Team:SDU-Denmark/Results
Results
Abstract
On this page, you can read about how we achieved a succesful production of psilocybin.
Introduction
PsiDKM
Assembly of PsiDKM
The genes PsiDK and PsiM were assembled using overhang PCR. The enzymes PsiD, PsiK and PsiM are necessary to produce psilocybin from 4-hydroxy indole. The successful assemble of PsiDKM was observed on a 1% agarose gel.
Agarose gel run with assembled PsiDKM: Bands at ~3500bp indicated PsiDKM. Lane 2, 4, 6 and 8 assembled PsiDKM
The expected length of PsiDKM is 3360bp, and since the observed bands have a size of ~3500bp, this indicates that PsiDKM has been successfully assembled.
Ligation into backbone
The backbone pSB1K3 was transformed into E. coli Top10 strain. Afterwards the backbone was ligated with LacI and the medium constitutive promoter (J23106) and transformed into E. coli Top10 strain. The insert of J23106-LacI was amplified using colony PCR and observed on a 1% agarose gel. The expected length of the LacI + medium constitutive promotor is 1598bp. The length of the bands is ~1600bp, which confirms the construction of pSB1K3+J23106+LacI.
Agarose gel with part pSB1K3+J23106+LacI: Bands indicate LacI + medium constitutive promoter inserted in backbone. Bands of Lane 2 to 12 indicates pSB1K3+J23106+LacI.
The assembled PsiDKM was ligated with the backbone pSB1K3+J23106+LacI and transformed into E. coli Top10 strain for the construction of pSB1K3+J23106+LacI+T7lac+PsiDKM. The successful assembly of pSB1K3-LacI-T7lac-PsiDKM and the transformation into E. coli Top10 strain were confirmed using colony PCR. The expected length of pSB1K3-LacI-T7lac-PsiDKM is 7022bp. The colony PCR confirm the assembly with bands ~6000bp, meaning that lane 11 does not confirm the assembly of pSB1K3-LacI-T7lac-PsiDKM.
Agarose gel with pSB1K3+J23106+LacI+T7lac+PsiDKM: Bands ~6000 indicate assembling of pSB1K3+J23106+LacI+T7lac+PsiDKM: The lanes correspond to the different colonies obtained from the transformation.
Sequecing
Sequencing by Eurofins confirm the correct assembly of PsiDKM. Sequencing of the assemble pSB1K3+J23106+LacI+T7lac+PsiDKM shows a 10bp deletion in PsiD. However, the results have later shown that pSB1K3+J23106+LacI+T7lac+PsiDKM is functional and can produce psilocybin. It is possible that the mutation has arisen during the PCR amplification used to prepare the sequencing samples.
Transcription and translation control
After successful transformation of pSB1K3+J23106+LacI-T7lac-PsiDKM into E. Coli ER2566, the successful transformants was grown and induced with IPTG to be able to test the expression of PsiDKM using real-time qPCR. The results showed the gene expression of PsiD, PsiK, PsiM and T7-polymerase which increased after IPTG induction. However, the expression of all genes decreased after 3 hours, which indicates that the cells might be stressed after induction with IPTG, which might affect gene expression. The optimal gene expression is therefore seen after either 1- or 2 hours.
qPCR analysis of PsiDKM: Gene expression of PsiD, PsiK and PsiM and the T7-polymerase.
Colour reaction
To test the initial production of psilocybin from 4-hydroxyindole with only PsiDKM, we performed a colour reaction using the Ehrlich reagent. This is a compound known for its ability to bind indoles, which is a functional group that characterizes psilocybin. Binding to different indoles results in different colors.1 However, every single intermediate of the pathway including the precursor 4-hydroxyindole consist of an indole. In addition, indoles are found in many naturally occurring indoles such as amino acids, such as tryptophan. As the first samples were not divided up between cell and media, a huge uncertainty is related to using this method. However, when conducting this test, we observed a distinct variation between our transformants and the control (normal ER2566). Both groups had undergone the exact same treatment (addition of 4-hydroxyindole, methionine, serine and induction with IPTG), which is why we found the outcome intriguing. The four experimental samples with ER2566 expressing PsiDKM turned instantly black once the Ehrlich reagent was added compared with the control constituting of wild-type ER2566 which remained red. This implied a significant difference in the occurrence of indoles between the two groups.
Color reaction with Ehrlich reagent: From left is shown 1: ER2566 with PsiDKM, 2: Wild-type ER2566, 3-5: ER2566 with PsiDKM.
LC/MS
To test whether the PsiDKM construct was able to produce psilocybin or not, 4-hydroxyindole was added to cultures containing our plasmid with PsiDKM and induced with IPTG. The freeze dried and prepared samples were analyzed via Triple Quad Liquid Chromatography-Mass Spectrometry. Fragment ions as well as standard curve used for quantification are described in detail in the LC/MS section within results.
Both fragment ions are present in all three PsiDKM samples, this is visualized on the figure below, indicating that the system can produce psilocybin when 4-hydroxy indole is added. Two out of three can be quantified using the standard curve, these calculations are shown below. These indicate that PsiDKM system #1 can produce 0.65ng/mL psilocybin and PsiDKM system #2 is able to produce 2.4ng/mL psilocybin.
LC/MS of PsiDKM: Arbitrary areas of three different PsiDKM samples of fragtment ions m/z=204 and m/z=160.
PsiH
PsiH/CPR
The PsiH and CPR biobricks were included in an overhang introducing PCR. As the biobricks did not include a T7 promotor or RBS, an overhang introducing PCR is run using PsiDK as template to obtain the T7 promoter and RBS from this biobrick. In this way, the T7-promoter and the RBS with an overhang are obtained without parts of PsiDK-gene.
As seen on Figure 1 it is seen that the overhangs 2 to 7 for PsiH have been successfully added to T7lac as bands are observed at 160 base pairs. In relation to CPR the following results are seen (Figure 2) for T7lac overhang-introducing PCR.
Here it seen that the overhang introducing an N-terminal-deletion as well as N-terminal 14 has been successful as bands are observed at 160 base pairs (see Figure 2 A and B, lane 2-5)
In the next overhang PCR different overhangs are introduced to the PsiH and CPR. The results can be seen on (Figure 3 A, B, C and Figure 4 A and B), as bands are observed at 1700 base pairs.
Based on this it is seen that the overhang introducing PCR for N-terminal 3, 4, 5, 6, 7 for PsiH have been successful (see Figure 4). In relation to CPR the N-terminal overhangs 5, 14 and deletion could be confirmed (see Figure 3).
This is seen based on the predicted band lengths simulated in Snapgene. The T7lac promoter with the introduced 5’ overhang was assembled with the corresponding PsiH or CPR 5’-overhang part using overhang PCR (see Figure 5 and 6). In this way, biobricks with T7-promoter, RBS and CPR or PsiH with all the different N-terminals, were obtained.
After assembling the T7lac promoter with the different PsiH and CPR variants, they were cut, and introduced in a backbone. These ligation products were transformed into E. coli Top10 and confirmed with colony PCR. Here it can be seen that for instance PsiH with the N-terminal 6 (BBa_K4059041) and CPR with N-terminals 5 ((BBa_K4059042) have successfully been introduced into Top10 (Figure 7, 8).
The plasmids were purified from the succesful E. coli Top10 transformants and transformed into E. coli ER2566 which functions as the protein production strain. These transformants were selected using negative selection with antibiotic plates. These single transformants are included in RT-qPCR experiments.
The results for PsiH demonstrate that the lacI inducible promoter system and thus the expression of PsiH was clearly induced when IPTG was added. However, the negative control which was not induced with IPTG showed expression as well. This indicated that the promotor system was leaky, resulting in continued low level expression of the target genes. Here it must be noticed that the induction level of for instance CPR with a N-terminal deletion (Figure 10) and PsiH with N-terminal 5 (Figure 9) decreased over time. This might be due to cell stress caused by heterogenous protein expression which resulted in reduction of induction and expression. For the transformants not demonstrating induction, this might be due to toxicity caused by the indoles resulting in induction deficiency.
The following figures show a progress overview of testing different N-terminals for both PsiH and CPR:
CPR Assay
The assay was used to test the activity of CPR alone in all the successful transformants – this means CPR with a deletion of the N-terminal and the two N-terminal modifications 5 and 14 (BBa_K4059056, BBa_K4059051 and BBa_K4059054).
Due to the limitations of the assay (this was originally made for eukaryotic CPR) combined with the duration in which the assay was performed (samples needed to be frozen several times due to lack of time), some adjustments and mistakes might have influenced the results (Figure 13). However, when testing the dilutions containing 10 µL of the given CPR, a coherence was observed when looking at the change of absorbance after exactly 8 minutes when normalized to T=0.
As depicted, the assay indicates that CPR5 is the most catalytic effective version.
GroEL/ES
The biobrick of GroES/EL was ligated into the backbone pSB1C3 + I0500 (pBad/araC promoter), which had chloramphenicol resistance. The resulting plasmid was transformed into E. coli Top10, and a colony PCR was performed on several colonies. The length of GroES/EL is expected to be 3671bp. The length of the bands from colony B, C, D, E and F were ~3600bp, which confirms the construction of pSB1C3 + BBaI0500 with GroES/EL. Further, colony A showed the same length as the control, which is pSB1C3 + I0500 that had not been cut. This indicates that colony A may be a relegation (see figure 22).
The assembly of GroES/EL with the backbone pSB1C3 + I0500 was send to sequencing by Eurofins. The sequencing of GroES/EL showed the expected sequence.
A SDS on GroES/EL was made to separate the proteins, and thereby show their protein expression. Before SDS, an overnight culture of GroES/EL in Top10 is induced with arabinose. The two proteins of GroES/EL were expected to have different lengths. GroES is expected to be 10.4 kDa, where GroEL is expected to be 57.3 kDa. The results showed heavy bands between 70 kDa and 55 kDa for the samples induced with arabinose. These bands may indicate that GroEL is present. However, GroES is not seen on the gel. This may be due to that the gel had run for too long, since the band for 10 kDa of the ladder is not seen.
Miniprep of GroES/EL was later used for triple transformation with PsiH and CPR in ER2566. GroES/EL is combined in triple transformants to improve the folding and thereby the function of PsiH.
Production of Psilocybin
Triple Transformation
The successfully assembled biobricks moved to different resistance backbones in order to allow the creation of the following triple transformant systems enabling de novo synthesis of psilocybin and analyzation of PsiH and CPR performance.
CPR with LacI and T7-promoter was moved from J61002 backbone to the backbones pSB1C3 and pSB1K3 and introduced into TOP10. These new plasmids were transformed into ER2566. In a stepwise approach the remaining two plasmids were introduced. Colony PCR was used to confirm the presence of all three plasmids in triple transformants, by using gene specific primers for the three plasmids. Three bands were seen on the correct respective lengths of around about 3200 base pairs for the psiH plasmid, 3800 base pairs for the CPR plasmid, and 5000 base pairs for the PsiDKM plasmid (Figure 14).
Double/Triple transformants MS analysis
The main goal of PsiloAid was to create a de novo biosynthesis of psilocybin in E. coli. We decided to use triple quad Liquid Chromatography-Mass Spectrometry (LC/MS) to analyse and possibly quantify the amount of psilocybin produced by our system as well as detecting if our PsiH construct could produce 4-hydroxy-tryptamine. To determine whether psilocybin was present in our samples we had to look for specific fragment ions in the spectrum. We chose to look for two distinct ions; m/z=204 which is the remaining fragment ion left after the loss of dihydroxy(oxo)phosphonium, this fragment ion is shown on figure 15 The other ion that we are looking for in the spectrum is m/z=160 this is the result of the loss of both the dihydrogen phosphate and two methyl groups (see figure 16). Both the m/z=202 and m/z=157 fragment ions are both characteristic for psilocybin. Both ions are present in 11 of our samples, and it is therefore concluded that the transformants in these samples could produce psilocybin. A retention time of 5,105 and m/z=157 and m/z=202 is used on figure 17. Due to a flaw in the instrument, each measurement was consequently 3 Daltons lower than expected which is why the m/z values don’t reflect the ones reported in the literature. This was established by using a standard of psilocybin. The flaw is also indicated on figure 21. The results did not indicate a specific pattern regarding which constructs were able to produce psilocybin, but they did indicate that the triple transformants, consisting of PsiDKM, PsiH and CPR, were able to successfully produce psilocybin, and our main goal (de novo biosynthesis of psilocybin) is therefore obtained. We were also able to quantify the amount of psilocybin, however this quantification was challenged by a standard that were delayed due to the Covid-19 pandemic and unfortunately also of poor quality. This standard was used to create a standard curve (figure 18) with varying dilutions with known concentrations of psilocybin. This was used to quantify the amount of produced psilocybin. As depicted the standard concentrations variesd between 0.5 ng/mL to 100 ng/mL. The coefficient of determination, which indicates the degree of correlation between the two parameters, is estimated to R² = 0.9893 which verified the assumption of a linear correlation, as it was above 0.95, making the correlation of statistical significance. The given equation is applied to calculate the concentration of the sample with PsiDKM with a measured area over of 74 Arbitrary area units when looking at only m/z = 157:
Due to the poor quality of the standard sample, we were only able to quantify the amount of produced psilocybin in the sample only containing PsiDKM and unfortunately not any of the constructs containing PsiH. The PsiDKM sample contained 2.4 ng/mL. Instead, we validated the remaining samples based only on the arbitrary area units. When comparing the original sequence of PsiH in double transformants designed in 2019, PsiH WT (J23106+I732820+BBa_K314004), and our double transformants containing PsiH7 (BBa_K4059043), a noticeable difference in the yield of psilocybin was found. The arbitrary area units for the modified PsiH were 44 and 28 whereas they were 14 and 9 for the wild-type. This is a threefold improvement and is visualized on figure 17.
Figure 15 illustrates the fragment ion located at m/z=204
Figure 16 Illustrates the fragment ion located at m/z=160
Figure 17 Illustrates the samples that contains psilocybin. The yellow bars indicate m/z=202 arbitrary area and the orange bars indicate m/z=157 arbitrary area.
Figure 18 Illustrates the standard curve with known concentrations of psilocybin ranging from 0,5ng/mL to 100ng/mL.
We tested several single transformants (only PsiDKM), double transformants (PsiDKM + PsiH) and triple transformants (PsiDKM + PsiH + CPR) which varied in the composition of modified enzymes. Of all the different modifications, these were the only ones that have successfully been transformed into E. coli ER2566. The different samples are shown in the table below as well as the result of the analysis.
Table 1 illustrates the different samples as well as the result of the analysis
A standard for 4-hydroxy tryptamine was not obtained as we were not able to receive the sample within the deadline due to the Covid-19 pandemic. We therefore used 5-hydroxy tryptamine (serotonin) as theoretical values of fragmentation ions are researched and available. We chose to focus on the three most significant fragment ions. The fragment ions are m/z=160, m/z=133 and m/z=117. As mentioned, the instrument had a fault and measured 3 Dalton less than the theoretical value when the m/z is above 118. This meant that we were looking for a retention time of 4.928 and m/z=157 and m/z=130 when we analysed the spectra. We concluded that there was 4-hydroxy tryptamine present in seven of our samples. Most of the results seemed to be random, although some improvement is associated with the addition of CPR to the system. However, none of the results look to be consistent. For instance, PsiH7 ((BBa_K4059043) together with CPR5 ((BBa_K4059051) produces a higher yield of 4-hydroxytryptamine compared to single transformants (this matches the results from the CPR assay), but when PsiH is combined with CPR Del (BBa_K4059056), it lead to a significant reduction of the yield even though the same modification of CPR worked quite well with PsiH. WT (J23106+I732820+BBa_K314004). This is visualized on figure 19. In general, the data indicates that CPR5 is our best candidate. This is also reflected in the CPR-assay in which CPR5 showed to possess the greatest activity. PsiH7 is also one of our best candidates so far, since it showed a fine yield in both the production of psilocybin as well as 4-hydroxyindole. Though, those two have not been tested together.
Figure 20 Illustrates the samples confirmed to produce 4-hydroxy tryptamine. As for the psilocybin producing system, we analyzed samples containing single transformants (PsiH), double transformants (PsiH + CPR) and triple transformants (PsiH + CPR + chaperone) in terms of the system synthesising the intermediate 4-hydroxytryptamin. Of all the different modifications, these were the only ones that have successfully been transformed into E. coli ER2566. The different samples are shown in the table below as well as the result of the analysis
Table 2 illustrates the different samples as well as the result of the analysis
We did experience several difficulties related to the measurements carried out by LC/MS. We were only able to detect psilocybin in the samples with supernatant containing only media. This may indicate that the centrifugation is too violent (3000 G for 20 min). It may also be due to the phase separation that we observed using a 50% acetonitrile and 1% formic acid solution. In general, just the processing of the samples may have been problematic.
Transport
Assembly of contructs
The GFP-terminal constructs were assembled through restriction and ligation. The first step in the assembly was to cleave Bba_J61002, and C-terminal GFP fusion protein and N-terminal GFP fusion protein with XbaI and PstI. We verified this cleavage by gel electrophoresis in 1 % TAE agarose gel. The simulated gel below in snapgene shows the expected outcome of the gels.
Well 1 shows the cleaved Bba_J61002 in which the fragments have lengths of 895 bp and 2053 bp. Well 2 shows the cleaved N-terminal GFP fusion protein with an expected length of 933 bp and two small fragments of 46- and 21 bp. Well 3 shows the cleaved C-terminal GFP fusion protein with an expected length of 936 bp and 46- and 21 bp.
Our results indicated that the cleavage was successful by showing the expected lengths of fragments except for the small fragments from the terminal GFP genes from IDT that were run out of the gel. Well 4 C-terminal GFP fusion cut Well 5 cut Bba_J61002. Well 6 cut C-terminal GFP fusion cut.
Well 2: N-terminal GFP fusion cut Due to the gel verification of our cleavage products we moved on to the next step of our project in which these products were ligated and transformed into E.coli K12.
Ligation into backbone
The cleavage products were ligated and transformed into E. coli Top 10 strain. The insertion of GFP-constructs (BBa_K4059034 and BBa_K4059035) were amplified using colony PCR and confirmed on a 1% agarose gel.
The simulated gel below in snapgene shows the expected outcome of the gels created in Snapgene. Well 1 shows N-terminal GFP fusion protein ligated to Bba_J61002 in which the insert was amplified between prefix and suffix to an expected length of 1145 bp. Well 2 shows C-terminal GFP fusion protein ligated to Bba_J61002 in which the insert was amplified between prefix and suffix to an expected length of 1148 bp.
The assembled C-and N-terminal constructs (BBa_K4059034 and BBa_K4059035) in Bba_J61002 was verified on an 1 % agarose gel as shown in figure 27 below created with BioRender.com.
The results showed that one colony of N-terminal GFP fusion ligated with Bba_J61002 in well 1 was relegated while the others showed at the expected length. Well 1-5 shows colonies from transformation with ligated Bba_J61002 and N-terminal GFP fusion amplified by prefix and suffix primers. Well 6-9 shows colonies from transformation with ligated Bba_J61002 and C-terminal GFP fusion amplified by prefix and suffix primers. Well 11 and 12 shows religated plasmids with Bba_J61002.
Insertion of transporters
The transporters were inserted into our GFP constructs (BBa_K4059034 and BBa_K4059035) through restriction and ligation. Before this cleavage the BamHI sites were introduced to our biobricks with transporters using the primers specific for introduction to either the N-terminal or C-terminal of the transporter:
The C-terminal and N-terminal transporters to be inserted into this construct were cut using BamHI and NdeI and ligated.
The N-terminal GFP construct (BBa_K4059035) and transporters to be inserted into this construct were cut using BamHI and SpeI but the fragments cleaved by this are non-detectable and therefore not simulated or shown on gel.
The small fragments are not detectable on an electrophoresis gel, wherefore these are not included as we had to purify the product and move onto ligation without seeing cleavage directly. Due to the gel verification of our cut products we moved on to the next step of our project in which these products were ligated and transformed into E.coli K12.
Ligation into backbone and transformation
The products from the restriction were ligated and transformed into E. coli Top 10 strain. The insertion of transporters were amplified using colony PCR and confirmed on a 1% agarose gel.
The simulated gel below in snapgene shows the expected outcome of the gels. Well 1 shows PsiT1 (BBa_K4059032) coupled to the N-terminal GFP construct (BBa_K4059035) with expected length of 2548 bp of the amplified insert. Well 2 shows PsiT2 (BBa_K4059033) coupled to the N-terminal GFP construct (BBa_K4059035) with expected length of 2704 bp of the amplified insert. Well 3 shows PsiT1 (BBa_K4059032) coupled to the C-terminal GFP construct (BBa_K4059034) with expected length of 2558 bp of the amplified insert. Well 4 shows PsiT2 (BBa_K4059033) coupled to the C-terminal GFP construct (BBa_K4059034) with expected length of 2714 bp of the amplified insert.
Gel with N-terminal GFP construct (BBa_K4059035) transformants. Well 1-5 shows PsiT1 transporter (BBa_K4059032) with this GFP construct (BBa_K4059035) but it appears that only well 1 is a successful transformation. Therefore, the PsiT1 coupled transporter was taken further to experiments. Well 6-9 are the PsiT2 transporter (BBa_K4059033) with GFP construct (BBa_K4059035) but all appear to be religations due to a shorter length than expected. Well 10-11 are religations of the backbone for control. Well 11-14 are C-terminal GFP construct (BBa_K4059034) transformants with PsiT1 transporters but all appear to be religations due to the small size.
Gel with C-terminal GFP construct (BBa_K4059034) transformants well 1-6 shows PsiT1 transporter (BBa_K4059032) with C-terminal GFP construct while well 8 is a religation. Only well 7 was a successful transformation with the expected length while the rest show either comparable length to control sample or shorter lengths indicating that something went wrong in these. No transformation with C-terminal construct (BBa_K4059034) and PsiT2 (BBa_K4059033) was successful but due to our microscopy results of the N-terminal fusion elaborated further in the next step it was decided to stop the construction of this transformant here. We moved on with the successful transformants to perform fluorescence microscopy.
Fluorescence microscopy
This fluorescence image shows PsiT1 (BBa_K4059032) with GFP coupled to the N-terminal (BBa_K4059035). It is evident that only one cell fluoresces while the rest do not. However, the one cell that fluoresces shows an aggregation of GFP intracellularly. This indicates that the transporter has not localized to the membrane.
This fluorescence image shows PsiT2 (BBa_K4059033) with GFP coupled to the N-terminal (BBa_K4059035). It is evident that most cells fluoresce. However, the fluorescence shows an aggregation of GFP intracellularly. This indicates that the transporter has not localized to the membrane.
This fluorescence image shows PsiT1 (BBa_K4059032) with GFP coupled to the C-terminal. It is evident that most cells fluoresce. However, the fluorescence shows an aggregation of GFP intracellularly. This indicates that the transporter has not localized to the membrane.
The microscopy images show that the transporters are not localized to the membrane. However, Clare Kirkpatrick made us aware that sometimes cleavage can occur intracellularly between GFP and the coupled proteins.
Western blot
We carried out a western blot with Anti-Green Fluorescent Protein from Roche Diagnostics GmbH and secondary polyclonal Goat Anti-Mouse Immunoglobins/HRP for GFP to see if this is cleaved from the transporters.
The image below shows an expected outcome if cleavage had not occurred showing the wells for easier understanding of the expected results. The gel shows PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa on the left. The first well contains a C-terminal construct (BBa_K4059034) linked PsiT1 (BBa_K4059032) which has a theoretical size of 79.2 kDa.
Well 2 contains normal Top10 which is not expected to contain any GFP that the antibody can bind to and therefore no blot results are shown. Well 3 contains a N-terminal construct (BBa_K4059035) linked PsiT1 (BBa_K4059032) which has a theoretical size of 79.2 kDa. Well 4 contains a N-terminal construct (BBa_K4059035) linked PsiT2 (BBa_K4059033) which has a theoretical size of 84.3 kDa according to snapgene.
The image below shows an expected outcome if cleavage had not occurred showing the wells for easier understanding of the expected results. The gel shows PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa on the left. The first well contains a C-terminal construct (BBa_K4059034) linked PsiT1 (BBa_K4059032) as it could theoretically be cleaved to leave PsiT1 and GFP. GFP has a theoretical size of 27.4 kDa according to Snapgene while PsiT1 (BBa_K4059032) has a theoretical size of 51.8 kDa according to snapgene.
Well 2 contains normal Top10 which is not expected to contain any GFP that the antibody can bind to and therefore no blot results are shown. Well 3 contains a N-terminal construct (BBa_K4059035) linked PsiT1 as it could theoretically be cleaved to leave PsiT1 and GFP. GFP has a theroetical size of 27.4 kDa while PsiT1 (BBa_K4059032) has a theoretical size of 51.8 kDa. Well 4 contains a C-terminal construct (BBa_K4059034) linked PsiT2 (BBa_K4059033) as it could theoretically be cleaved to leave PsiT2 (BBa_K4059033) and GFP. GFP has a theoretical size of 27.4 kDa according to Snapgene while PsiT2 (BBa_K4059033) has a theoretical size of 56.8 kDa according to snapgene.
Our actual western blot is shown below with the wells corresponding to the theoretical images.
It is evident that no PsiT1 (BBa_K4059032) linked to C-terminal construct was present in neither cleaved or non-cleaved form. It is likely that something went wrong with this sample but due to time limitations, it was not possible to repeat the experiment. Well 3 and 4 shows binding of antibody at around 27 kDa, which corresponds to GFP. However well 4 also shows a large amount of other sizes corresponding to other fragments. These fragments were not identified but as it is a known fact that the E. coli genome encodes serine proteases, it would be likely that these correspond to cleavage by these. The linker chosen BBa_K1486004 contains serines that could also be cleaved and therefore show the free GFP in the cytosol.
Knockout of E. coli native transporters
P1 phages were used to create wild type (WT) phage lysate of ER2566 E. coli. With the created WT phage lysate, specific lysates were made of the mutants arcA and yhjV from the two knockout strains JW0452 and JW3508, respectively. Transduction was performed with different amounts of lysates and seen below are results of colony PCR. The colonies are named as follows: Knockout mutant-Lysate amount–Duplicant number.
Gel electrophoresis of colony PCR products did not show a significant change in bp size, this is as expected since the removed gene and inserted kanamycin cassette is approximately the same length. O/N cultures of the colonies were created, however as acrA mutant showed no growth in LB media, this mutant was excluded from the subsequent knockout experiments.
For this reason, only yhjV mutants were used for transformation with the FLP recombinase pPC20. From the colony PCR we expected a size of 300bp for the knocked-out gene, exactly 1200bp less that the WT of 1500bp.
Transformation of PsiDKM into knockout strain
Next goal was a knockout mutant capable of producing psilocybin from 4-hydroxyindole.
Transformation was performed with different samples of BBa_K4059010. By inducing with IPTG we were able to test the enzymatic activity of PsiDKM using cDNA synthesis and real-time qPCR. RNA was purified prior to this and confirmed on a 1% agarose gel. Results showed the RNA purification was successful, as 3 bands can be seen but with the first band being very faint. The three bands are 5S, 16S and 23S ribosomal RNA.
The new ΔyhjV ER2566 strain containing BBa_K4059010 was supplied with 4-hydroxyindole to test the potential effect on the psilocybin production. Ideally for the knock-out mutants, no amount of psilocybin should be detected in the media whereas all should be located inside the cells. LC/MS was used for this, however, there were no reads from the samples of PsiDKM knockout strain. This indicated the knock-out strain was unable to produce psilocybin. Further optimization and testing need to be done before a conclusion can be reached.
Future perspectives:
Due to time constraints, our iGEM team was not able to create the ΔacrA ER2566 mutant, which would have been used to limit E. coli’s natural transportation of indole intermediates in the pathway out of the cell. Results also indicated the ΔyhjV strain containing BBa_K4059010 cannot produce psilocybin, however, this experiment should be redone to further confirm if the lack of yhjV stops the production of psilocybin or if there was an error in our transformant.
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