data from figure 5 would suggest that a 5% solution would not have an effect. However, figure 6 shows that a 5% solution has an adverse effect on cell growth, similar to that of a 10% solution. This difference is likely due to the diethyl ether evaporating and escaping the 96 well plates during the 16-hour overnight assay because they did not have lids on. The 20-hour incubation involved growing the bacteria in a sealed Eppendorf tube, continuously exposing the bacteria to the solvent. From this data, we decided a 1% solution to dissolve 1-eicosanol would be suitable.
Figure 5. The effect of varying percentages of diethyl ether on E. coli DH5-alpha growth over 16 hours. Bacteria was grown at 37 degrees Celsius at 200 rpm.
Figure 6. The effect of different percentages of diethyl ether on E. coli DH5-alpha growth after 20 hours incubation. Bacteria was grown at 37 degrees Celsius at 200 rpm.
Eicosanol-RFP characterization
After a method of dissolving 1-eicosanol had been established and the percentage solvent to use where E. coli DH5-alpha growth was not affected, the biosensor was to be characterized by observing RFP fluorescent output when the transformed bacteria was exposed to varying concentrations of 1-eicosanol.
Firstly, 0.4435 mg of 1-eicosanol was first dissolved in 10 mL of diethyl ether to give a final concentration of 147.980 mM. One milliliter of this solution was added to 99 mL of deionized water to make a 1/100 dilution, giving a final concentration of 1.4798 mM 1-eicosanol for the stock solution. A 96-well plate assay was performed with 1-eicosanol varying from 1000 uM to 0.001 uM suspended in LB.
An overnight culture of E. coli DH5-alpha transformed with the 1-eicosanol biosensor was grown. E. coli DH5-alpha transformed with Dvk-AF was used as a control to compare fluorescent intensity as there should be no fluorescent output compared to the transformed bacteria. Both overnight cultures were diluted to an OD600 of 0.01 and used to inoculate each well. There were 3 repeats for each 1-eicosanol condition and non-inoculated LB with 1-eicosanol at each concentration were used to blank the readings. Optical density was recorded at 600 nm, and the conditions for excitation and emission was 550 nm and 610 nm respectively. From this, it would be expected that there would be an increase in fluorescent intensity as the concentration of 1-eicosanol increased. There should be no increase in fluorescence for the control as the biosensor is not present.
Figure 7 (A1 and A2) shows that the concentration of 1-eicosanol had little effect on the growth on E. coli- DH5-alpha. Figure 7 (B1 and B2) suggests that there was no RFP produced by E. coli DH5-alpha transformed with the 1-eicosanol biosensor. This is because there was no increase in RFP fluorescence as 1-eicosanol concentration increased. Additionally, when comparing the fluorescent output to E. coli DH5-alpha transformed with Dvk-AF, the data was essentially the same as there was no linear increase. Similar results are found in figure 7 C1 and C2 as data was similar which was not expected. Additionally, the pattern of data for fluorescence/OD600 (figure 7 C1 and C2) did not match expectations as there was no linear increase of RFP fluorescence/OD600 for the E. coli containing the 1-eicosanol biosensor.
1-eicosanol is a long chain fatty acid consisting of 20 carbons: because of the size of the molecule, it may not be able to pass the cell membrane of E. coli and activate the promoter, therefore not producing RFP and fluorescence. Because of this, we wanted to try to activate the promoter using shorter chain fatty alcohols that could pass through the cell membrane such as ethanol, butanol and propanol.
Figure 7. A- The optical density (OD600) of E. coli DH5-alpha grown in different concentrations (uM) of 1-eicosanol over 22 hours (1= E. coli DH5-alpha transformed with 1-eicosanol biosensor. 2= E. coli DH5-alpha transformed with Dvk-AF). B- The level of RFP fluorescence overtime at different concentration (uM) of 1-eicosanol (1= E. coli DH5-alpha transformed with 1-eicosanol biosensor. 2= E. coli DH5-alpha transformed with Dvk-AF). C- Relative AFUs (RFP fluorescence/ OD) over time for E. coli DH5-alpha transformed with 1-eicosanol biosensor (1) and E. coli DH5-alpha transformed with Dvk-AF (2) with different concentrations of 1-eicosanol.
For ethanol, a 50 mM stock concentration of ethanol was made in deionized water and a 96-well plate at varying concentrations of the organic solvent was performed. Similar controls were used (E. coli DH5-alpha with Dvk-AF) and wells with just LB and ethanol at each concentration was used as blanks. For this assay, we wanted to see if varying concentrations of ethanol would produce a dose dependent response in RFP output.
Figure 8 (A1 and A2) showed that ethanol had little effect on the growth of both forms of E. coli used. Figure 8 (B1/2 and C1/2) shows that ethanol was unable to trigger an RFP response with our bacteria transformed with the eicosanol biosensor because no increase in fluorescence was observed when ethanol concentration was increased. Overall, the results suggest that ethanol was unable to activate the biosensor.
Figure 8. A- The optical density (OD600) of E. coli DH5-alpha grown in different concentrations (uM) of ethanol over 22 hours (1= E. coli DH5-alpha transformed with 1-eicosanol biosensor. 2= E. coli DH5-alpha transformed with Dvk-AF). B- The level of RFP fluorescence overtime at different concentration (uM) of ethanol (1= E. coli DH5-alpha transformed with 1-eicosanol biosensor. 2= E. coli DH5-alpha transformed with Dvk-AF). C- Relative AFUs (RFP fluorescence/ OD) over time for E. coli DH5-alpha transformed with 1-eicosanol biosensor (1) and E. coli DH5-alpha transformed with Dvk-AF (2) grown with different concentrations of ethanol.
Figure 9 (A1 and A2) shows that the concentrations of butanol had little effect on the growth of E. coli. Comparison of the fluorescent output between E. coli transformed with the biosensor and the control shows that the butanol concentration had no effect on RFP output. Like the previous results, there was no increase in RFP fluorescence over time as butanol concentration increased. The values of fluorescence in figure 9 B and C can be attributed to noise within the system.
Figure 9. A- The optical density (OD600) of E. coli DH5-alpha grown in different concentrations (uM) of butanol over 22 hours (1= E. coli DH5-alpha transformed with 1-eicosanol biosensor. 2= E. coli DH5-alpha transformed with Dvk-AF). B- The level of RFP fluorescence overtime at different concentration (uM) of butanol (1= E. coli DH5-alpha transformed with 1-eicosanol biosensor. 2= E. coli DH5-alpha transformed with Dvk-AF). C- Relative AFUs (RFP fluorescence/ OD) over time for E. coli DH5-alpha transformed with 1-eicosanol biosensor (1) and E. coli DH5-alpha transformed with Dvk-AF (2) grown with different concentrations of butanol.
Figure 10 (A1 and A2) shows that propanol concentrations used had little effect on E. coli growth. Like the previous results, propanol was unable to trigger an RFP response in our E. coli transformed with the eicosanol biosensor as there was no increase in fluorescence as propanol concentration increased (Figure 10 B and C).
Discussion
The muninn project attempted to design a method of detecting eicosane levels by degrading eicosane into 1-eicosanol and detecting this product using a 1-eicosanol biosensor.
A 1-eicosanol biosensor was designed using an adhEp2 and an RFP reporter gene, which was thought to become active in the presence of 1-eicosanol. This part was successfully designed to be compatible with type IIs restriction and built into a DVK-AF destination vector.
It was found that 1-eicosanol was difficult to dissolve and solubilise; a range of organic solvents were used and it was found that diethyl ether worked most efficiently. Furthermore, kill curves were used to determine what percentage of diethyl ether E. coli grew best at; this was determined to be around 1%. Characterisation of this part was done by growing the Escherichia coli DH5-alpha transformed with the biosensor at different concentrations of 1-eicosanol to determine if this could RFP transcription relative to these concentrations. It was found that 1-eicosanol was not able to cause the biosensor to become active by producing RFP. A variety of fatty alcohols, such as butanol, propanol and ethanol, were used in an attempt to activate the biosensor at different concentration: however, no RFP response occurred.
Upon designing the model for this part, research suggested that 1-eicosanol would not interact with the adhEp2 promoter as expected. Therefore, the proposed biosensor would not be able to detect varying levels of 1-eicosanol. Future research should focus on designing a construct that is able to detect eicosane or 1-eicosanol more effectively.
Attributions
- Content: Matthew Rogan
- Figures: Matthew Rogan
- Proofreading: Connor Trotter, Jasmine Bird, Dr Alice Banks
References
- Trivedi DK, Sinclair E, Xu Y, Sarkar D, Walton-Doyle C, Liscio C, et al. Discovery of volatile biomarkers of Parkinson’s disease from sebum. ACS Central Science. 2019.
- Kim GK (2009) Seborrheic Dermatitis and Malassezia species: How Are They Related? The Journal of clinical and aesthetic dermatology .
- National Centre for Biotechnology Information. PubChem Database. 1-Eicosanol, CID=12404, https://pubchem.ncbi.nlm.nih.gov/compound/1-Eicosanol (accessed on Oct. 12, 2019)