Team:Newcastle/Results/Glutathione
Glutathione Detection
Summary
New Parts: BBa_K3206000, BBa_K3206001, BBa_K3206002, BBa_K3206003, BBa_K3206004, BBa_K3206005, BBa_K3206006, BBa_K3206007, BBa_K3206008, BBa_K3206009,
Glutathione is a biomarker associated with Parkinson's disease; it has been found that these levels decrease in the Substantia nigra and the peripheral blood with people living with Parkinson's disease. The muninn project attempted to detect detect glutathione by using the glutathione degradation pathway to produce glycine, which would be detected by a glycine biosensor.
Using the promoter gcvBp, a promoter involved in the glycine cleavage pathway that becomes activated when glycine levels are increased, we have combined this promoter with a RFP gene to form a glycine biosensor. The part has been successfully designed and built using type IIs assembly into a DVK-AF destination vector, which has been sequence verified. The constructed has been tested, showing that is able to respond effectively to higher concentrations of glycine when grown over 48 hours, but not until after 24 hours. For a future diagnostic tool, we would use this part to detect glycine produced from glutathione degradation.
Additionally to the glycine biosensor, we have also designed and built constructs constitutively expressing the GcvA transcription factor, GcvR transcription factor and pepD using the Bba_J23100 promoter found on the iGEM registry of Basic Biological Parts. Characterisation of GcvA and GcvR was attempted to see how the two interact in low/high levels of glycine and how this interaction affects expression of the glycine biosensor by binding to the gcvBp promoter in a cell free system. However, no interaction was observed using this cell free method. Furthermore, a ChaC encoding construct was designed under constitutive expression of the Bba_J23100 promoter: however assembly was unsuccessful.
We found that glycine is toxic to the cells at high concentrations. The model suggested that all glutathione would be converted to glycine in 584.6 seconds; this could be toxic to the cells as shown from our experiments. Therefore, to delay this level of glycine production, the PepD construct (BBa_K3206007) was redesigned to have lower expression in an attempt to lower to amount of glycine the cell would be exposed to. The new PepD construct (BBa_K3206015) was designed to have lower expression using a Bba_I1051 promoter.
muninn has designed a glycine biosensor that can be used for future detection of glutathione. Future work should focus on the degradation of glutathione using ChaC and PepD, and if this can be detected using out biosensor. Furthermore, the glycine biosensor should be optimised to detect lower glycine concentrations at an earlier time.
Introduction
Glutathione is an antioxidant involved in helping with oxidative stresses that occur in metabolic reactions. It helps reduce stress caused by reactive oxygen species which can cause damage to cellular structures and metabolic processes. Glutathione has been shown to reduce in neurons and the peripheral blood with people living with Parkinson’s which could contribute to the degeneration of dopaminergic neuronal cells in the Substantia nigra [1, 2].
For this biomarker, we have designed a biosensor used to levels of glutathione in the blood. The biosensor consists of 3 parts as shown in figure 1; glycine produced by glutathione degradation will activate the transcription of RFP which will be used as a reporter. We should see that increased levels of glutathione would result in higher levels of fluorescent output due to increased glycine produced.
For this system, we aim to assemble the glycine biosensor gene construct using type IIs restriction, transform our chassis Escherichia coli DH5-alpha and determine whether elevated levels of glycine would result in an RFP output.
Figure 1. Glutathione degradation pathway to produce fluorescent reporter. 1. chaC- Glutathione-specific gamma-glutamylcyclotransferase protein hydrolyses glutathione to produce a cysteine-glycine dipeptide and 5-oxo-L-proline. 2. pepD- cytosol non-specific dipeptidase will catalyse the hydrolysis of the cysteine-glycine to produce cysteine and glycine. 3. Glycine will bind to GcvR, causing it to release GcvA; this allows GcvA to bind to the promoter gcvBp and activate transcription of an RFP reporter.
Glycine biosensor gene design
The glycine biosensor took inspiration from glutathione degradation and the glycine regulatory pathway. Glycine levels are regulated by the glycine cleavage system. When glycine levels are normal GcvR interacts with GcvA, rendering GcvA inactive; however, when glycine levels increase, GcvR stops interacting with GcvA allowing the transcription factor to be released and bind to the promoter region gcvBp [3, 4]. This activates transcription of the gene gcvB, which encodes a transcription factor involved in amino acid transport and glycine cleavage [4].
For the glycine biosensor, we took advantage of this system by swapping out the gcvB gene with an RFP reporter to produce a glycine biosensor. Theoretically, we would expect elevated glycine levels to trigger higher RFP production and a greater response. Therefore, we attempted to characterise the biosensor to see if there was a response to glycine within the growth media [5] .
gcvR and gcvA are already present in E. coli DH5-alpha [6]; because of this, we wanted to see the effect of elevated levels of glycine with just the glycine biosensor present. If there was little response with elevated levels of glycine, then these genes with constitutive expression would be added to our chassis.
We designed the glycine biosensor using benchling to make it compatible with Type IIs assembly, with BsaI restriction sites and fusion sites compatible with a DVK-AF destination plasmid (Figure 2). Additionally, separate transcriptional units encoding chaC, pepD, gcvA and gcvR under the constitutive expression of a BBa_J23100 promoter were made.
Figure 2. Glycine biosensor transcriptional unit assembled using benchling. This construct includes a gcvBp promoter which becomes activated when GcvA transcription factor binds, causing transcription of an RFP reporter.
Figure 3. chaC, pepD, gcvA and gcvR under the constitutive expression by the promoter BBa_J23100. Gene constructs were designed using benchling (above) and basic parts making up transcriptional units (below).
The glutathione model found that all glutathione would be converted to glycine in 548.6 seconds. Because the experiments conducted have found that glycine is toxic to the cells, we decided to re-think the design for the glutathione degradation pathway in order to delay the time until glycine is produced. This is so the cells are not overburdened with high levels of glycine. This was done by redesigning our original PepD construct to have a weaker expression using a Bba_I1051 promoter instead of a Bba_J23100 promoter. The new designed PepD part can be found here. Link to our glutathione page can be found here.
Assembly and transformation
Once gene constructs had been designed, they were ordered as g-Blocks from IDT, resuspended in TE buffer at a concentration of 10 ng/uL DNA and stored at -20 degrees Celsius.
All g-Blocks were assembled into individual DVK-AF destination plasmids using the MoClo procedure. Initially, a 1:1 ratio of g-Block to DVK-AF plasmid was used in the MoClo procedure, with a total of 20 fmol of DNA. This worked successfully for the GcvA construct but was unsuccessful for the other constructs (Figure 4).
Figure 4. 1% agarose gel electrophoresis of assembled DNA constructs after MoClo experimental procedure. GcvA, Eicosanol-RFP biosensor and nitroxoline have a higher band compared to Dvk-AF plasmid DNA (the positive control). No bands are present for GcvR, PepD, ChaC or the glycine biosensor, indicating a failed assembly.
In attempt to rectify this issue, a number of MoClo reactions were performed using a range or ratios. These varied from 1:1 to 1:4 g-Block vs DVK-AF plasmid. Figure 5 shows that MoClo was successful as samples had higher base pair band then the DVK-AF plasmid. This would indicate that the band had inserted itself into the plasmid successfully, thus making the plasmid larger in base pairs. In order to confirm that the assembly had worked correctly, a transformation of the chassis was needed. Unfortunately, after numerous attempts we were unable to assemble the ChaC part into the DVK-AF vector.
Figure 5. 1% agarose gel electrophoresis of assembled DNA constructs after MoClo experimental procedure. Left gel shows the glycine biosensor construct. Right image shows GcvR and PepD g-Block to DVK-AF ratio of 1:1 to 1:4.
Once constructs had been assembled into the DVK-AF destination vector, transformation of our chassis Escherichia coli DH5-alpha. For this, we used a heat-shock method to transform our bacteria with each of the constructs. Once the bacteria had been heat-shocked, they we plated on plates containing kanamycin, IPTG and x-gal. The DVK-AF plasmid contains a kanamycin resistance gene and a LacZ gene, which is induced by IPTG and causes the expression of beta-galactosidase. This protein then metabolizes x-gal to produce a blue colour. E. coli which have taken up the plasmid will either present as blue, indicating that the plasmid has taken out and the LacZ gene is still intact, or white, indicating that the bacteria has taken up the plasmid, but the g-Block has been inserted into the LacZ gene. This indicates a successful transformation. Figure 6 shows a successful transformation as colonies grew on kanamycin plates and did not turn blue. Additionally, the control plates showed that our chassis was able to uptake the DVK-AF plasmid (Figure 6.2) and was not naturally resistant (Figure 6.1).
Figure 6. 1) Negative control- E. coli DH5-alpha treated with water. 2) Positive control- E. coli DH5-alpha transformed with DVK-AF. Successful transformation of E. coli DH5-alpha with glycine biosensor (3), GcvA gene construct (4), GcvR gene construct (5) and PepD gene construct (6).
Once transformations were completed, overnights of each transformed bacteria were made, and each assembled plasmid was isolated using the Miniprep procedure. After plasmid purification and isolation, the plasmid was sent for sequencing Eurofins Scientific and the sequence data was aligned to the designed construct in benchling (Figure 7).
Figure 7. Sequence alignment of pepD (1), glycine biosensor (2), gcvA (3) and gcvR (4) constructs assembled into DVK-AF plasmids. Sequence alignment was performed in Benchling.
50% glycerol stocks were made for all transformed bacteria and stored at -80 degrees Celsius for future experimental work.
Glycine biosensor characterisation
Once it had been determined that the transformation was successful, it had to be shown that glycine would result in RFP production. To do this, E. coli was grown in LB with elevated levels of glycine to see if there was an increased fluorescent output. Initially, we wanted to see if our E. coli transformed with glycine biosensor was able to respond differently to varying concentrations of glycine.
The effect of elevated levels of glycine in LB media on RFP output
The transformed E. coli was grown overnight in 10 mL LB containing kanamycin overnight. This overnight culture was then used to inoculate 100 mL of LB the next day and grown to an optical density ranging between 0.3 and 0.6. A 96 well plate was then performed at glycine concentrations at 50, 1 and 0 mM glycine and reading were taken over 40 hours. The wells were excited at a wavelength of 550 nm and emission was detected at 610 nm (readings taken every 5 min). OD600 was read every 5 mins too in order to determine the relative fluorescent units (RFU). For this experiment, we would expect there to be an increase in relative florescent units over time as the concentration of glycine increased.
Figure 8 shows that higher levels of glycine results in impaired growth of E. coli after around 18 hours for E. coli exposed to 50 mM glycine. This is likely due to osmotic stress affecting the growth. Despite this, figure 8 suggests that transformed E. coli exposed causes a higher level of fluorescent output when exposed to higher levels of glycine. This is because RFP continues to increase for the E. coli treated at 50 mM glycine and the relative fluorescent units are higher when comparing to the other concentrations. Although there is high variation seen in the data presented, it is clear that there is a trend over time as concentration increases.
Figure 8. The optical density (OD600), RFP fluorescence and RFP fluorescence/OD600 (+/- S.E) for E. coli DH5-alpha transformed with the glycine biosensor when grown in LB at different concentrations of glycine (mM). X-axis starts at 12h point as this is where RFP response appears to begin. Excitation wavelength= 550 nm. Emission wavelength= 610 nm. Measurements were taken in a BioTek synergy H1 microplate reader.
Charecterising the specificity of the biosensor
To confirm that glycine was activating the biosensor and not any other amino acid, the transformed bacteria was exposed to varying concentrations of an amino acid that should not initiate a response. For this we used the amino acid cysteine. Additionally, to this experiment, a range of concentrations (mM) was used to establish whether there was more of a relationship between glycine concentration and fluorescence.
Like the previous experiment, an overnight culture of the glycine biosensor bacteria was made and used to inoculate a flask LB the following morning. This was grown to an optical density of around 0.3 to 0.6 and then diluted to an OD600 of 0.1. This was then used to inoculate wells containing varying concentrations of either glycine or cysteine. The concentrations for each were 50, 40, 30, 20 and 10 mM of the amino acid, and just LB by itself. A blank was determined using uninoculated LB containing each concentration of glycine or cysteine. OD600 and fluorescence (excitation= 550 nm, emission= 610 nm) was measured every 5 min for to determine growth, fluorescence and RFU.
For this experiment, we would expect to see a greater increase in fluorescence for transformed bacteria exposed to higher concentrations of glycine. Furthermore, there should be some increased fluorescence for the cysteine containing wells due to glycine already present in the media. However, this level of fluorescence should be similar to the inoculated well containing no added amino acids.
Figure 9 shows that increased concentration of glycine continues to have an effect on the growth of the transformed bacteria until around 20 mM. Interestingly, this effect is not observed with cysteine at similar concentrations. Figure 9 also shows that for both treatments, fluorescence begins at around 6 hours. When looking at fluorescence alone, it is hard to distinguish whether there is a difference in the level of fluorescence. There may be some increase in relative fluorescence units when the transformed bacteria is exposed to glycine: however, this is difficult to distinguish. For the biosensor containing bacteria exposed to cysteine, relative fluorescence increases, which is expected because there is glycine in the LB media: however, data points for the higher concentrations of cysteine has a similar fluorescence response to the lower concentrations. It could be argued that bacteria grown in 50 mM glycine has higher RFU than 50 mM cysteine and LB containing no added glycine. This is not completely clear so it cannot be concluded that higher levels of glycine induces a higher response. This is likely due to the short experimental time and a greater response may be observed if the experiment went on for a longer period like figure 8.
Figure 9. The optical density (OD600), RFP fluorescence and RFP fluorescence/OD600 (+/- S.E) for E. coli DH5-alpha transformed with the glycine biosensor when grown in LB at different concentrations of glycine (top row) and cysteine (bottom row) (mM). Measurements were taken every 5 min for 24 hours. Excitation wavelength= 550 nm. Emission wavelength= 610 nm. Measurements were taken in a BioTek synergy H1 microplate reader.
To get a better picture of how our glycine biosensing bacteria responds to elevated levels of glycine and whether this response was specific to glycine, the bacteria was left to grown for 48 hours. Flacon tubes containing 10 mL of LB containing kanamycin and either 100 mM, 50 mM, 10 mM and 0 mM glycine or cysteine were inoculated with the transformed bacteria. This was left for 48 hours at 37 degrees Celsius rotating at 200 rpm. After this time, 200 uL of culture was placed in a 96 well plate. OD600 and fluorescence (Excitation= 582 nm, emission= 607 nm) was measured for each condition and were taken for each concentration. This was done using a Thermo Fisher Varioskan™ LUX multimode microplate reader.
Figure 9 shows similar results to that of the previous in that higher concentrations of glycine effects the growth of E. coli DH5-alpha and cysteine does not cause a similar response. Figure 9 shows that elevated levels of glycine results in higher levels of fluorescence and that this response is specific to this amino acid. This is because the level of fluorescence increases when glycine concentration increases, and fluorescence remains constant when cysteine levels are increased. The levels of fluorescence for the bacteria grown with elevated levels of cysteine can be attributed to the glycine already present to the growth media. Furthermore, when looking at the fluorescence levels relative to OD600, it is clear that fluorescence increases exponentially when glycine concentration increases.
The scale of RFP fluorescence differs between previous figures; this is due to using a different plate reader, which uses different parameters and detection levels.
Figure 10. The optical density (OD600), RFP fluorescence and RFP fluorescence/OD600 (+/- S.E) for E. coli DH5-alpha transformed with the glycine biosensor when grown in LB at different concentrations of glycine and cysteine (mM). Measurements were taken after 48-hour incubation at 37 degrees Celsius rotating at 200 rpm. Excitation wavelength= 582 nm. Emission wavelength= 607 nm. Measurements were taken in a Thermo Fisher Varioskan™ LUX multimode microplate reader (data not normalised for background fluorescence).
The effects of elevated levels of glycine in minimal media on RFP output
The previous experiments showed that E. coli DH5-alpha was capable of responding to different levels of glycine when grown in LB media by producing RFP and this response was specific to this amino acid. There is glycine already present within LB which results in a background level of fluorescence, which could limit the detection of lower levels of glycine. In an attempt to see if glycine could be detected at lower concentrations, the transformed bacteria was grown in M9 minimal media at a range of glycine concentrations (100,000 uM/L-0.01 uM/L) over 24 hours to determine if RFP could be produced and if there was a relationship between glycine concentration and fluorescence. The minimal media also contained 25 uL/L glycerol as a carbon source and 5 mM thiamine as E. coli DH5-alpha is unable to produce this amino acid [7].
E. coli DH5-alpha containing the glycine biosensor was grown in an overnight culture in LB containing kanamycin. After 16-hour growth, this overnight culture was used to inoculate LB and grown until an optical density of 0.3-0.6 was reached. Once reached, 1 mL of culture was centrifuged at 4000 x g to obtain a cell pellet and the supernatant was decanted. The pellet was washed in 1 mL of M9 minimal media and centrifuged to obtain a cell pellet. The supernatant was decanted, and the previous step was repeated 3 times. This was to remove any residual LB media. The optical density of the washed cells was measured and then adjusted to an OD600 of 0.1. A 96 well plate was used containing 198 uL of M9 minimal media at a range of glycine concentrations and 2 uL of bacteria culture. Three repeats were made for each glycine concentration. OD600 and fluorescence (Excitation= 550 nm, emission= 610 nm) was measured every 5 minutes using a BioTek Synergy H1 microplate reader. The incubation conditions were set at 37 degrees Celsius at 200 rpm.
Data from figure 11 shows that the bacteria was not able to grow within the M9 minimal media after 24 hours. This experiment was repeated another time using the same reaction conditions and an additional time containing 2% Casamino: however, no growth was observed. Despite nutrients being supplemented within the M9 minimal media, the data would suggest that this was not optimal for bacterial growth. Future experiments should attempt to find an optimal growth media with known and controlled levels of nutrients and amino acids, particularly glycine.
Figure 11. The optical density (OD600) and RFP fluorescence (+/- S.E) for E. coli DH5-alpha transformed with the glycine biosensor when grown in M9 minimal media containing glycerol, thiamine and different concentrations of glycine (uM/L). Measurements were taken every 5 min for 24 hours at 37 degrees Celsius rotating at 200 rpm. Excitation wavelength= 550 nm. Emission wavelength= 610 nm. Measurements were taken in a BioTek Synergy H1 microplate reader.
Cell free protein synthesis experiments
Previous research has shown that the transcription factors GcvR interacts with GcvA when glycine levels are low, preventing GcvA binding to promoter regions found on the gcv operon. However, when glycine levels increase this interaction is disrupted, allowing GcvA to be released and bind to these promoter regions and activate transcription. In an attempt to observe this transcriptional interaction, a cell free experiment was attempted. These experiments were done under the expertise and supervision of Dr. Alice Banks.
To do this, each plasmid containing either gcvA, gcvR and the glycine biosensor were purified individually. For a control, a plasmid containing a mCherry gene under constitutive expression was isolated. A QIAGEN midiprep was used to purify these plasmids at a high concentration, so a total of 3 ug DNA could be used per reaction. Each reaction contained the following components: 10 uL cell free extract (44.5 mg/ml), 25 uL 2 x buffer (containing 10 mM Mg-glutamate, 240 mM K-glutamate, 3 mM each amino acid, 100 mM HEPES, 3 mM ATP and GTP, 1.8 mM CTP and UTP, 0.4 mg/ml tRNA, 0.52 mM CoA, 0.66 NAD, 1.5 mM cAMP, 0.136 mM folinic acid, 2 mM spermidine, 60 mM 3-PGA), 2.5 uL 40 % polyethylene glycol-8000, 3 ug DNA, inducers (if required) and ddH2O up to 50 uL. Seven reactions were used for this experiment as shown in Table 1. Three repeats were made for each reaction condition in a 384 well plate.
Table 1. Reaction conditions used for cell free protein synthesis reactions| Reaction | Inducers | Expected fluorescent output |
|---|---|---|
| 1 | GcvA + glycine biosensor - glycine | Fluorescence |
| 2 | Glycine biosensor - glycine | No fluorescence |
| 3 | GcvR + glycine biosensor - glycine | No fluorescence |
| 4 | GcvA + GcvR + glycine biosensor - glycine | No fluorescence |
| 5 | GcvA + GcvR + glycine biosensor + glycine (50 mM) | Fluorescence |
| 6 | mCherry (positive control) | Fluorescence |
| 7 | water (negative control) | No fluorescence |
For this experiment, it was expected that reaction 1 would produce RFP because the GcvA transcription factor would be able to bind to the gcvBp promoter region and activate transcription of the reporter. It was also expected that reaction 4 would not have a fluorescent output and reaction 5 would. This is because the elevated glycine would disrupt the interactions between GcvR and GcvA and allow expression of the glycine biosensor to occur (reaction 4), whereas there is no elevated glycine in reaction 5 so this interaction would not be disrupted. Furthermore, reaction 6 should result in fluorescence as a positive control as it contains an mCherry gene under constitutive expression.
RFP fluorescence (excitation= 584 nm, emission= 607 nm) and mCherry fluorescence (excitation= 587 nm, emission 610 nm) was measured every 5 min for 13 h. The 384 well plate was incubated at 30 degrees Celsius for this reaction.
Figure 12.1 shows that the reaction condition was able to elicit a response has there was a fluorescent output for the positive control as expected. Figure 12.2 and 12.3 shows that a cell free reaction containing plasmids encoding GcvA and the plasmid containing the glycine biosensor was unable to cause the expression and synthesis of RFP. This may be due to issues with folding for the transcription factor as reaction conditions may not be optimal. Because of this, cell lysates from cultures of E. coli DH5-alpha containing GcvA and GcvR constructs were made to substitute the GcvA and GcvR plasmids for each reaction. Furthermore, it may take longer for the system to become active as shown in previous experiments before a district signal-to-noise ratio appears. In a cell free protein synthesis system, resources may deplete for a signal appears (after 600 min). Therefore the system should be optimised to last longer to see if there is a change in signal.
Figure 12. Cell free protein synthesis reaction of fluorescent reporter proteins. Wells contained different combinations of plasmids (3 ug) which encode either GcvA, GcvR or glycine biosensor. Negative control= reaction containing water and no DNA inducer. Positive control= reaction containing mCherry under constitutive expression. 1. RFU over 13 h for all reaction conditions. 2. RFU over 13 h for all reaction conditions apart from positive control. 3. RFU over 13 h for reactions 4 and 5. RFP fluorescence (excitation= 584 nm, emission= 607 nm) and mCherry fluorescence (excitation= 587 nm, emission 610 nm) was measured using a Thermo Fisher Varioskan™ LUX multimode microplate reader.
Transformed E. coli DH5-alpha constitutively expressing either GcvA or GcvR were grown overnight in 100 mL of LB containing kanamycin. After 16 h of growth, each inoculated media was centrifuged at 4000 x g to obtain a cell pellet. These pellets were then resuspended in dH20 and centrifuged again to wash any residual LB media. Once washed, each pellet was resuspended in 10 mL of dH20 and sonicated to lyse open the bacterial cell wall. Sonicated products were then centrifuged at 20,000 x g to obtain cell lysates. The protein concentration (ug/mL) was then determined using a bicinchoninic acid assay. The same reaction conditions were used for this experiment, but 5 uL of cell free extract was used instead of 10 uL. Furthermore, a total of 2.5 ug of GcvA and GcvR cell lysate was used instead of 3 ug of GcvA plasmid and GcvR plasmid respectively.
The expected outcomes for this experiment are the same for the previous and can be found in table 1.
Figure 13 shows that addition of cell lysate obtained from GcvA was not activate transcription of the glycine biosensor. It was expected there would be an RFP output in wells containing GcvA and the glycine biosensor (Table 1- reaction 1). Additionally, it was expected that there would be RFP production in reaction 5 as the glycine would prevent GcvR binding to GcvA, allowing GcvA to activate transcription of the glycine reporter. However, none of these outcomes were observed. There are a number of reasons that could attribute to this, mainly the low concentration of protein in the cell lysate used for each reaction. Future experiments should focus on optimisation by increasing the amount of protein for the reaction.
Figure 13. Cell free protein synthesis reaction of fluorescent reporter proteins. Wells contained plasmid with glycine biosensor(3 ug DNA) and different combinations of of GcvA lysate and GcvR cell lysate. Negative control= reaction containing water and no DNA inducer. Positive control= reaction containing mCherry under constitutive expression. 1. RFU over 13 h for all reaction conditions. 2. RFU over 13 h for all reaction conditions apart from positive control. 3. RFU over 13 h for reactions 4 and 5. RFP fluorescence (excitation= 584 nm, emission= 607 nm) and mCherry fluorescence (excitation= 587 nm, emission 610 nm) was measured using a Thermo Fisher Varioskan™ LUX multimode microplate reader.
Conclusion
Using the promoter gcvBp, a promoter involved in the glycine cleavage pathway that becomes activated when glycine levels are increased, we successfully designed a glycine biosensor by combining this promoter with an RFP reporter. The part has been successfully designed and built using type IIs assembly into a DVK-AF destination vector, which has been sequence verified. The constructed has been tested, showing that is able to respond effectively to higher concentrations of glycine when grown over 48 hours, but not after 24 hours. Furthermore, when grown in M9 minimal media conatining varying concentrations of glycine, no bacterial growth or an RFP response was observed. For a future diagnostic tool, we would use this part to detect glycine produced from glutathione degradation.
Additionally to the glycine biosensor, we have also designed and built constructs constitutively expressing the GcvA transcription factor, GcvR transcription factor and pepD using the BBa_J23100 promoter found on the iGEM registry of Basic Biological Parts. Characterisation of GcvA and GcvR was attempted. This was to see how GcvA initiates transcription of the glycine biosensor by binding to the gcvBp promoter, how GcvR inhibits GcvA binding to the promoter and how glycine stops GcvR binding to GcvA. Characterisation of this interaction was done using a cell free system: however, no interaction was observed using this cell free method because there was no RFP fluorescence observed. Furthermore, a ChaC encoding construct was designed under constitutive expression of the BBa_J23100 promoter: however assembly was unsuccessful.
muninn has designed a glycine biosensor that can be used for future detection of glutathione. Future work should focus on the degradation of glutathione using ChaC and PepD, and if this can be detected using out biosensor. Furthermore, the glycine biosensor should be optimised to detect lower glycine concentrations at an earlier time.
Attributions
- Content: Matthew Rogan
- Figures: Matthew Rogan
- Proofreading: Connor Trotter, Dr Alice Banks, Bradley Brown, Jasmine Bird, Matthew Rogan
References
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