Team:Sydney Australia/Results

Aims

To express psiD, psiK and psiM genes individually in E.coli, replicating their known in vivo expression (Fricke, Janis, Blei, Felix, and Hoffmeister, Dirk. “Enzymatic Synthesis of Psilocybin.” Angewandte Chemie International Edition 56.40 (2017): 12352–12355. Web.).
To characterise the functionality and kinetics of these psi genes in vitro.

Constructing psi gene Expression Vectors

Our first step successful step in the experiment was cloning of psiD, psiK and psiM genes individually into pET28 vectors (Figure 1). It was important for us to generate stable recombinant pET28 vectors as this would enable us to transform any T7 polymerase containing E.coli cell line and induce strong expression of our proteins with an added N terminal 6xHIS tag.

Figure 1. Double restriction digestion of psi genes psiD, psiK and psiM in the pET28c vector. Constructs were digested for 2 hours at room temperature alongside an empty pET28 vector and no template control using NEB's HindIII-HF and EcoRI-HF enzymes. Products were loaded onto a 1% Agarose, 40mL TAE gel prestained with Gel Green. The gel was run for 40 minutes at 100V before being imaged by UV transillumination using a BioRad XRS+ Chemidoc and QuantityOne's basic imaging software.

After using our constructs to transform BL21 E.coli cells we started testing our cell lysates for the presence of our proteins after induction. For this, we used SDS-PAGE gel electrophoresis as our diagnostic tool. In our first series of investigations, we encountered a noticeable problem. When we attempted expressing Psi proteins they appeared highly insoluble under our expression conditions (figure 2). To improve the solubility of our protein we began revising and troubleshooting our expression conditions and enlisted assistance from the Macquarie iGEM team (Figure 3) (See more about their help here!( See more about their help here!)

Achieving soluble Psi protein expression

Figure 2: SDS PAGE of cell lysates from BL21(DE3) cells containing pET28-psi gene constructs grown the presence or absence of IPTG (1mM). Psi proteins highlighted in red, (A) PsiM, (B) PsiK, (C) PsiD. Cells were grown to OD600 of ~0.5 at a temperature of 37℃ with shaking (200rpm) before IPTG induction at room temperature. Cultures were then returned to incubate with shaking overnight. Lysates were collected via bead beating. Insoluble lysate fractions were collected by beating cells in 1x SDS sample buffer (4% SDS, 20% glycerol, 0.002% Bromophenol Blue, 5% fresh B-mercaptoethanol, 62.5mM Tris-HCL pH6.8). Soluble fractions were collected by beating cells in 1x TE buffer (10mM Tris, 1mM EDTA pH 8). Gels were run at 200V for 40min. Gels were imaged using BioRad’s stain free UV imaging technology.)

Insoluble expression at 22°C

Figure 3. SDS PAGE of cell lysates from BL21(DE3) cells containing pET28-psiD gene constructs grown the presence or absence of IPTG (1mM). Cells were grown to OD600 of ~0.5 at temperature of 37℃ with shaking (200rpm) before IPTG induction at room temperature. Cultures were then returned to incubate with shaking overnight at 22℃ . Cells were lysed via sonication (60% amplitude, 6 cycles, 10 seconds on, 10 seconds off) and soluble and soluble fractions were separated by centrifugation (30 minutes, 20,000g) before being run on SDS PAGE)

With the results obtained from Macquarie and our own data we determined that it might be necessary to modify not only our expression conditions but also our system to hopefully increase the soluble expression of our proteins.

We decided to co-transform our pET28 vectors alongside an additional vector (pGro7) encoding the molecular chaperone complex groES-groEL into new BL21 cells. We believed the added chaperone and an incubation temperature around 15C after induction would reduce the stress on our cells and increase the solubility of our proteins. After generating new SDS PAGE gels of our cell lysates we observed evidence of our Psi proteins appearing relative to our uninduced controls in our soluble cell extracts.

Figure 4.SDS PAGE of cell lysates from BL21(DE3)(pGr07) cells containing psi genes present in pET28 vectors in the presence or absence of IPTG (1mM). Soluble Psi proteins highlighted in red boxes. Cultures were grown to OD600 of ~0.5 at 37 degrees before induction at room temperature. Cultures were then left to incubate at 15C overnight. Cells were lysed by bead beating in 1xTE buffer (10mM Tris, 1mM EDTA, pH 8). Samples were then denatured (3 minutes at 95C in 1XSDS sample buffer: 4% SDS, 20% glycerol, 0.002% Bromophenol Blue, 5% fresh B-mercaptoethanol, 62.5mM Tris-HCL pH6.8) before being run on SDS PAGE. Gel was run at 120V for 60min. Gels were imaged using BioRad’s stain free UV imaging technology.)

Confirmation of Soluble Psi Proteins

Figure 5. SDS PAGE gel containing soluble cell lysate profiles from cultures believed to be expressing soluble PsiK and PsiM was stained with coomassie blue and sent for MS analysis. The bands highlighted in red were cut, destained with 40% acetonitrile/60% 20mM ammonium bicarbonate, dehydrated, rehydrated with 12ng/ul porcine trypsin at 4C for 30min, excess trypsin was removed and 10ul 20mM am.bic was added and incubated overnight at 37C. Peptides masses were determined using MS and samples were identified using a Mascot peptide mass fingerprint search against all si genes.)

We were successful in selecting and identifying two gel spots we believed to be Psi proteins. Mascot returned significant matches with PsiM and PsiK for these bands and although we were unable to identify PsiD, and in the interest of time, we were confident that it was also expressing at our new conditions based on the results for the other two proteins. Using our new expression conditions we grew large cultures of cells containing our constructs and attempted to nickel column purify our Psi proteins from cell lysates. Due to technical errors in the lab, we were only able to recover column purified fractions we believed contained PsiD and PsiK. In the interest of time we decided to test the functionality of these Psi proteins using a qualitative LC/MS in-vitro assay modelled off Frick et al (Figure 6) (Figure 7).

Figure 5: Qualitative LC/MS profile of PsiD in vitro showing conversion of tryptophan to tryptamine. Ni+ Column purified fractions of cell lysate believed to contain PsiD were incubated at room temperature for 2-3 in the presence and absence of L-tryptophan (2.5mM). Ion peaks of interest from in vitro samples, tryptophan (209.0572 m/z) and tryptamine (161.1073 m/z), were identified against known 1mM standards also identified using LC/MS.

Figure 6: Qualitative LC/MS profile of PsiK in vitro showing conversion of 4-hydroxytryptamine to norbeocystin.Ni+ Column purified fractions of cell lysate believed to contain PsiK were incubated at room temperature for 2-3 in the presence and absence of 4-hydroxytryptamine (2.5mM), ATP (5mM), MgCl2 (5mM) and B-mercaptoethanol (5mM). Ion peaks of interest from in vitro samples, tryptophan (209.0572 m/z) and 4-hydroxytryptamine (177.1022 m/z), were identified against known 1mM standards also identified using LC/MS. Norbaeocystin standards could not be obtained.

Did we meet our aims?

Our LC/MS data yielded exciting results! We were able to confirm catalytic activity for both PsiD and PsiK with PsiD successfully converting L-tryptophan to tryptamine and psiK converting 4 hydroxytryptamine to norbaeocystin in vitro. We noticed the presence of a hydroxylated tryptamine compound in our PsiD assay supplemented with tryptophan. Because we also observed the formation hydroxytryptamine compound in our 1mM standard of tryptamine we suspect that tryptamine may spontaneous oxidize to form this compound and therefore its presence in our in vitro assay is not the result of any conversion by endogenous E.coli proteins. Along with our peptide mass fingerprint identification, we made considerable headway in achieving our aims as outlined below.

Table 1. Summarised PsiD, PsiK and PsiM Results

Future Directions?

Going forward with this experiment there are several avenues we wish to explore. We would first want to confirm soluble PsiD expression using LC/MS and peptide mass fingerprint identification. Next we would work towards purifying and quantifying all psi genes once more and use LC/MS again to characterize the functionality of PsiM. We would also like to quantify the amount of Psi protein present in our purified protein fraction. This would enable us to make estimations on the amount of protein present in each cell. Following this, we could then characterize the kinetics of our proteins using HPLC LC/MS. Sampling our in vitro assay containing a known amount of protein during a time course, after the addition of respective substrates, should help us build a kinetic profile for our enzymes. This information would be highly valuable in helping model systems designed using these parts.

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Aim

To take the three genes psiD, psiK and psiM from the psilocybin enzymatic pathway that we had previously expressed using the PK18 vector and clone them into our in-house PUS250 vector which has a tightly regulated cumate promoter system and a chromoprotein allowing easy selection. We built the new construct using Golden Gate assembly and validated our results by Sanger sequencing and LCMS of metabolites.

Figure 1: We completed a colony PCR on transformants from Golden gate assembly using primers that spanned the insert. The above gel (1% agarose 1x TAE 40 mM. Tris-acetate and 1 mM EDTA pH 8.5, 100V, 1 hour) shows DNA from the two colonies numbers 14 and 16 amplified with forward primer MVS48 reverse primer CFM23 these were approximately the right size at ~3500bp so we grew up cultures of these colonies and purified plasmid DNA for sequencing.

Figure 2: We completed resting cell metabolism assays on our two PUS387 constructs by growing up cultures of DH5a cells including the PUS387 vectors then transferring them to a Phosphate glucose solution and providing the cells with the intermediate 4-hydroxy tryptamine then allowing them to metabolise at 15C with shaking (150rpm) for 24 hours. The supernatant was then filtered and sent for LCMS analysis. The LCMS results showed peaks at 161.10732 m/z corresponding to Tryptamine the product of PSID activity on endogenous Tryptophan. The Psilocybin precursor Baeocystin was identified as a peak at m/z of 271.08422. This demonstrates the activity of the PsiD, PsiK and PsiM enzymes in the synthesis pathway from 4-hydroxytryptamine.

Sequencing of pUS387 plasmid

The result of our sequencing shows a large ~400 bp deletion in the psiM part of the construct. This is concerning, as the activity of PsiM must be intact for the production of Baeocystin, which appeared in our LCMS results. This deletion also appears in the sequence of both of our PUS387 constructs which were derived from separate colonies. It seems unlikely that such a large truncation would still result in a functional enzyme and that it could appear in both Golden Gate constructs when we had confirmed the sequence of the inserts was correct. However, diagnostic restriction digest of the PUS387 constructs shows a degraded band of ~500bp which corresponds to the appropriate size of a truncated PSIM protein. There is also the possibility that E. coli contains an endogenous methyltransferase that can catalyse the conversion of Norbaeocystin to Baeocystin. The ability of this protein to still be catalytically active requires further investigation.

Figure 3: Double restriction digestion of pUS250 and pUS387 clones (14 and 16) Constructs were digested for 30 seconds using a 900W microwave alongside a no template control using NEB's HindIII-HF and EcoRI-HF enzymes. Products were loaded onto a 1% Agarose, 40mL TAE gel prestained with Gel Green. The gel was run for 40 minutes at 100V before being imaged by UV transillumination using a BioRad XR+ Chemidoc and QuantityOne's basic imaging software.

Future Directions

Although we have demonstrated that the enzymes in the pathway are active in the construct we were unable to demonstrate the production of Psilocybin. We postulate that this is due to low levels of activity and insufficient production of Baeocystin to drive the equilibrium of the reaction towards the product. Future work will need to focus on the optimisation of the conditions for protein production. This may involve the optimisation of growing media, conditions and the use of chaperone proteins. One potential optimisation pathway could include the use of error-prone PCR and to screen for and improved protein production phenotype.

Dual Plasmid Assay

We have cloned both the PUS382 and PUS383 plasmids which contain the gene for psiH into DH5 pGro7 and performed a resting metabolism assay but are yet to receive the LCMS results. This is a key part of our experiment to recreate the entire Psilocybin synthesis pathway in a single organism and brings us closer to our aim of bulk production.

Considerations for replication of experiments

The unusual sequencing results for the construct will need to be resolved by designing primers to span the putative region of the deletion. Additionally, we need to purify the PSIM protein from our constructs and test its activity with a quantitative assay such as HPLC-MS. This will confirm whether there is a true deletion, some other mutation or an issue with our primer design and will allow us to better characterise the construct. If the deletion is confirmed we will need to rebuild the construct with the correct PSID insert and compare their activities to see if we can improve the efficiency of the construct.

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Aim

This part of the project aimed to express and characterise the activity of the PsiH enzyme in vivo before putting it into our single-cell system.

Plasmid Construction

The first step was to excise the human p450 gene from the pCW plasmid and then re-ligate the plasmid back together. This was achieved, and the plasmid pUS381 was created. From there, we could clone in our wild type PsiH gene, making pUS382, and the codon optimised PsiH gene, making pUS383. The maps of these plasmids can be seen in Figure 1, and restriction digests can be seen in figure 2.

Figure 1. Plasmid maps of pCW, pUS381, 382 and 383. (a) PCW-CYP2A6 is the parental plamid of the other plasmid constructs. It contains a human p450 (in red) and cytochrome p450 reductase (in brown). (b) pUS381 is a PCW-CYP2A6 plasmid derivative lacking the human p450 gene but still containing the p450 reductase, therefore it was used as the base plasmid from which pUS382 and pUS383 were constructed. (c) pUS382 contains the wild-type psiH from *Psilocybe cubensis* (in purple), while (d) pUS383 contains the IDT codon-optimised psiH (in purple).

Figure 2. PCR of pUS381, pUS381 and pUS383. Spanning PCR products in the left four lanes show psiH insert in pUS382 and pUS383. Junction PCRs used primers that bound internally to psiH genes. The middle four lanes show products of PCR with primers specific to wild type psiH gene. The right four lanes show products of PCR with primers specific to codon optimised psiH. ntc - no template control.

After constructing each plasmid (pUS381, pUS382 and pUS383), they were sent off for Sanger sequencing. It was confirmed that pUS381 and pUS382 were correct. However, we found out that in pUS383, there was a single nucleotide deletion at the start of the codon optimised PsiH gene. This results in a frame-shift mutation and a truncation of the protein. Unfortunately, this sequencing data only came back after we had performed all of the assays, and we did not have time to re-do them.

Protein Expression of psiH

To confirm that our protein could be expressed in E. coli, we set up cultures of cells containing the plasmids pUS381, pUS382, and pUS383. Each plasmid had two cultures – one that was induced with IPTG and one that was uninduced. The insoluble fraction from these proteins was run on an SDS PAGE gel and imaged with stain-free imaging.

Figure 3.SDS PAGE gel showing insoluble fraction of cell lysate from induced and uninduced cultures of pUS381, pUS382 and pUS383. Cells were grown in TB for 43 hours after induction with IPTG. Insoluble fraction was collected by lysing cells at 95°C for 5 minutes in SDS-containing loading buffer, then supernatant was loaded onto precast 8-16% PAGE gels. Gel was run at 120V for 70 minutes and imaged with stain-free imaging. U = uninduced. I = induced. Red boxes show probable PsiH protein.

The SDS PAGE gel clearly shows bands (highlighted with red boxes) that appear in the cell lysate from induced cultures containing PsiH – pUS382 and pUS383. They do not appear in the cell lysates of uninduced cells, or cells that had the pUS381 plasmid with no PsiH. These bands are therefore probably PsiH. PsiH is a 57kDA protein, but because it is a membrane-bound protein, it can appear on gels at a slightly different size. This explains why the PsiH bands on this gel appear to be approximately 50kDa.

As mentioned earlier, the pUS383 construct had a single nucleotide deletion, resulting in a frameshift mutation. This frameshift results in a truncation and brings the molecular weight of the protein down to 52kDa. It can be seen that the PsiH band in the pUS383 induced fraction is slightly lower on the gel than the band in the pUS382 fraction

Figure 4. SDS PAGE gel sent for Mass Spec analysis. The insoluble fraction of induced cell culture lysates were run on a gel, then stained with Coomassie blue. The bands suspected to be PsiH were cut out, destained with 40% acetonitirile/60% 20mM ammonium bicarbonate, dehydrated, re-hydrated with 12ng/ul porcine trypsin at 4C for 30min, excess trypsin was removed and 10ul 20mM am.bic was added and incubated overnight at 37C. Peptides masses were determined using MS and samples were identified using a Mascot peptide mass fingerprint search against all psi genes.

Another SDS PAGE gel of the same samples was run and stained with Coomassie blue (shown in Figure 4), to be sent off Mass Spectrometry analysis of the protein. The Coomassie staining makes it harder to see the PsiH proteins. However, it does show other relevant proteins. As indicated in Figure 4, the 70kDa cytochrome p450 reductase partner protein, which is also under IPTG inducible control, can be seen in the induced cell lysates. There is also a very large band which is likely to be the 60kDa chaperone proteins, groES, that we are using to help with PsiH expression.

Despite being able to see bands on the gel, the Mass Spectrometry results were unable to identify PsiH. This does not mean that PsiH was not present - rather, it means that PsiH was not the most abundant protein in the portion of gel that was analysed.

Testing PsiH enzyme function - Indole Assay

Figure 5. Cultures of induced cells after indole assay had been performed. From left to right: pCW, pUS381, puS382 and pUS383

After seeing that our PsiH proteins were being expressed we wanted to test their functionality, so we decided to do an indole assay. Many p450 monooxygenases can oxidize indole to make coloured compounds such as indigo. E. coli cells contain endogenous indole, so when grown in the right conditions, cells containing functional p450 monooxygenases can turn blue. This assay takes around 5 days and has many complicated components. We used a positive control, the pCW plasmid containing a human p450 monooxygenase which is known to turn indole to indigo so that we could tell if we had performed the assay correctly. We also used a negative control, the pUS381 plasmid that had no p450 monooxygenase. For each plasmid type, we grew up two cultures, and then induced one and left the other uninduced so we could compare the colours. The results of this experiment show that the induced culture of cells containing pCW turned blue, but none of the other induced cultures changed colour (as shown in figure 5). We did not expect to see a colour change in the pUS381 culture, as it contains no p450 enzyme. Retrospectively, it was also impossible that there would have been a colour change in the pUS383 culture either, because of the frameshift mutation in the codon optimised PsiH in this plasmid. Therefore, this assay only gives us information about the wt PsiH gene in our pUS382 plasmids

This assay could reveal one of two things about that wt PsiH gene - either it was expressing but was not functional, or was functional but could not oxidise indole or is doing so in such a small amount that it was impossible to see. In order to determine which of these conclusions is correct, we needed to perform another assay.

Testing PsiH enzyme function - Tryptamine assay

This assay was performed to test the ability of PsiH to catalyse the reaction that it performs in the psilocybin biosynthesis pathway, the oxidation of tryptamine to 4-hydroxytryptamine.

Figure 6. LCMS results analysing compounds in media of resting cell assay. Cells containing pUS381, pUS382 and pUS383 were grown up as per indole assay and induced with IPTG. 8 hours after induction, cells were transferred to phosphate buffer containing 2% glucose, and supplied with 1mM tryptamine dissolved in DMSO. Samples were collected 24 hours after addition of tryptamine, pelleted, and substrate sent off for LCMS analysis. 1mM samples of tryptamine standard and 4-hydroxytryptamine were also analysed with LCMS for comparison.

The LCMS results were difficult to analyse. The reason for this was that a substance of the same molecular mass and composition as 4-hydroxytryptamine was found in our ‘pure’ tryptamine standard. We also saw this compound in our negative control (cultures with pUS381) and in our culture with pUS383, which we now know has a mutated psiH. However, we did not see any of this compound in our culture expressing the correct PsiH. This compound may have been 4-hydroxytryptamine, but may also have been 5 or 6-hyroxytryptamine - LCMS is unable to distinguish between them. These results are unexpected and contrary. Firstly, they show that our PsiH gene was probably non-functional. Secondly, they may show that tryptamine can auto-oxidise to some compound that is the same or very similar to 4-hydroxytryptamine. This can potentially confuse any attempted analysis of PsiH function with this assay.

Conclusions

Ultimately, while we were able to induce E. coli cells to express PsiH, it was not found to be functional on either indole or its substrate, tryptamine. This result is not surprising, as we thought that PsiH would have to be codon optimised or harmonised to work in E. coli. Unfortunately, we were unable to test the codon optimised PsiH function in E. coli due to a frameshift mutation in the gene that drastically changed the coding sequence.

Future directions

Future directions of PsiH should be focused on further adapting the sequence of the gene for better expression in E. coli. This could be achieved by modifying the N-terminal of the gene and trying different harmonisation and optimisation methods. Additionally, different p450 reductase partner enzymes could be tested along with PsiH, such as fungal variants.

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Aim

Express PsiD, PsiK, PsiM and PsiH in DH5-alpha and assess enzyme function using LC/MS.

Figure 1: Colonies on LB-Km-Cm-Carb plates for PUS387-PUS382 cells.

We were able to successfully transform our chassis with our two plasmids, pUS387 and pUS382. With this transformation, we now had our first iteration of Magi.coli! - An organism with all wild type psi genes. This was as far as we got with our two plasmid system. We set up metabolic resting cell assays using cultures containing these constructs, however we have yet to gather LC/MS data for this assay.

While we have not achieved our ultimate aim with this experiment so far, we are happy with our progress :D.

Future directions

The most immediate direction we can take with this experiment would be performing LC/MS on our resting cell assays in the presence of excess tryptophan and determine how functional the system is.

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Aim

The aim of this experiment was to investigate and compare the growth of our chassis organism in two separate systems:

Fermenter: The culture was grown in batch mode using a BIOSTAT A fermenter (Sartorius). The fermenter had two six-bladed Rushton impellers, a stirrer speed of 750 rpm was used, the airflow rate was 1 vvm (1.5 L/min). A 1.5 L working volume was used, foaming was controlled via the addition of a 2% (v/v) solution of Antifoam A (Sigma) using an automated foam probe. The temperature was maintained at 37 ℃. Temperature and pH were logged using the in-built data-logging software at a rate of one sample per minute (Figure 1).
Lab culture: The culture was grown at 37oC with shaking at 200rpm in a 2L conical flask. A 1.5L culture volume was used.

Figure 1. Dr Dale McClure's Fermentor. Ain't it a beauty? A heavy duty beauty but still a beauty.

Figure 2. Figure 2. 2L fermenter vs 2L conical flask culture growth. 1.5L of TB broth supplemented with phosphate buffer, trace metal elements and thiamine in each system were inoculated with 100mL of DH5-alpha cells grown overnight in glucose supplemented LB. OD600 was sampled periodically and used to determine density (cells/L)

The results of this exercise demonstrated that the fermentation system is vastly superior in cultivating the growth of our chassis in our media of choice. The number of cells per litre of culture grown was 7.43 times higher in the fermenter than the lab culture over 7 hours. Growth in the fermented culture was so effective the culture had exhausted its nutrient supply by hour 6 resulting in a decline steady decrease in cell density.

Going forward we would like to optimize our Magi.coli for expression at these conditions to take advantage of this stark difference in growth.

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Aim

The aim of this experiment was to determine which type of codon harmonisation/ optimisation (rank-order method, absolute frequency method, relative frequency method and IDT codon optimisation) would improve the expression of the VVD protein compared to the wild-type codon usage. CH1 was harmonised by rank order, CH2 by nearest frequency, CH3 by relative proportion and CH4 used the IPT codon optimisation tool available here. The harmonisation method descriptions can be found under the documentation for software. The results of this experiment would then be used to codon harmonise our psi genes to improve their expression.

Figure 1. Spanning PCR of the codon harmonised/optimised VIVID inserts in the pk18 vector showing band of correct size at ~624bp. Agarose gel electrophoresis was conducted on 1% agarose gel in 1X TAE for 50 min at 100 V.

Figure 2. Wild type, CH1, CH2, CH3, and CH4 compared to M130I under long-wave UV (~350nm) on charcoal LB agar demonstrating fluorescence of all the Vivid protein types.

Figure 3. We conducted a fluorescence assay using a TECAN Spark microplate reader Escherichia coli TOP10 cultures containing WT, CH1, CH2, CH3, and CH4 in pK18. As shown the codon harmonisation and optimisation did not improve fluorescence. The wild type vivid remains significantly (p<0.01 post hoc anova with Bon Ferrony test) more fluorescent than the variants we created.

Considerations for Replication

Through this experiment, we have concluded that codon harmonisation and optimisation may not be the most efficient method for improving the activity of these fluorescent proteins. We had significantly more success with our error-prone PCR improvement (see Error-Prone PCR of Vivid). Codon harmonisation and optimisation are essential for other expression applications in particular where the speed of translation may need to be limited to ensure appropriate protein folding.

Future Directions

We experienced some difficulty in maintaining the stability of the pk18 VIVID constructs in TOP10 cells. This may be since pk18 is a high copy number plasmid with constitutive expression and may have caused stress to the TOP10 cells. Stability may be improved using an Inducible rather than constitutive vector.

Literally 3 months of painful work with failures left and right...

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Aim

The aim of this experiment was to use error-prone PCR to generate a more fluorescence version of the weakly fluorescent Vivid protein from the fungi, Neurospora crassa.

Error prone PCR

Error prone PCR was performed on the weakly fluorescent codon optimised vivid gene. The PCR products, when ligated in plasmids and transformed into E. coli, yielded 12 colonies that were brighter than the rest (Figure 1).

The sequencing results of the vivid genes from these bright colonies revealed that there was a common mutation in the majority of these genes (see Figure 2). This mutation modified the methionine at residue 130 to either isoleucine (found in 5 of the mutants), a threonine (found in three of the mutants), or a leucine residue (found in one of the mutants).

Figure 2: Sequence alignment of error-prone PCR mutants with original VVD gene, with the region of common M130 mutations circled.

When a G-Block of the vivid gene containing a Methionine to Isoleucine change at residue 130 was cloned into E. coli and compared to vivid genes without the mutation, it was seen to be much brighter (Figure 3). This confirmed that it was the mutation away from methionine at residue 130 that improved the fluorescence of the protein.

Figure 3: The Met130Ile vivid mutant compared to the weakly fluorescence wild type vivid and 4 codon harmonised vivid variants (CH1, CH2, CH3 and CH4) under long wave UV light.

A fluorescence assay was performed that compared the original codon harmonised fluorescence Vivid, the wild type fluorescent Vivid, an empty plasmid, and the new brighter Met130Ile Vivid protein (see Figure 4). This shows the extent to which the single amino acid mutation increased the fluorescence of the protein.

Figure 4: Graph of TECAN fluorescence data from the VVD-Met301 g-block cells and the original VVD_wt and VVD_CH4 cells.

Lastly, the excitation and emission spectra of the new Met130I Vivid protein was measured (figure 5). This spectrum is remarkably similar to the spectra of CFP, meaning that our fluoroprotein can be used as a substitute for this fluoroprotein.

Figure 5: Excitation and Emission spectra of the Met130Ile Vivid protein.

Conclusion

Error prone PCR was used to create a more fluorescent version of the fungal protein, Vivid.

Future directions

Future work should focus on characterising the protein so that its usefulness can be compared to other fluoroproteins can be established.