iGEM 2021 — Team SDU-Denmark

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# Team:SDU-Denmark/Notebook

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# Notebook

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# Abstract

Here we describe our weekly notebook regarding what we’ve done in the lab, from the beginning of the project and all the way till Wikifreeze week!

## Introduction

This is our notebooks for the entire project.

## June

*Week 23 – June 7th to June 13th*
Learned how to streak bacteria from freeze stock onto a LA plate and how to make ON cultures from the bacteria grown on the LA plate. From this ON culture we made our first freeze stock of 250µL 50% glycerol and 750µL ON culture.
This was our first freeze stock, named freeze stock 1.
This week a ON culture of an AMP resistant Top10 strain from SDU-iGEM2019’s part 11 was prepared, which was miniprepped to make Part 1 (Bba\_J61002 with Bba\_J23102) and made a freeze stock from freeze stock 2.
To learn how to preform gel electrophoresis, part 1 (Bba\_J61002 with Bba\_J23102) was loaded onto a 1% agarose gel.

*Week 24 – June 14th to June 20th*
It was taught how to use restriction enzymes to cut a plasmid, which in the future will be used for transformation.
 After cutting the plasmid it was loaded on a gel and purified afterwards. Solutions of 0.1M MgCl2 and 0.1M CaCl2 was prepared for use in CaCl2 competent cells.
Also learned how to make our own LA-plates with selection, here using kanamycin.

*Week 25 – June 21st to June 27th*
To further prepare for transformation, it was taught how to make TSB buffer. ON culture of SDU-iGEM 2019’s part 10 was miniprepped to create part 2 (Bba\_J61002 with Bba\_J23106).
From the iGEM distribution plate 3 2019 well 2M, which contained the LacI gene (Bba\_I732820), part 3 (Bba\_I732820 in pSB1C3) was taken. This week the first transformation was made, here part 3 (Bba\_I732820 in pSB1C3) was transformed into a Top10 culture. After transformation the bacteria was plated on LB plates with ampicillin, however, no colonies formed due to using the wrong antibiotics.
We cleaved the part 2 (Bba\_J61002 with Bba\_J23106) we had created this week. First, we cleaved using SpeI and XbaI, which was not correct, hereafter it was redone correctly.

*Week 26 – June 28th to July 4th*
This week we transformed part 3 into a Top10 culture again and plated the bacteria out on chloramphenicol plates. A freeze stock was made of the Top10 containing part 3 (Bba\_I732820 in pSB1C3).
Using Taq PCR and electrophoresis we also confirmed our primers oligo 3 (suffix primer) and oligo 4 (prefix primer) are correct by testing them on our part 2 (Bba\_J61002 with Bba\_J23106).
We learned how to ligate two parts together, here with part 2 and part 3 (Bba\_I732820 in pSB1C3) to a new part called part 4 (Bba\_J61002 with Bba\_I732820). The ligation product was transformed into Top10 culture.

**Group DKM:**
This week we also received our first biobricks PsiDK and PsiM, they were amplified and purified so they were ready for use (BBa\_K4059010).

## July

*Week 27 – July 5th to July 11th*
**Group PsiDKM:**
This week, using Phusion PCR mix, we put together our biobricks PsiDK and PsiM to create PsiDKM (BBa\_K4059010). We cleaved our part 4 (Bba\_J61002 with Bba\_I732820) (vector) and PsiDKM (insert) and ligated them together. The ligation product was transformed into Top10 and named part 5 (BBa\_K4059037). The bacteria was plated and haven harvested for use in colony PCR. Correct transformed bacteria from the colony PCR was made into a ON culture. The cultures were induced with IPTG as it should induce PsiDKM. To test the induction, we peformed SDS-page.
The SDS-page results were unfortunately not conclusive as the expected bands were present both in IPTG induced samples and non-induced samples.
**Group PsiH:**
We transformed the future backbone for our chaperone proteins into Top10 (SOP14). The backbone was pSB1C3 containing biobrick BBa\_I0500, there BBa\_I0500 consists of a pBad/araC promoter system for *E. coli*. The plasmid was obtained from the iGEM 2019 plate 5 well 18M. From the successful transformation of pSB1C3+BBa\_I0500, an overnight culture was grown (SOP4) and used for miniprep (SOP7) and freeze stock preparation (SOP5) for future use. Furthermore, we prepared a freeze stock of *E. coli* strain ER2566, our future protein expression strain.

*Week 28 – July 12th to July 18th*
**Group PsiDKM:**
Gel electrophoresis showed part 5 (BBa\_K4059010) did not have the correct length, therefore we redid the ligation to recreate part 4 (Bba\_J61002 with Bba\_I732820) and ligated it together with our PsiDKM insert again. The ligation product was transformed into Top10 again.
**Group PsiH:**
pSB1C3 backbone with BBa\_I0500 was cut with the FastDigest enzymes SpeI and PstI (SOP12) to prepare for insertion of GroES/EL biobrick. The gel-electrophoresis (SOP 8) showed a band just above 3000 bp (expected length 3270 bp), which was purified.
Furthermore, the backbone J61002 which contains medium cons. promoter (J23106) and LacI (I732820) was miniprepped from ON-culture (SOP 7) and cut with SpeI and PstI (SOP 12). The gel-electrophoresis (SOP 8) showed bands at just above 3000 bp (expected length 3310bp), which was purified (SOP 6). The purpose was to prepare a backbone for PsiH and CPR, before their arrival.

*Week 29 – July 19th to July 25th*
**Group PsiDKM**
To test if insert and vector length was correct after the ligation and transformation, we cleaved part 5 with EcoRI + PstI, EcoRI + XbaI, SpeI + PstI, PstI and EcoRI. Gel electrophoresis did show bands were not the correct length.
**Group PsiH:**
We received the biobricks for PsiH, CPR and GroES/EL. We resuspended the biobricks to receive a stock concentration of 50 ng/μL and further diluted the biobricks to working concentrations of 0,5 ng/μL. We also received overhang oligo sets for PsiH N-terminal 2-7 and DK N-terminal 2-7, which were diluted to working concentrations of 10 µM.
We amplified GroES/EL by Phusion PCR (SOP 11). The PCR product was run on an agarose gel, purified, and cut with FastDigest enzymes XbaI and PstI. The cut products were also run on an agarose gel and purified (SOP12, SOP8 and SOP6). Afterward, GroEL/ES was ligated into pSB1C3 + BBa\_I0500 (SOP 13), and the resulting plasmid was transformed into *E. coli* Top10 (SOP 14) (BBa\_4059062). Single colonies from transformation with GroEL/ES were streaked out on agar plates and colony-PCR was performed the following day (SOP 9).
To obtain the T7-promoter + RBS (BBa\_K4059009) from the DK biobrick, we performed PCR with different overhang oligos (N-terminal 2-7) on the DK-biobrick. The gel showed PCR-products of expected length ~160 bp, which were cut from gel and purified (SOP 10, step 8.1-8.5). We now had a T7-promoter + RBS with overhangs complementary to N-term. 2-7 (named T7-overhang 2-7).
For PsiH, we performed PCR with different overhang oligos (N-terminals 2-7), which initially did not show any bands. We then performed gradient-PCR (different annealing temperatures) on PsiH with oligo N-terminal 2, which also showed no results, which probably was due to hairpin formation in the long single stranded overhang oligos. We overcame this problem by adding DMSO (Dimethyl-sulfoxide). The results for PsiH oligo N-term 2 showed that 7% DMSO and 60°C annealing temperature was appropriate. These conditions were then used to amplify PsiH with the remaining oligos (N-term. 3-7). This worked for N-terminal 3-7. We now had PsiH with overhangs containing N-terminal 2-7 (named PsiH-overhang 2-7).
We assembled T7-overhang 2 with PsiH-overhang 2 by overhang gradient PCR (SOP10, step 8.6-8.8). The best result was observed for annealing temperature at 64℃, where a clear band was seen at ～1800bp’s . The PCR-product was purified (SOP 6) and another PCR with our forward and reverse primer (annealing before iGEM standard prefix and after standard suffix, respectively). This showed amplification, which confirmed that our T7- and PsiH-overhang 2 had successfully been assembled. We then repeated the above T7- and PsiH-overhangs for 3-7. This gave us T7+RBS+PsiH with N-terminals 2-7 (BBa\_numbers below). All of the biobricks were cut using FastDigest enzymes XbaI and PstI (SOP12) to prepare for ligation with backbone J61002 containing med. cons. promoter + lacI (J23106 + I732820). The T7+RBS+PsiH N-term 2 was ligated with this backbone (SOP13) and transformed into *E. Coli* Top10 (SOP14). Single colonies from transformation were streak plated and colony-PCR was performed the following day (SOP9) and showed no bands in the expected length of ～3100bp’s (LacI + PsiH). We decided to redo the backbone with LacI (J61002 + J23106 + I732820) and also the assembling of T7- and PsiH-overhang 2 by overhang PCR next week.

![](https://static.igem.org/mediawiki/2021/9/98/T--SDU-Denmark--TabelPsiH.svg)

*Week 30 – July 26th to August 1st*
**Group PsiDKM**
Transformation was performed of Top10 cells.
**Group PsiH**
In order to remake the backbone for PsiH and CPR (J61002+J23106+I732820), we cut J61002+J23106 with FastDigest enzymes SpeI + PstI and pSB1C3 + I732820 (SOP12). The cut products were run on gel (SOP 8) and showed bands at expected lengths of ～2100 bp’s (for J61002+J23106) and ～1300 bp’s (for I732820). The cut products were purified (SOP6), ligated (SOP13) and transformed into *E. coli* Top10 (SOP14). Single colonies were streak plated and colony-PCR was performed (SOP9), showing correct lengths at around 1500bp’s for at least four colonies, D, F, G and H. ON-cultures were grown from these four colonies.
We transformed our GroEL/ES plasmid (BBa\_K4059062) into *E. Coli* ER2566 to prepare for gene expression assays. From the overnight culture of this transformation, we prepared a freeze stock (SOP5) for later use. Using the same overnight culture, we performed SDS-page (SOP21). The result showed an over-expression of proteins around 57 kDa and 10kDa in the Arabinose-induced cultures compared to the non-induced culture and the Wild type ER2566. These protein sizes are consistent with our GroEL (57.3 kDa) and GroES (10.4 kDa), respectively.

## August

*Week 31 – August 2nd to August 8th*
**Group PsiDKM**
To figure out what was wrong, our PsiDKM biobrick was amplifided using Taq RED mastermix PCR mix and send in for sequencing using primer sets seq\_1, seq\_2, seq\_7 and seq\_8.
Meanwhile, two new PsiDKM was made using Taq RED mastermix PCR and Phusion PCR, using Taq RED mastermix gave no results and using phusion the bands were too short when run on a gel.
**Group PsiH:**
Oligos for N-terminal modification of CPR (CPR overhang oligo N-terminal 2-5) and further N-terminal modifications of PsiH (PsiH overhang oligo N-terminal 7 (with different annealing sequence) and Deletion) were received and diluted to 10µM.
For PsiH, a new overhang PCR was run to assemble T7- and PsiH-overhang 2 again (SOP10). This showed good results, with bands at ～1800bp’s i gelelectrophoresis. The PCR-product was cut out and purified (SOP6). Then, the product, T7+RBS+PsiH-Nterm 2 (BBa\_K4059039), was cut with FastDigest restriction enzymes XbaI and PstI to prepare for ligation into the backbone (SOP12).
With the new overhang oligos for PsiH (Nterm 7 and Nterm-Deletion), the first PCR on the IDT PsiH-biobrick was performed (SOP10, step 8.1-8.5). PsiH amplified with overhang oligo N-term-Deletion showed results at annealing temperatures of 60℃, and the band was cut and purified to use for secondary PCR reaction (SOP10, step 8.6-8.8) later.
Initially no bands were observed for PsiH amplified with overhang oligo N-term-7, probably due to too high annealing temperature (since the annealing sequence was redesigned to be shorter in order to prevent hairpin-formation). PCR-reactions were repeated as gradient-PCRs, which showed convincing bands at 54°C and 55°C. These bands were cut and purified and used right away for the secondary PCR reaction (SOP10, step 8.6-8.8) to assemble T7- and PsiH-overhang 7. The following gel-electrophoresis showed bands at ～1800bp’s as expected, and these were cut out and purified (SOP8 and SOP6). Now, T7+RBS+PsiH-Nterm 7 (BBa\_K4059043) were assembled, but unfortunately, the concentrations were too low for restriction cutting.
For CPR, the initial overhang PCR’s (SOP10, step 8.1-8.5) with overhang oligos Nterm-2-5 showed no results, probably also due to too high annealing temperature. The same PCR was repeated as gradient-PCR, which resulted in bands of correct length at ～2300bp’s for CPR-overhang 2 and 5 at lower annealing temperatures, which were cut and purified (SOP10 and SOP6). The secondary overhang PCR to assemble T7- + CPR-overhang 2 and T7- + CPR-overhang 5, showed bands at ～2500bp’s as expected, and these were cut and purified. Thereby, we had assembled T7+RBS+CPR Nterm 2 (BBa\_K4059048) and T7+RBS+CPR 5 (BBa\_K4059051). Unfortunately, the concentrations obtained from gel-purification were too low to cut, so PCR will be repeated.
The PCR on CPR with overhang oligo N-term 3 and 4 (SOP10, step 8.1-8.5) was repeated several times with different annealing temperatures and DMSO-concentrations but no good results. We decided to discontinue the use of overhang oligos N-term 3 and 4 for CPR.

*Week 32 – August 9th to August 15th*
**Group PsiDKM**
A new attempted at making PsiDKM was made, here the bands was the correct length, but they were smeared.
The same precedor was done, this time using phusion mastermix. Here bands were of the correct size and no smears were visible.
Both part 4 and the new PsiDKM was cleaved. Part 4 with SpeI and PstI, PsiDKM with XbaI and PstI. The cleaved products were run on a gel and then purified, as the bands are the correct size. Part 4 and PsiDKM was used for ligation and then transformed into Top10. The transformation was plated and then streak plated the next day.
**Group PsiH:**
The new backbone for PsiH and CPR (J61002 + J23106 + I732820) was miniprepped from ON-culture to obtain enough for cutting (SOP7). It was then cut with FastDigest enzymes SpeI and PstI, run on gel and purified (SOP12). The backbone was ligated with different previously prepared inserts (SOP13), T7+RBS+PsiH-Nterm 2 (BBa\_K4059039) (both old, from week 29 and new version from week 31) and T7+RBS+PsiH-Nterm 3 (BBa\_K4059040) (from week 29). The plasmids were then transformed into *E. coli* Top10 (SOP14). Streak plating of single colonies, and then colony-PCR the next day (SOP9), which showed bands at the expected lengths of ～3100bp’s (LacI + PsiH). From ON-cultures (SOP4) of the successfully transformed colonies, freeze-stocks were prepared, and plasmids were miniprepped and sent to sequencing (SOP7 and SOP17).
For PsiH, overhang PCR to assemble T7- and PsiH-overhang 7 was repeated to obtain more product (T7+RBS+PsiH-Nterm 7) (SOP10).
The same was done for T7- and CPR-overhang 2 and T7- and CPR-overhang 5. None of the mentioned overhang PCR reactions showed any correct bands on the following gel-electrophoresis, and a lot of smear was seen on gel-pictures. For this reason, we decided to repeat all of the primary PCRs (SOP10, step 8.1-8.5) using overhang oligos N-term 2-7+Deletion for PsiH, overhang oligos N-term 2 + 5 for CPR, and overhang oligos N-term 2-7+Deletion.
For DK (to obtain T7-polymerase and RBS from this biobrick), the PCR products were run on agarose gels (SOP8), which were much longer than the regular gels, and also had different concentrations of agarose (0,7% for CPR and PsiH and 2% for T7). This way, we hoped to better separate the PCR products from any unspecific bands this way. The following gel-electrophoresis showed bands of correct length for PsiH-overhang 3, 5 and Deletion (～1800bp’s) and for T7-overhang 2-7 + Deletion (～160bp’s). The bands were cut out and purified (SOP6). For CPR with overhang oligos N-term 2 and 5, no bands were observed on agarose gel.

*Week 33 – August 16th to August 22nd*
**Group PsiDKM**
We did a colony PCR over the transformation of Part 5, part 5 was afterwards miniprepped and transformed into ER2566. Part 5 in ER2566 was induced with different concentrations of IPTG and samples was taken during different time stamps. These were run on an SDS-page gel but PsiD, PsiK, and PsiM are all similar in size and hard to tell apart. Therefore, qPCR preparation will begin next week.
**Group PsiH:**
Following the same procedure as last week, the backbone for PsiH and CPR (J61002 + J23106 + I732820) was miniprepped from ON-culture (SOP7), and cut with FastDigest enzymes SpeI and PstI, run on gel and purified (SOP6). The backbone was ligated with different previously prepared inserts, this time T7+RBS+PsiH-Nterm 4 (BBa\_K4059036) (from week 29) and T7+RBS+PsiH-Nterm 5 (BBa\_K4059042) (from week 29) (SOP13). The plasmids were then transformed into *E. coli* Top10 (SOP14). Streak plating of single colonies, and then colony-PCR the next day (SOP9), which showed bands at the expected lengths of ～3100bp’s (LacI + PsiH) for the T7+RBS+PsiH-Nterm 4 insert. For the Nterm 5 insert, the colony PCR was inconclusive.
The miniprepped plasmids from last week’s transformations, T7+RBS+PsiH-Nterm 2 (BBa\_K4059039) (both old and new version) and T7+RBS+PsiH-Nterm 3 (BBa\_K4059040) were cut with FastDigest enzymes X and P to verify the insert (SOP12), which showed successful results on the gel electrophoresis. insert lengths of around 3100 bp’s as expected, and entire plasmids at around 5200bp’s.The plasmids, which were now confirmed both by colony-PCR and restriction cutting, were transformed into our *E. coli* protein expression strain, ER2566 (SOP14). We then performed SDS-page on the successfully transformed strains (SOP21) and inducing with IPTG. The SDS-page-gels showed no conclusive results.
For CPR with overhang oligos N-term 2 and 5, we repeated the PCR (SOP10, step 8.1-8.5), and finally obtained good results by increasing the denaturation time to 20 seconds instead of 10. The CPR-overhang 2 and 5 were then assembled with the corresponding T7-overhang 2 and 5 by overhang-PCR (SOP10, step 8.6-8.8), and bands were observed at expected lengths of ~2500bp’s. We now had T7+RBS+CPR-Nterm 2 (BBa\_K4059048) and 5 (BBa\_K4059042) with concentrations high enough to cut, which was performed using FastDigest enzymes XbaI and PstI (SOP12). The cut products were ligated into the backbone J61002+J231060+I732820 (SOP13), and the plasmids were transformed into *E. coli* Top10 (SOP14). Thereafter colony PCR was performed (SOP9). For CPR-Nterm2 (BBa\_K4059048), the plates showed no colonies, while single colonies for the CPR-Nterm 5 (BBa\_BBa\_K4059051) transformation were streak plated the next day. The following colony-PCR showed correct insert lengths for the CPR-Nterm 5 (BBa\_BBa\_K4059051) transformation. The ON-cultures from successful colonies were used for freeze-stock preparation and plasmid miniprep (SOP4, SOP5 and SOP7).

*Week 34 – August 23rd to August 29th*
**Group PsiDKM**
RNA purification was performed, and samples of both not induced and IPTG induced part 5 in ER2566 (BBa\_K4059037). After RNA purification, cDNA synthesis was performed form the purified RNA.
**Group PsiH:**
Cultures of J61002 + J23106 + I732820 with PsiH+Nterm 2 (new) (BBa\_K4059039) and PsiH+Nterm 3 (BBa\_K4059040) in ER2566 were grown in an overnight culture (SOP4) in later use for qPCR. The cultures were grown to OD = 0.5. Here, 5 mL were removed to function as a negative control for qPCR. The rest of the overnight cultures were then induced with IPTG. The induced cultures were harvested at 1 hour, 2 hours and 3 hours. RNA purification of all the harvested samples for the three different cultures were performed (SOP24), where the RNA qualities were controlled by gel-electrophoresis (SOP8). The gel of J61002 + J23106 + I732820 with PsiH+Nterm 2 (new) (BBa\_K4059039) general showed better quality compared to J61002 + J23106 + I732820 with PsiH+Nterm 3 (BBa\_K4059040). Here, only very weak bands were seen.
Further, overnight cultures of GroES/EL (BBa\_K4059062) and PsiH-Nterm 2 (BBa\_K4059039) and CPR-Nterm 5 (BBa\_K40590) were made in order to miniprep and send to sequencing (SOP7 and SOP17). In addition, CPR-Nterm 5 (BBa\_K4059051) was transformed into ER2566 (SOP14). The transformation showed lots of colonies, and an overnight culture was prepared to make a freeze stock (SOP14 and SOP5).
An overnight culture of ER2566 containing CPR-Nterm 5 (BBa\_K4059051) were made for SDS (SOP4). The SDS was prepared for next week (step 7.1 - 7.11 SOP21).
At the end of the week, individual PCR reactions of N-terminal oligo 5, 10 and 11 for PsiH and oligo 33, 34 and 35 for CPR were performed. Oligo 10 and 11 are forward primers to PsiH-Nterm 5 and PsiH-Nterm 6, where oligo 33 is reverse primer to DKM with Deletion for PsiH. Oligo 34 and 35 are forward primers to CPR-Nterm 6 and CPR-Nterm 7.
**Group Transport:**
This week a new group was formed with the project of trying to get *E. Coli* to transport the substrate into the cell and transport the end-product out. Amplification and gel purification was made on PsiT1, PsiT2, GFP C-term, and GFP N-term.
Part 2 (Bba\_J61002 with Bba\_J23106) was minipreped and afterwards cut along with GFP C-term and GFP N-term. Part 2.2 was cut with PstI and XbaI and GFP C- and N-term was cut with XbaI and SpeI. Part 2.2 was ligated together with GFP N-term and GFP C-term. All the ligation products were transformed into Top10 and then plated on AMP.

*Week 35 – August 30th to September 5th*
**Group PsiDKM**
Due to trouble getting our part 5 in ER2566 to work properly, it was decided to redo all part making. Biobrick PsiDK and PsiM was put together again to create PsiDKM (BBa\_K4059037).
Part 5 was miniprepped from freeze stock and sent in for sequencing.
**Group PsiH:**
The RNA samples of PsiH+Nterm 2 (new) (BBa\_K4059039) and PsiH+Nterm 3 (BBa\_K4059040) from week 34 were used for cDNA synthesis (SOP18).
SDS of ER2566 containing CPR-Nterm 5 (BBa\_K4059051) was also prepared in the previous week. The SDS was run (SOP21), which showed no bands at all. Therefore, the experiment was repeated but showed the same result.
Additionally, overnight cultures of J61002 + J23106 + I732820 with PsiH+Nterm 2 (old), PsiH+Nterm 2 (new) (BBa\_K4059039), PsiH+Nterm 3 (BBa\_K4059040), PsiH+Nterm 4 (BBa\_K4059036) and CPR+Nterm 5 (BBa\_K4059051) were made for miniprep (SOP4 and SOP7).
Two Colony PCRs were also performed with the purpose of sending the samples for sequencing (SOP9). The first colony PCR were made on pSB1C3 + I732820 and pSB1C3 + I732820 with the J61002 backbone. Gel-purification was performed, but the concentrations were too low. Therefore, overnight cultures were prepared and miniprep for sequencing (SOP4 and SOP7).
The second colony PCR were made on GroES/EL (BBa\_K4059062), PsiH+Nterm 4 (BBa\_K4059036) and CPR+Nterm 5 (BBa\_K4059051). Gel-purification was performed on all samples (SOP6) and stored at -20°C before sending them to sequencing the next week (Monday September 6th).
At the end of the week, PCR reactions on several Nterm oligos for CPR, PsiH and DK were performed. 18 different PCR reactions were run (SOP11). No bands were observed for all CPR except CPR-Nterm 15. Also, only DK-Nterm WT showed a band.(SOP6). Bands observed for PsiH-Nterm 8, PsiH-Nterm 9 and PsiH-Nterm “S”. All the seen bands were cut out and purified (SOP6).
**Group Transport:**
We streaked colonies from the transformation the previous week on plates and checked on a colony PCR. A colony from part 2 (Bba\_J61002 with Bba\_J23106)+ GFP C-term and part 2 (Bba\_J61002 with Bba\_J23106) GFP N-term was chosen and observed under a flourescent microscope to see expression of GFP. The two colonies were miniprepped and cut along with T1\_F, T2\_F, T1\_R and T1\_R in which the BamHI sites are introduced by specific primers for N-terminal and C-terminal. The cutting was done using BamHI + SpeI and BamHI + NdeI. The cleaved products were ligated with GFP C-term with T1\_R and T2\_R and GFP N-term with T1\_F and T2\_F. The ligation products were transformed into Top10. The negative control however grew and AMP plates, colony PCR was used to check the size of the colonies. We discarded the plates due to a negative control that showed colonies.

## September

*Week 36 – September 6th to September 12th*
**Group PsiDKM:**
This week prep work was done for qPCR, this included mixing the master mix containing SYBR green master mix 2x, forward, and reverse primers. A total of 6 different primer sets were used, so the expression of different genes could be quantified. The 6 different genes were PsiD, PsiK, PsiM, GyrA housekeeping gene, RpoB housekeeping gene, and T7 polymerase.
The master mix was mixed with the cDNA from week 34 in a 384-well plate. This plate was placed in a qPCR machine.
During this week, another transformation was also preformed, here transforming part ? Into Top10. This was tested in a colony PCR.
**Group PsiH:**
In the beginning of this week, the prepared samples from last week (week 35) were sent to sequencing (SOP17).
qPCR of the cDNA samples PsiH+Nterm 2 (new) (BBa\_K4059039) and PsiH+Nterm 3 (BBa\_K4059040) were performed (SOP18). Primer sets for GyrA, Rpo, PsiH and T7 were used. The housekeeping genes GyrA and Rpo are used for normalization of the gene expression of PsiH and T7. The expression of the housekeeping genes should remain constant between the cells and are used for normalization to check errors between the samples.
Furthermore, a new backbone of J61002 containing the medium cons. promoter and LacI (J23106 + I732820) was made, since we still thought there was something wrong with LacI. Overnight culture of J23106 + J61002 and I732820 + pSB1C3 were miniprepped (SOP7). Afterward, J23106 + J61002 was cut with FastDigest enzymes SpeI and PstI and I732820 + pSB1C was cut with the enzymes XbaI and PstI (SOP12). The two parts J23106 + J61002 and I732820 were ligated to create the new backbone J23106 + J61002 + I732820 (SOP13). The new backbone was transformed into Top10 and colony PCR was performed (SOP14 and SOP9) with good results (bands around 1500 Bp’s). An overnight culture of the transformant (SOP4) was made, miniprepped (SOP7) and sent to Eurofins for sequencing (SOP17).
We repeated many of last week’s non-successful PCR-reactions, this time repeated as gradient-PCR’s with different annealing temperatures.
For PsiH we did overhang PCR (SOP11, step 8.1-8.5) with overhang oligo N-term 10 and the overhang oligo meant to introduce a peptide-linker between CPR and PsiH. Both of these overhang-PCR’s were successful with bands at gel-electrophoresis ~1800 bp’s - cut out and purified (SOP 6 + 8). We also assembled T7-overhang Deletion with PsiH-overhang Deletion and T7-overhang “S” with PsiH-overhang WT through overhang-PCR (SOP 11, step 8.6-8.8). The gel-electrophoresis showed bands of correct length of ~1800bp’s, which were cut and purified. This gave us T7+RBS+PsiH-Nterm Deletion (BBa\_K4059047) and Nterm WT (J23106+I732820+BBa\_K314004).
For the T7 Lac promoter + RBS, we did overhang PCR with overhang oligo Nterm 8, 9, 10, 14, 15 and CPR\_Deletion. All worked and showed bands ~160bp except the one with overhang oligo Nterm 15.
For CPR we did overhang PCR with overhang oligos 14 and 15. Overhang oligo 14 worked, with band on gel-electrophoresis at ~2300 bp’s. 15 did not work. We also assembled T7-overhang 2, 14 and Deletion with CPR-overhang 2, 14 and Deletion through overhang PCR (SOP11, step 8.6-8.8). The gel-electrophoresis showed bands of correct length of ~2500bp’s for all three reactions, which were cut and purified. This gave us T7+RBS+CPR-Nterm 2, 14 and Deletion.

*Week 37 – September 13th to September 19th*
**Group PsiDKM**
Another attempt at starting over with assembling PsiDKM, but in a backbone containing kanamycin resistence. Here PsiDk and PsiM was amplified using phusion PCR. These were run on a gel and then purified using GFX-gel purification. Phusion PCR was used to assemble PsiDKM, it was then run a gel. The bands were too big; however, it will be sent off to sequencing and the experiment will continue.
In order to create a backbone with Kanamycin resistance, a transformation of psb1k3 into top10 was made and called part 14.
**Group PsiH:**
Part 4.2A is sent for sequencing. Overhang PCR on DKH2, DKH8, DK+H9 and DK+H10. Gave inconclusive results - DMSO might be the issue, amount of DK and H is adjusted for next reactions.
**Group Transport**
Ligation of C-term + T1\_R was made and transformed into Top1, however no colonies except negative control grew. A new transformation was made, and streak plated.
Freezestock 25 and 26 (N+PsiT1 and N+PsiT2) was miniprepped and sent in for sequencing.
**Group Knock-out:**
Preparation of solutions MgCl2 and CaCl2 of the different molarities, that were needed for the phage experiments. Culture preparation of pCP20, a flippase, and miniprepped it to obtain its plasmid.

*Week 38 – September 20th to September 26th*
**Group PsiDKM**
This week, part 4.2 , part 14, and PsiDKM was all cleaved with restriction enzymes, run on gel, and purified. Miniprep was performed of several part 4.2 O/N cultures.
Ligation product was made, in which part 14 and part 15 was used. Hereafter the ligation product was transformed into Top10. The Transformation was plated onto kanamycin plates and then streak plated. A colony PCR was performed on the streak plate colonies. This new part was called part 16.
**Group PsiH:**
From our old version of the LacI backbone J61002+J23106+I732820, we moved T7+RBS+CPR-Nterm 5 (BBa\_K4059042) (which is confirmed by sequencing) to the newest version of the LacI backbone , since the last backbone showed Deletions in LacI in sequencing data. This was done by cutting both the new backbone and the T7+RBS+CPR-Nterm 5 (BBa\_K4059051) with the FastDigest enzyme SpeI and the New England Biolabs enzyme BstEII (unique cutter in the middle of LacI), which enabled us to move the last half of the mutated LacI gene (the part AFTER the mutation, therefore unaffected) and the T7+RBS+CPR-Nterm 5 (BBa\_K4059051) to the new backbone, where LacI was not mutated. The colony-PCR from this transformation showed good results, indicating that T7+RBS+CPR-Nterm 5 (BBa\_K4059051) had been successfully ligated into the new backbone and transformed into Top10. The plasmid was then moved to ER2566.
A gradient overhang PCR on T7 with overhang oligo N-terminal 15 was performed (SOP11, step 8.1-8.5), showing bands at expected length of ~160 bps. We tried multiple times to assemble T7-overhang 15 and CPR-overhang 15 (SOP 11, step 8.6-8.8) with unsatisfying results, but finally found that equal amounts of the two template DNA’s (2ng’s of each) and no DMSO at 60oC annealing temperature worked. DNA purified from gel (SOP6).
We also cut our biobricks T7+RBS+PsiH Nterm 7 (BBa\_K4059043), Nterm “S” and Nterm (BBa\_K4059047) Deletion and T7+RBS+CPR Nterm 14 (BBa\_K4059054) + Nterm-Deletion (BBa\_K4059056) using the FastDigest enzymes XbaI and PstI to prepare for ligation with the new LacI backbone (J61002+J23106+I732820), which was cut with SpeI and PstI. The ligation products were transformed into *E. Coli* Top10 and confirmed by colony-PCR. To make sure of getting constructs without mutations, we also ligated all of the inserts into our initial version of the LacI backbone (from week 26) and transformed these ligation products into Top10 as well. For all of the inserts, at least one colony showed correct length of bands on colony-PCR (~3000bp’s for the PsiH transformants and around 3700 bps for the CPR-transformants). The old versions of T7+RBS+PsiH Nterm 3 (BBa\_K4059040), 5 (BBa\_K4059042) and 6 (BBa\_K4059041) (from week 29) were also cut with XbaI and PstI and ligated into the new LacI backbone (J61002+J23106+I732820), which was then transformed into top 10. These showed good results on the following colony-PCR.
From all of the Top10 transformants confirmed by colony-PCR, plasmids were miniprepped and transformed into our protein expression strain ER2566. We now have the following transformants of ER2566 (all in the backbone J61002+J23106+I732820, either old or newest version):
For PsiH:

* New LacI-backbone with T7+RBS+H-Nterm 3 (BBa\_K4059040)
* New LacI-backbone with T7+RBS+H-Nterm 5\* (BBa\_K4059042)
* New LacI-backbone with T7+RBS+H-Nterm 6\* (BBa\_K4059041)
* Old LacI-backbone with T7+RBS+H-Nterm 7\* (BBa\_K4059043)
* New LacI-backbone with T7+RBS+H-Nterm 7\* (BBa\_K4059043)
* Old LacI-backbone with T7+RBS+H-Nterm-WT (J23106+I732820+BBa\_K314004)

For CPR::

* New LacI-backbone with T7+RBS+CPR-Nterm 5 (BBa\_K4059051)
* New LacI-backbone with T7+RBS+CPR-Nterm 14 \* (BBa\_K4059054)
* New LacI-backbone with T7+RBS+CPR-Nterm-Deletion (BBa\_K4059056)
* Old LacI-backbone with T7+RBS+CPR-Nterm-Deletion \* (BBa\_K4059056)

**Group Transport:**
We ran colony PCR on the transformants from the previous week and one correct colony of C-term + T1\_R was found.
**Group Knockout:**
We made O/N culture of ER2566 and used to make phage lysate from using P1 phages. We acquired colonies of JW0452 and JW3508 and created phage lysate of these too using P1 phages, in order to knockout the genes acrA and yhjV.
A WT culture of ER2566 was transduced with the P1 lysate of ACRA and YHJV and then plated onto Kanamycin plates. However, due to an error, the wrong plates were used, and normal LA plates were used for plating.

*Week 39 – September 27th to October 3rd*
**Group PsiDKM**
The new part 16 was minipreped, cut with SpeI and PstI. The cut part 16 was run on a gel and then purified. The cut part 16 was ligated with PsiDKM. The ligation product of part16 + PsiDKM was transformed into Top10. The transformation was plated on kanamycin plates. The colonies were named part 19.
**Group PsiH:**
On most of the below listed ER2566 transformants, SDS prep and qPCR-prep was performed with ER2566 Wild Type as negative control. This was done by following SOP21 for SDS-prep. Cells for RNA-purification were harvested from the same cultures, which were induced with IPTG. The only differences were that cells for qPCR was harvested from the cultures at 0H (minus IPTG), at 1 hour (post-induction) AND 2 hours (post-induction), and that the harvested volumes were 1.5mL instead of 1.

For PsiH:

* New LacI-backbone with T7+RBS+H-Nterm 5\* (BBa\_K4059042)
* New LacI-backbone with T7+RBS+H-Nterm 6\* (BBa\_K4059041)
* Old LacI-backbone with T7+RBS+H-Nterm 7\* (BBa\_K4059043)
* New LacI-backbone with T7+RBS+H-Nterm 7\* (BBa\_K4059043)

For CPR:

* New LacI-backbone with T7+RBS+CPR-Nterm 14 \* (BBa\_K4059054)
* Old LacI-backbone with T7+RBS+CPR-Nterm-Deletion \* (BBa\_K4059056)

Cell pellets were stored in -20 freezer overnight.
SDS gels did not show any significant results, may be repeated due to loading of too little of the uninduced (0H) samples.
**Group Transport:**
Initial production experiments were carried out by us. These involved the old version of PsiDKM in which a deletion of LacI had been detected by Sanger sequencing. We also freeze dryed this.
This week we made preparations for SDS and O/N cultures of Freezestock 25, 26, 39 and 1. The O/N cultures were miniprepped.
**Group Knockout:**
A new transduction of WT ER2566 with the two phage lysates were made and plated onto 25γ Kanamycin plates. Colonies grew and a colony PCR was performed on YHJV and ACRA. O/N cultures of the colonies were also made, however only YHJV grew in liquid medium and ACRA did not grow. The O/N cultures of YHJV was used for transformation.
The flippase pPC20 was transformed into ER2566 with the knockout of YHJV and then plated onto 25γ Kanamycin plates. The plates were incubated at 30℃ as pPC20 is temperature sensitive. The colonies were run on a colony PCR and results indicated a knockout.

## October

*Week 40 – October 4th to October 10th*
**Group PsiDKM and knockout:**
We did Colony PCR on the transformation from the previous week. Multiple of the part 19s were miniprepped and transformed into ER2566 and then plated. Two of the part 19s were chosen and cut with E+PstI, SpeI+PstI and just E. The cut products were run on a gel, but no results were obtained.
Different part 19s from week were transformed into ER2566 containing the YHJV knockout (ER2566 ∆YHJV) and plated on Kanamycin plates.
Part 19 + ER2566 and part 19 + ER2566 ∆YHJV were made and induced with IPTG, cells were harvested at different timepoints after induction. RNA purification was performed on the cells to prepare for qPCR the following week.
**Group PsiH:**
From last weeks harvested cells RNA purification was performed (SOP24).cDNA synthesis and qPCR were made to investigate the gene expression of the different samples of PsiH and CPR. (SOP18).
At the beginning, both PsiH and CPR were transformed into the backbone J61002+J23106+I732820, which has an ampicillin resistance. However, we need to move CPR over to another backbone with a different resistance to be able to test CPR together with PsiH and GroES/EL in the same bacteria. Also, we want to test CPR with PsiH and PsiDKM. Therefore, CPR was transformed into a backbone with kanamycin resistance (pSB1K3 + J23106 + I732820) and a backbone with chloramphenicol resistance (pSB1C3 + J23106 + I732820). The CPR samples CPR-Nterm-Deletion (BBa\_K4059056), CPR-Nterm 5 (BBa\_K4059051) and CPR-Nterm 14 (BBa\_K4059054) were ligated and transformed with both backbones (SOP13 and SOP14). Colonies from the transformations were streaked out on agar plates. Also, colony PCR and gel-electrophoresis was performed (SOP9 and SOP8). Further, overnight cultures were prepared, miniprepped and freeze stocks were made (SOP4, SOP7 and SOP5). We now had CPR in a backbone with kanamycin resistance and a backbone with chloramphenicol resistance.
In addition, our group made overnight cultures of two different PsiDKM (BBa\_K4059037) with kanamycin resistance (SOP4). The samples were miniprepped to make freeze stocks (SOP7 and SOP5). These are later used for transformation in ER2566 to combine PsiH, CPR and DKM. A freeze stock of GroES/EL was made in week 30.
Later in the week, transformations of PsiH-Nterm 3 (BBa\_K4059040), 5 (BBa\_K4059042), 6 (BBa\_K4059041), 7 (BBa\_K4059043) and “S” with PsiDKM (BBa\_K4059037) in ER2566 were performed (SOP14). The PsiH samples came from miniprep. These transformations succeeded and overnight cultures were made for freeze stocks (SOP4 and SOP5). We had combined PsiDKM and PsiH with different N-terminals in ER2566. All PsiH samples had ampicillin resistance and DKM had kanamycin resistance.
Furthermore, transformations of PsiH-Nterm 3 (BBa\_K4059040), 5 (BBa\_K4059042), 6 (BBa\_K4059041), 7 (BBa\_K4059043) and “S” with CPR-Nterm Deletion (BBa\_K4059056), 5 (BBa\_K4059051) and 14 (BBa\_K4059054) in ER2566 were performed (SOP14). The CPR samples came from miniprep. These transformations were also successful and overnight cultures were made for freeze stocks (SOP4 and SOP5). We had combined PsiH and CPR with different N-terminals in ER2566. All PsiH samples had ampicillin resistance and all CPR had kanamycin resistance.
At the end of the week, overnight cultures of the PsiH combined with respectively PsiDKM and CPR were made (SOP4). These cultures were used next week to make triple transformants.
**Group Transport:**
More production experiments were done. These included both PsiH and PsiDKM. For PsiH tryptamine turned out not to be dissolved when added directly to the media without any other processing. All samples were freeze dried. We didn’t succeed unfortunately due to overload of the freeze dryer.
Preparations for CPR-assay were made, O/N cultures of CPR DEL (4.0), CPR DEL (4.2), CPR 5 (4.2), and CPR 14 (4.2) were diluted and induced with IPTG. Cells were harvested after some time when exponential growth was obtained. The samples were afterwards frozen for later use. Some days later the actual assay was performed, following SOP number 25.

*Week 41 – October 11th to October 17th*
**Group PsiDKM and knockout:**
cDNA synthesis were made from the purified RNA from both ER2566 + part 19 and ER2566 ∆YHJV + part 19 and gel electrophoresis were run with the purified RNA.
qPCR was performed using the master mix containing SYBR green master mix 2x, together with forward, and reverse primers. 5 different primer sets were used to quantify the gene expression for PsiD, PsiK, PsiM, GyrA housekeeping gene, and T7 polymerase.
The master mix was mixed with the cDNA from week 40 for both ER2566 + part 19 and ER2566 ∆YHJV + part 19 in two 384-well plate. Both plates were placed in a qPCR machine.
To prepare for LC/MS 100mL cultures containing both ER2566 + part 19 and ER2566 ∆YHJV + part 19 were grown to OD600=0,3 and induced with 4-Hydroxyindol as well as methionine and serine. The cultures were left overnight in a shaking incubator at 37oC. The cultures were then split into 4x 25mL and placed in 50mL falcon tubes and spun in a centrifuge to separate media from cells. The media were then distributed into 5x 5mL in 15mL falcon tubes and freeze-dried overnight. The cells were transferred to Eppendorf tubes and freeze dried overnight and stored at -80oC.
**Group PsiH:**
Your overnight cultures of PsiH-Nterm 3 (BBa\_K4059040), 5 (BBa\_K4059042), 6 (BBa\_K4059041), 7 (BBa\_K4059043) and WT (J23106+I732820+BBa\_K314004) with CPR-Nterm Deletion (BBa\_K4059056), 5 (BBa\_K4059051) and 14 (BBa\_K4059054) combined with PsiDKM (BBa\_K4059037) from last week were used for transformation with CPR-Nterm Deletion (BBa\_K4059056), 5 (BBa\_K4059051) and 14 (BBa\_K405905414 (chloramphenicol resistance) (SOP14). Meanwhile, we also transformed PsiH-Nterm 3, 5, 6, 7 and WT combined with CPR-Nterm Deletion, 5 and 14 together with GroES/EL (SOP14). Both transformations occurred in ER2566.
At first, we thought that our transformation did not work. We chose to incubate our transformation for a longer time, and after 2 days we saw colonies on our triple transformants. For both of our triple transformations, they had only succeeded for PsiH-Nterm 5 (BBa\_K4059042), 7 (BBa\_K4059043) and WT (J23106+I732820+BBa\_K314004). From here, we went on with the transformation of PsiH-Nterm 5, 7 and “S” in both cases.
We made overnight cultures of the successful transformations(SOP4). However, they grow very slowly and it took 2 days before they were grown. In addition, we performed colony PCR and gel-electrophoresis on the triple transformants (SOP9 and SOP8). At first, we used our forward and reverse primers to try to separate PsiH, PsiDKM, CPR and GroES/EL from each other. The expected lengths were PsiH ～3200bp’s, PsiDKM ～5000bp’s, CPR ～3800bp’s and GroES/EL ～3600bp’s. However, we failed to separate fragments from each other and we repeated the colony PCR. For the next colony PCR, we tried to separate the fragments by using specific primer sets for PsiH, PsiDKM, CPR and GroES/EL respectively. We still failed to separate PsiH, CPR and GroES/EL, although we also tried to change the percentage of the gel in gel electrophoresis. This may be because the lengths of the three fragments are very close. We managed to separate PsiH, PsiDKM and CPR for some colonies with the correct lengths. In addition, freeze stocks of the successful triple transformants were made (SOP5).
**Group Transport:**
Lots of production experiments were performed according to sub. This were done with both single, double and triple transformants. In relation to the solubility problem, it was tested whether dissolving tryptamine in ethanol could be a solution, as it was described in the literature that this method could reach a concentration of 10 mg/ml. This was possible but resulted in the death of the cells when added. Later it turned out that the problem could be overcome by simply dissolving the compound in the medium when this was prepared from the beginning. This should take place at a warm temperature with stirring using a magnet stirrer.

*Week 42 – October 18th to October 24th*
**Group PsiDKM and knockout:**
The products from freeze-drying were resuspended in 250µL 50% acetonitril+1% fomic acid solution. These solutions were centrifuged and 150µL supernatant were transferred into micro-insert placed in a vial used in mass spectrometry. To obtain mass spectrometry results the samples were run on Triple Quad LC/MS.
**Group PsiH:**
In our last week in the lab, we did SDS on several samples of PsiH, CPR and GroES/EL (SOP21). The samples from previous SDS experiments were stored and used for these.
SDS was performed on PsiH+Nterm 5 (BBa\_K4059042), 6 (BBa\_K4059041) and 7 (BBa\_K4059043) with the newest backbone of J61002 + J23106 + I732820. Also, the oldest backbone of PsiH+Nterm 7 (BBa\_K4059043) was included.
Further, SDS was performed on CPR+Nterm Deletion (BBa\_K4059056) and 14 (BBa\_K4059054) with backbone J61002 + J23106 + I732820.
In the end, SDS was made on GroES/EL (BBa\_K4059062) with a backbone containing pSB1C3 and BBa\_I0500.
None of the SDS results showed any results.

![](https://static.igem.org/mediawiki/2021/1/15/T--SDU-Denmark--frontpagemushroom.svg)

#### Protocols

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# Team:SDU-Denmark/Engineering

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# Engineering

![]()

# Abstract

On this page, you can read about how we have successfully engineered the part, pSB1K3-LacI-T7-PsiDKM

## Engineering success

To make a system to produce psilocybin from the substrate 4-hydroxy indole the three enzymes PsiD, PsiK and PsiM are needed. The enzymes were designed as two bricks containing PsiDK and PsiM, because IDT were unable to synthesize biobricks containing >3000bp. For allowing an inducible gene expression LacI and the medium constitutive promoter (J23106) were inserted into the backbone, pSB1K3, containing kanamycin resistance.

![](https://static.igem.org/mediawiki/2021/7/77/T--SDU-Denmark--billede1.png)

PCR amplifications were used to assemble pSB1K3-LacI and PsiDKM respectively by using designed primers, which generates overhang between the separate gene parts. Finally, PsiDKM was cloned into pSB1K3-LacI in an E. coli Top10 strain, finalizing the construction of pSB1K3-LacI-T7lac-PsiDKM.

![](https://static.igem.org/mediawiki/2021/b/bc/T--SDU-Denmark--billede2.png)

*Agarose gel with pSB1K3-LacI-T7lac-PsiDKM: Bands ~6000 indicate successful assembly of pSB1K3-LacI-T7lac-PsiDKM. The lanes correspond to the different colonies obtained from the transformation of pSB1K3-LacI-T7lac-PsiDKM.*

The synthesised plasmid encoding pSB1K3-LacI-T7lac-PsiDKM (BBa\_K4059037) were successfully transformed into E. coli ER2566 strain, which is suitable for protein expression. Afterwards the culture was induced with 1mM IPTG enabling to test the enzymatic activity of PsiDKM using real-time qPCR.

![](https://static.igem.org/mediawiki/2021/thumb/f/f3/T--SDU-Denmark--PsiDKMgeneExpression.svg/800px-T--SDU-Denmark--PsiDKMgeneExpression.svg.png)

*qPCR analysis of PsiDKM: Gene expression of the enzymes PsiD, PsiK and PsiM and the T7 promoter.*

After inducing with 1mM IPTG it is generally confirmed that the gene expression of PsiDKM and T7 promoter increase after 1- or 2 hours confirming the function of the inducible system. However, the gene expression decreases after 3 hours, indicating that the cells might be stressed after induction of proteins, which could affect the gene expression.

To test whether the PsiDKM construct was able to produce psilocybin or not, 4-hydroxy indole was added to cultures containing our plasmid with PsiDKM. These cultures were separated into media and cells. This was the analyzed via Triple Quad Liquid Chromatography-Mass Spectrometry. Fragment ions and standard curve used for quantification are described in the LC/MS section within [Results.](https://2021.igem.org/Team%3ASDU-Denmark/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.

![](https://static.igem.org/mediawiki/2021/3/3b/T--SDU-Denmark--billede3.png)

*qPCR analysis of PsiDKM: Gene expression of the enzymes PsiD, PsiK and PsiM and the T7 promoter.*

![](https://static.igem.org/mediawiki/2021/f/f6/T--SDU-Denmark--billede4.png)

*LC/MS of PsiDKM: Arbitrary areas of three different PsiDKM samples of fragment ions m/z=204 and m/z=160.*

All things considered we conclude that the assembly of pSB1K3-LacI-T7-PsiDKM satisfy the requirements for engineering success, due to its ability to produce psilocybin from 4-hydroxy indole.

![](https://static.igem.org/mediawiki/2021/1/15/T--SDU-Denmark--frontpagemushroom.svg)

#### Model

![](https://static.igem.org/mediawiki/2021/1/1c/T--SDU-Denmark--Next_pil.svg)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:SDU-Denmark/Experiments

[![](https://static.igem.org/mediawiki/2021/b/b6/T--SDU-Denmark--TeamPsiloAid_logo.svg)](https://2021.igem.org/Team%3ASDU-Denmark)

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# Experiments

![](...)

# Abstract

On this page you can read more about the experiments we carried out throughout our project for the production of psilocybin.

## Introduction

Psilocybin is naturally produced by a family of mushrooms, called Psilocybe. This biosynthesis involves four key enzymes, which in five steps convert L-tryptophan into psilocybin.[1] Here L-tryptophan decarboxylase (PsiD) catalyzes the decarboxylation of L-tryptophan to tryptamine. Tryptamine is hydroxylated into 4-hydroxytryptamine by 4-monooxygenase (PsiH). 4-hydroxytryptamine kinase (PsiK) phosphorylates 4-hydroxytryptamine thereby forming nor-baeocystin. Psilocybin synthase (PsiM) catalyzes the transfer of a methyl to the amino group of norbaecystin, which results in the intermediate baeocystin, and a subsequent methyl transfer to the secondary amin of baeocystin, producing psilocybin psilocybin.[1]

![](https://static.igem.org/mediawiki/2021/5/5a/T--SDU-Denmark--1.svg)

For this project, the four genes from *Psilocybe cubensis* are used as all the enzymes have previously been expressed successfully in eukaryotic organisms as for instance *Saccharomyces cerevisiae*. Here it must be noticed that it is only PsiD, PsiK, and PsiM which have successfully been expressed in prokaryotes in previous studies.[2] This approach makes the psilocybin production dependent on the expensive precursor 4-hydroxy indole but avoids PsiH which is extremely difficult to express in prokaryotes.[2]

In order to create the de novo synthesis system of psilocybin in *E. coli*, our experimental approach is based on multiple research papers as well as the work of the Sydney iGEM team of 2019.[3]

The enzymatic pathway for the biosynthesis of psilocybin from substrates such as tryptophan, tryptamine or 4-hydroxytryptamine, was first characterized by (1).[1] The year after Hoefgen et al. 2018 succeeded in the production of psilocybin in a heterologous system.[4] In 2020 further work was conducted with the expression of the PsiD, PsiK, PsiM, and PsiH eukaryotic cells, in this case, *Saccharomyces cerevisiae*, which was successful but with a concerning 51% of psilocybin being degraded into psilocin passively within the yeast.[5]

 **The psilocybin producing system**

 In order to introduce the de novo synthesis system into a proaryotic host, *E. coli* is used as a production strain. Using *E. coli* enables a broad and easy biotechnical modeling and cloning due to its characteristics. Implementing the entire system in prokaryotes has never been done successfully before. This is due to PsiH being a cytochrome P450 monooxygenase. These enzymes are usually anchored to the endoplasmic reticulum by a large proportion of their N-terminals in eukaryotic cells, which is also the case for PsiH.[6] Here they catalyze hydroxylation in cooperation with specific reductases such as the NADPH-cytochrome P450 reductase (CPR) that delivers the electrons necessary for the reactions. Like PsiH, CPR is anchored to the endoplasmic reticulum by its N-terminal. Due to the hydrophobic nature of the N-terminals, CPR and PsiH will most likely result in misfolded proteins when expressed in bacteria, as they lack an endoplasmic reticulum.

 **Modification of the N-terminals and expression of GroEL/ES**

 Firstly, different kinds of modifications of the enzyme’s N-terminals have been created, to produce more hydrophilic proteins which are soluble in the cytosol of the bacterium. Secondly, previous studies have shown that the overexpression of the native chaperone GroEL/ES is beneficial concerning the expression of P450 monooxygenases. Taking this approach GroEL/ES is overexpressed by co-expressing it in *E. coli* along with CPR and PsiH to ensure correct folding and optimal catalytic activity.In relation to the N-terminals, there is currently no standard procedure, and it is mostly based on a trial-and-error approach. [7]

In this project, N-terminal variants demonstrating promising results in other studies are used as well as different modifications such as point mutations, deletions, and - most importantly - substitutions of entire N-terminals from other proteins, to make chimeric enzymes. [7] The modified PsiH and CPR enzymes were analyzed in silico by the software RoseTTAFold which uses deep learning to predict how the introduction of the different modifications affects the structure of the enzymes.
[Model](https://2021.igem.org/Team%3ASDU-Denmark/Model)[8]

![](https://static.igem.org/mediawiki/2021/7/71/T--SDU-Denmark--PsiHNT.png)

![](https://static.igem.org/mediawiki/2021/1/14/T--SDU-Denmark--CDRNT.png)

CPR showed to be very tricky as well because the original one from *Psilocybe cubensis* (*P. cubensis*) turned out to consist of several transmembrane segments, thereby making it very difficult to modify using the same technique as described above. Therefore, we have used an orthologue from the other mushroom Phanerochaete chrysosporium, which has a transmembrane domain in the N-terminal only that is much easier to handle using the same approaches as for PsiH.

Point mutations: We decided to replace phenylalanine with alanine because removing big aromatic amino acids and insert alanine instead of has been done with great success before.[9][10] This modification has only been applied on PsiH.

Deletions: For both PsiH and CPR, the signal peptide defined on UniProt was removed as they constitute the only hydrophobic transmembrane domain.[10]

Substitutions: This approach requires that the entire segment of the N-terminal can be identified as we don’t want to affect the catalytic domains. In terms of P450, this is quite easy as it only demands that one determine the proline rich region near the N-terminal, which in our case is PPGPP.[7] For CPR, the end of the N-terminal region was defined by range of the signal peptide.[11] The sequence before these segments can be replaced with N-terminals from other proteins. For PsiH these have been taken from the studies of Ichinose and Wariishi in which lots of different N-terminals from other cytochrome P450s were found to enhance solubility.[7],[12] Some of these have been used for CPR as well, although a few sequences of new N-terminals from water soluble cytochrome b5 reductases have been found using UniProt.

## General Theory

## Inducible Promoter-systems

Inducible promoter systems are used to regulate the protein expression from the psiH, psiDKM, CPR, and groEL/ES genes. There are multiple advantages of using inducible promoters rather than constitutively active ones. Constitutive expression of proteins will often slow the growth of bacteria, as it is energy-consuming and products may be toxic for cells, and thus it is of in-terest that the production does not begin until the exponential growth phase. In the project, two different inducible promoter systems will be used. The aim of this is to be able to express the chaperone GroEL/ES prior to the expression of PsiH, so that the chaperone will be ready to help PsiH fold correctly.[13]

![](https://static.igem.org/mediawiki/2021/c/c3/T--SDU-Denmark--Billede6.png)

One of the inducible promoter systems used is the pBad/araC system. The repressor protein AraC regulates the transcription of genes controlled by the pBad promoter. In the presence of the monosaccharide L-arabinose, AraC activates the transcription while it actively represses the transcription in the absence of L-arabinose. Thus L-arabinose can be used to induce the tran-scription as desired of GroEL/ES, which is controlled by the pBad/araC promoter.[14]

The other system used is the T7 Lac promoter system. Only the bacteriophage T7-polymerase can initiate transcription from the T7 promoter. In the protein expression strain used (E. coli, ER2566), the T7-polymerase gene is included in the genome, and its expression is controlled by a Lac-promoter and an associated operator site. The T7-promoter controls the genes of interest which are also associated with the lac-operator site. The repressor protein LacI binds to this operator site, and thus inhibits the expression of both the T7-polymerase and the genes of interest. In the presence of the inducer isopropyl-β-D-1-thiogalactopyranoside (IPTG), the LacI repressor protein will let go of the operator site, the T7-promoter will be expressed and thus be able to initiate transcription from the genes of interest. Thus, IPTG can be used to induce the expression of PsiH, PsiDKM, and CPR, when these genes are controlled by a T7-promoter (2).

## Plasmid Construction

For the construction of a plasmid backbone, a resistance cassette carrying re-sistance genes for either, kanamycin-, ampicillin- or chloramphenicol is needed. The use of different antibiotic-resistance cassettes in each plasmid is necessary to ultimately be able to test different combinations of PsiDKM, PsiH, GroEL/ES, and CPR in the same bacteria strain.

For the assembly of an inducible T7 LacI promoter system, LacI and a medium constitutive promotor should be inserted into the respective plasmid backbone. This can be achieved by using the standard iGEM restriction enzyme system: EcoRI, XbaI, SpeI, and PstI. Starting with the J61002 backbone carrying ampicillin resistance and containing a medium constitutive promoter (BBa\_J23106), it is possible to insert the LacI gene (BBa number). (Figur) Afterward, the medium constitutive promoter and LacI can be moved together to a kanamycin resistance carrying backbone (pSB1K3) or a chloramphenicol carrying backbone (pSB1C3) as shown in figure. After transformation of ligation products into *E. coli* Top10, the insert length can be verified by colony-PCR, and the resulting plasmids will now be ready for insertion of biobricks (PsiDKM, PsiH, or CPR).

For GroEL/ES, the plasmid pSB1C3, containing araC and the inducible pBad/araC promoter system (BBa\_I0500), is used.

![](https://static.igem.org/mediawiki/2021/1/12/T--SDU-Denmark--Billede7.png)

 **SOPs used:** *SOP6, SOP7, SOP8, SOP9, SOP12, SOP13*

## Transformation - Two system approach

**Creation of single, double, and triple transformants**

 To create double and triple transformants, the different plasmids are first individually trans-formed into *E. coli* Top10 and afterward into *E. coli* ER2566. This two-step approach was used as the Top10 strain distinguishes itself by being good for cloning while ER2566 is good for protein expression. The ligation product-based transformation into Top10 is quality controlled by colony PCR. Here it is possible to distinguish between successful and relegation-based transformations.

![](https://static.igem.org/mediawiki/2021/a/a8/T--SDU-Denmark--billede8.png)

![](https://static.igem.org/mediawiki/2021/a/a4/T--SDU-Denmark--billede9.png)

The finished ER2566 single transformants were then included in transcription and translation control experiments and in addition sent to Eurofins for sequencing. Following this, triple transformants were created in a stepwise approach, by transforming one plasmid into the bacteria at a time. The ER2566 triple transformants were quality controlled by primer specific colony PCR. Taking these triple transformants further the production strain is changed to two different efflux pump knockout strains. Succeeding, the whole de novo synthesis system can be introduced into the photoautotroph *R. sulfidophilum* [Hardware](https://2021.igem.org/Team%3ASDU-Denmark/Hardware).

![](https://static.igem.org/mediawiki/2021/thumb/9/95/T--SDU-Denmark--6.svg/320px-T--SDU-Denmark--6.svg.png)

![](https://static.igem.org/mediawiki/2021/thumb/f/fb/T--SDU-Denmark--gelT7lac-PsiDKM.svg/800px-T--SDU-Denmark--gelT7lac-PsiDKM.svg.png)

*Agarose gel with pSB1K3-LacI-T7lac-PsiDKM: Bands ~6000 indicate successful assembly of pSB1K3-LacI-T7lac-PsiDKM. The lanes correspond to the different colonies obtained from the transformation of pSB1K3-LacI-T7lac-PsiDKM.*

 **SOPs used:** *SOP9, SOP14, SOP17*

## Transcriptional and translational control experiments

Confirmation of enzymatic activity is crucial for optimal production of psilocybin. Adding IPTG to the LacI system will induce the gene expression of the four enzymes PsiD, PsiK, PsiM, PsiH, and CPR. This can be confirmed on a transcriptional level using quantitative PCR (qPCR). By using qPCR, it is possible to confirm the activity of both the enzymes and the promotor system.

Furthermore, the gene expression of the enzymes can be confirmed on a translational level using SDS-PAGE, in which an increased expression can be seen on the SDS-Page gel as an intensified band, compared to a sample that has not been induced with IPTG.

## PsiDKM

**Approach**

 Our main point of interest is the assembly of the three enzymes PsiD, PsiK, and PsiM, which previously have been shown to produce psilocybin from the substrate 4-hydroxy indole.

**Assembly of PsiDKM**

 The enzymes PsiD, PsiK, and PsiM biobricks were designed as two separate bricks - one containing PsiD and PsiK and one containing PsiM. The design of a two biobrick systems was necessary due to the ability for IDT to only synthesize 3000bp at a time. For amplification of the two bricks PCR can be used, using the Phusion High-Fidelity PCR Master Mix. For the amplification of the bricks primers with complementary overhangs were used. The amplified bricks were separated with gelelectrophoresis followed by purification from gel.

For the assembly of PsiDKM the two bricks were merged and amplified using overhang PCR and the Phusion High-Fidelity PCR Master Mix. Gel electrophoresis confirms the assembly of PsiDKM, due to the knowledge of an expected length contained from Snap Gene and afterward purified from gel. Furthermore, the assembly of PsiDKM can be confirmed by sequencing.

 **SOPs used:** *SOP6, SOP8, SOP10, SOP17*

**Assembly of plasmid backbone and PsiDKM**
 For the assembly of the plasmid backbone containing kanamycin resistance (pSB1K3-LacI) and PsiDKM (BBa\_K4059037), the plasmid backbone should be digested using the restriction enzymes SpeI and PstI. The digestion can be confirmed using gelelectrophoresis and afterward purified from gel. Furthermore, PsiDKM should be digested with the restriction enzymes XbaI and PstI and afterward confirmed by gelelectrophoresis followed by purification from gel.

![](https://static.igem.org/mediawiki/2021/f/f4/T--SDU-Denmark--PsiDKM1.png)

**Assembly of pSB1K3-LacI and PsiDKM:**: The expected results for gel electrophoresis for backbone, pSB1K3-LacI, and PsiDKM

For the assembly of pSB1K3-LacI and PsiDKM a ligation mixture of digested pSB1K3-LacI and PsiDKM should be prepared followed by transformation into *E. coli* Top10 strain. Colony PCR can be used to confirm the assembly. The plasmid DNA from the transformed cells can be purified using miniprep resulting in pSB1K3-LacI-T7lac-PsiDKM. For the confirmation of pSB1K3-LacI-T7lac-PsiDKM sequencing was used.

![](https://static.igem.org/mediawiki/2021/a/a8/T--SDU-Denmark--PsiDKM2.png)

**Assembled pSB1K3-LacI-T7lac-PsiDKM**: The expected results for gel electrophoresis for pSB1K3-LacI-T7lac-PsiDKM.

pSB1K3-LacI-T7lac-PsiDKM should be transformed into *E. Coli* ER2566 strain. To analyze the expression of PsiDKM after inducing with IPTG, RNA purification followed by Real-Time Quantitative PCR (qPCR) was performed. The results of Real-Time qPCR were used to confirm the activity of PsiDKM.

 **SOPs used:** *SOP6, SOP7, SOP8, SOP9, SOP12, SOP13, SOP14, SOP17, SOP18, SOP24*

## PsiH + CPR + GroEL/ES

**Our approach**

Wild type PsiH and CPR have hydrophobic N-terminal domains which enable them to be associated with the endoplasmic reticulum membrane in the eukaryotic fungi cell. However, these intracellular membrane structures are not present in the prokaryotic *E. coli* cell. Due to this the N-terminals must be modified, making them more hydrophilic and thus enabling the expression of the enzyme in the cytosol without altering the 3° structure of the entire enzyme.
In the PsiH and CPR biobricks ordered from IDT the *Psilocybe cubensis* wild type gene sequences have been codon optimized for *E. coli*. Furthermore, the N-terminal encoding WT gene regions have been deleted.

The different N-terminals were introduced by overhang PCR. This method was used to conveniently produce different CPR and PsiH N-terminal variants with the use of designed over-hang oligoes rather than using complete individual biobricks for each modification. The overhang PCR products were inserted in different backbones and transformed following the general two step transformation approach.

The successful single ER2566 transformants of CPR and PsiH N-terminal variants, and Gro-EL/ES were moved to different antibiotic resistance carrying backbones, enabling the creation of double and lastly triple transformant systems.
The three plasmids were introduced stepwise into ER2566. Lastly, the triple systems were included in production experiments, aiming to produce psilocybin from either 4-Hydroxyindole or Tryptophan, which yield was quantified using liquid chromatography-mass spectrometry.

## The experiments - PsiH, CPR and GroEL/ES

**Creation of N-terminal variants of PsiH and CPR**

To be able to conveniently create numerous different N-terminal modified PsiH and CPR enzymes, overhang PCR is used. By this approach, it is possible to substitute the N-terminals. The standard PsiH and CPR biobricks from IDT were resuspended. Following this step, two PCR reactions were run to introduce the new N-terminals following. Here it must be noted that as the PsiH and CPR biobricks do not contain a T7 promotor and an RBS site, two overhang introducing PCR reactions had to be run. By using a forward primer specific for the T7lac promoter and an N-terminal variant-specific reverse overhang primer, a 5’-overhang could be introduced. For the PsiH or CPR biobrick a normal reverse primer together with a variant-specific forward overhang primer was used. This resulted in the creation of a 5’-overhang for the PsiH biobrick. The products of the overhang introducing PCR reactions were quality controlled by gel electrophoresis. The expected length of the different bands was predicted using SnapGene and compared to the experimental data.

![](https://static.igem.org/mediawiki/2021/1/1f/T--SDU-Denmark--billede10.png)

 **SOPs used:** *SOP16, SOP10, SOP8*

![](https://static.igem.org/mediawiki/2021/thumb/3/38/T--SDU-Denmark--1222.svg/262px-T--SDU-Denmark--1222.svg.png)

The bands confirming a successful introduction of overhangs were prepared for PCR reaction number two with the aim to assemble overhang DNA 1 and 2.
Following this step, the N-terminal modified CPR or PsiH products are inserted in different antibiotic resistance carrying backbones.

 **SOPs used:** *SOP6, SOP7, SOP12, SOP13*

## Transport and knockout

**Our approach - Why transport?**

 Most heterologous production relies on the lysis of cells to liberate their product. Lysis requires the continuous restart of growth of the production organism, but this is dependent on many resources and adds a workload. Therefore, we decided on a different approach to accommodate the green future.

Our approach relies on the use of transporters to transport psilocybin out of the cell thereby skipping the lysis step in manufacturing. This allows for continuous production of psilocybin in a [bioreactor](https://2021.igem.org/Team%3ASDU-Denmark/Hardware) ,where only the media would need to be changed and no lysis of the cells would be necessary.

Furthermore, transporting the product out of the cell hinders a possible buildup of psilocybin that could potentially slow production.
To actualize our vision of continuous transport out of the cell we decided upon two different approaches; one, which includes testing of two *P. cubensis* theoretical native transporters and second, a native *E. coli* transporter.

**P. cubensis transporters**

In literature we found a description of a genetic island containing two genes that were hypothesized as transporters for psilocybin.[1] The genes were named psiT1 and psiT2 and the protein products Major facilitator-type transporter psiT1 (BBa\_K4059032) (PsiT1 for short) and Major facilitator-type transporter psiT2 (BBa\_K4059033) (PsiT2 for short) respectively. The transporters were still on the theoretical basis wherefore we examined the amino acid sequence in Uniprot. We used Swiss-Model for protein structure homology-modelling and found that Ma-jor facilitator-type transporter psiT1 and Major facilitator-type transporter psiT2 show similarity with known ion coupled transporters. This indicated that the proteins are transporters which drove us to continue with experiments.
However, as we did not know how these transporters would be oriented in a membrane of *E. coli* or even if they would localize to the membrane, we designed our experiments around these questions.
We designed a method for investigating if a protein localizes to the membrane, as well as the orientation.
This method relies on coupling GFP to the N-terminal and C-terminal of the protein in two different plasmid constructs. The constructs consist of GFP modified from BBa\_K125500 from the iGEM registry linked to PsiT1 (BBa\_K4059032) and PsiT2 (BBa\_K4059033). The linker chosen was BBa\_K1486004 from the iGEM registry which was incorporated together with BBa\_K125500 in our BBa\_K4059034 and BBa\_K4059035 parts.

 **Plasmid design**

In designing our biobricks we thought about possible use in future connections with membrane proteins. To assemble our biobricks we chose a backbone BBa\_J61002 from the iGEM registry with an ampicillin resistance marker for ease in our transformations.
Our primary design was the designing of GFP-constructs that can be coupled to C-terminal of transporters or N-terminal of transporters. The GFP sequence was taken from BBa\_K125500 in the iGEM registry.
The constructs were designed to include a constitutive promoter BBa\_J23100 to ensure contin-uous expression of the inserted proteins (PsiT1 and PsiT2).
We then designed N-terminal- and C-terminal GFP coupled biobricks specific sequences to be ligated to our specific transporters by use of non-standard iGEM restriction enzymes NdeI and BamHI as described further in assembly of constructs for microscopy.
In order to introduce the specific NdeI restriction site, it was necessary to use a filler for this. We chose the filler 5'-TATACAT prior to the start codon of the transporters for the introduction of this site.

![](https://static.igem.org/mediawiki/2021/b/b4/T--SDU-Denmark--PT1.png)

This plasmid shows the GFP-C-terminal construct (BBa\_K4059034) coupled to our PsiT2 transporter.

![](https://static.igem.org/mediawiki/2021/3/3d/T--SDU-Denmark--PT2.png)

This plasmid shows the GFP-N-terminal construct (BBa\_K4059035) coupled to our PsiT2 transporter.

![](https://static.igem.org/mediawiki/2021/1/13/T--SDU-Denmark--PT3.png)

This plasmid shows the GFP-C-terminal construct (BBa\_K4059034) coupled to our PsiT1 transporter.

![](https://static.igem.org/mediawiki/2021/a/af/T--SDU-Denmark--PT4.png)

This plasmid shows the GFP-N-terminal construct (BBa\_K4059035) coupled to our PsiT1 transporter.

**E. coli native transporter**

As the *P. cubensis* native transporter might cause issues with expression or localization to the membrane as was our concern with the PsiH enzyme. As the first approach relies on the use of eukaryotic membrane proteins, we decided to look for possible candidate transporters in *E. coli*.
During extensive literature research we found an article describing the transport of different dyes containing extensive ring systems through the native *E. coli* transporters; YhjV, YihN and TolC. [15] We also found that a patent was given in 2020 concerning YhjV as a transporter of indole derivatives. [16]
As for PsiT1 and 2, we performed protein structure homology-modelling using Swiss-Model, which indicated that YhjV is an antitransporter - and even more intriguing; antitransporter of amino acids. We found this quite interesting as an ideal transport mechanism for our psilocybin producing system would be a scenario in which tryptophan, our start substrate, would be carried inside the cell while psilocybin should be exported.
The transporter YhjV is described to be involved in the transport of the dyes with extensive ring systems as the cells express significantly more fluorescence when this is upregulated, and less fluorescence when this is knocked out.[15] Since this transporter was also described in the patent application as a transporter of indole derivatives, we chose to investigate this further.

 **AcrA E. coli native efflux pump**

The intermediates in our pathway all share a common feature of being indoles, wherefore it would be an issue if these are exported from the cell. This can be done through efflux pumps of which we chose to focus on AcrA which is part of the multi-drug efflux pump along with AcrB and TolC. The pump is composed of the components in a 1:2:1: AcrB:AcrA:TolC.[17]
This efflux pump has broad substrate specificity but has been described in the patent as a transporter of indole derivatives.[16] As the pump is an efflux pump it is logical to assume that it could therefore export our intermediates from the cell thereby eventually lessening our output of psilocybin.
It is further described in the literature that indoles slightly upregulate the expression of AcrA, which could therefore potentially cause issues in our production of psilocybin and a lower yield.[18]

**Knockout experiments**

Investigating the effect of both YhjV as a possible transporter for our psilocybin and AcrA as an exporter of our intermediates we constructed knockout mutants in *E. coli*. We constructed the knockouts from strains in the keio collection specifically JW0452 and JW3508.
P1 phages are involved in the transfer of the knockout of genes from JW0452 by replacing the AcrA gene with the kanamycin cassette from JW0452 in *E. coli*. The same procedure was carried out for yhjV from JW3508.
We then utilized the ∆yhjV strain for mass-spectrometry to see if this lowered the yield of psilocybin and intermediates as described [further](https://2021.igem.org/Team%3ASDU-Denmark/Results)

## Transport

**The experiments – Transport Assembly**

 To assemble transporters (BBa\_K4059032 and BBa\_K4059033) with the GFP-constructs (BBa\_K4059034 and BBa\_K4059035) the following non-standard restriction enzymes are in-volved with the specified restriction sites:
NdeI: 5'-CATATG'-3 and BamHI: 5'-GGATCC-'3.

**Assembly of backbones**

The N-terminal and C-terminal backbones were designed as two separate biobricks, both con-taining GFP sequence from BBa\_K125500 and then each containing either the specific N-terminal or C-terminal. The bricks are amplified using Phusion PCR followed by gel electro-phoresis and purification from gel.

*The two biobricks*
![](https://static.igem.org/mediawiki/2021/7/75/T--SDU-Denmark--PT5.png)

![](https://static.igem.org/mediawiki/2021/4/47/T--SDU-Denmark--PT6.png)

The GFP-constructs are inserted into our backbone Bba\_J61002 with Bba\_J23106 through ligation of these two parts. Cutting of both N-terminal and C-terminal and Bba\_J61002 should be carried out with Xba1 and Pst1 followed by gel electrophoresis and gel purification. This cutting allows ligation to be carried out between gel purified C-terminal GFP protein and Bba\_J61002, and N-terminal GFP protein and Bba\_J61002.
The cut with XbaI and PstI removes both ribosomal binding site and promoter as this can be introduced later. The assembly can be carried out through ligation of which the products can then be used further for TSB transformation.

 **SOPs used:** *SOP6, SOP7, SOP8, SOP11, SOP13, SOP14*

**Assembly of constructs for microscopy**

 Once the C-terminal GFP construct (BBa\_K4059034) and N-terminal GFP construct (BBa\_K4059035) have been successfully assembled through ligation these can then be cut once again. This time the cutting can be done with different restriction enzymes which are not standard enzymes in iGEM. These enzymes are NdeI and BamHI.

The transporters PsiT1 and PsiT2 (Bba\_K4059032 and Bba\_K4059033) must have the BamHI restriction site introduced through PCR with Phusion in which the two transporters are amplified with both a suffix and prefix specific primer for the introduction of the BamHI restriction site.

N-terminal fusion to GFP construct: The transporter should be amplified using a suffix primer and primer that introduces BamHI site at the N-terminal of the protein. Then both the plasmid with construct and protein of interest are cut with BamHI + SpeI so that they will be compatible and the cutting is verified by gel electrophoresis followed by purification from gel.

C-terminal fusion to GFP construct: The protein of interest should be amplified with a prefix primer and the primer that introduces the BamHI site at the C-terminal, after which both the plasmid with the construct and the protein of interest are cut with NdeI + BamHI and the cutting is verified by gel electrophoresis followed by purification from gel. The ligation products can then be transformed into *E. coli* and selected based on the ampicillin resistance marker present in Bba\_J61002. This is verified by Colony PCR. The expected colony PCR results would be that the amplification with suffix and prefix primer would show the insert of 1145 bp and 1148 bp of N-terminal GFP construct and C-terminal GFP constructs (BBa\_K4059035 and BBa\_K4059034) in well 1 and well 2 below respectively.

![](https://static.igem.org/mediawiki/2021/6/62/T--SDU-Denmark--WesternBlotCleaved.svg)

![](https://static.igem.org/mediawiki/2021/b/b8/T--SDU-Denmark--WesternBlotNonCleaved.svg)

 **SOPs used:** *SOP6, SOP8, SOP9*

**Fluorescence microscopy**

Once the GFP has been successfully coupled to N-terminal and C-terminal GFP constructs part of our transporters, the constructs can be used to investigate the orientation of the transporters as well as their ability to be integrated in the membrane.
If the transporter was oriented with the terminal coupled to GFP intracellularly, only a halo of florescence occurs as seen schematically below:

![](https://static.igem.org/mediawiki/2021/f/fc/T--SDU-Denmark--GFPbacteriaOuter.svg)

If the transporter was oriented in such a way that the GFP would be found extracellularly, no fluorescence will be seen, since GFP cannot fold correctly outside the cell due to different pH.[19]

![](https://static.igem.org/mediawiki/2021/0/09/T--SDU-Denmark--GFPbacteriaInner.svg)

However, in case of misfolding or incorrectly expressed transporter, this results in a general florescence of the cell, seen as small dots or granulates consisting of aggregated proteins as seen below:

![](https://static.igem.org/mediawiki/2021/5/5c/T--SDU-Denmark--GFPbacteriaNone.svg)

This allows visualization of whether the transporter was integrated into the membrane as fluo-rescence would then be localized to the cell membrane and not generally in the cytosol.

 **SOPs used:** *SOP19*

**Western Blot**

Western blot is carried out to investigate potential cleavage of GFP and transporters in the link-er region as this region contains a lot of serine along with glycine to produce a very flexible peptide chain. *E. coli* is a bacteria-rich in serine proteases.[20] This is carried out with GFP antibody and secondary HRP conjugated antibody.
If cleavage had occurred, it could be seen as GFP in its size of 27.4 kDa according to Snapgene and possibly the transporters at their respective sizes. However, it is possible that the cleavage could occur in other places as well. This is shown in the figure below:

![](https://static.igem.org/mediawiki/2021/6/62/T--SDU-Denmark--WesternBlotCleaved.svg)

If no cleavage had occurred, it would be seen as the full size of the transporters with the GFP-constructs added at 79.2 kDa and 84.3 kDa for PsiT1 and PsiT2. This is shown in the figure below:

![](https://static.igem.org/mediawiki/2021/b/b8/T--SDU-Denmark--WesternBlotNonCleaved.svg)

 **SOPs used:** *SOP22*

## Knockout

**The experiments – Knockout**

To test the transporters acrA and yhjV’s role in the efflux of intermediates and of psilocybin respectively, a knockout of the transporters will be performed.
P1 phages are obtained and used for the making of phage lysate. Phage lysate is made of the two knockout mutants from the keio collection, acrA, and yhjV. When the lysate is transduced into *E. coli*, genes for acrA or yhjV are replaced with an FRT-flanked kanamycin resistance cassette.[21] The transduced bacteria are selected for kanamycin to test if the cassette has been inserted correctly.
Once the kanamycin cassette is introduced, it needs to be excised. Here a plasmid containing pCP20 and an ampicillin marker is introduced into the bacteria. pCP20 is an FLP recombinase that mediates the recombination between the FRT sites flanking the kanamycin cassette. The recombinase is temperature-sensitive and must be grown at temperatures below 30oC to introduce the plasmid into the cell. To complete the knockout the recombinase is cured at 42℃, removing it from the bacteria.[21] By performing a colony PCR over the created knockout mutants, the length should indicate a smaller fragment size than the WT ER2566.

![](https://static.igem.org/mediawiki/2021/f/f2/T--SDU-Denmark--kanamycin.svg)

 **SOPs used:** *SOP20*

## Production and detection

To create De novo synthesis system cell factories of psilocybin or cell factories producing it from 4-Hydroxyindol, triple transformants were created. The triple transformants were created in a stepwise process through a double transformant. The double ER2566 transformants were confirmed by negative selection on antibiotic LA-plates, whereas the triple transformants were confirmed using colony PCR with gene specific primers. If all there plasmids are correctly present in the cell, all three primer stets are able to bind in their respective PCR reaction. This results in the formation of three PCR products which the SnapGene predicted lengths.

![](https://static.igem.org/mediawiki/2021/c/cb/T--SDU-Denmark--Billede11.png)

![](https://static.igem.org/mediawiki/2021/f/f7/T--SDU-Denmark--psihhh.svg)

**Future Experiments**

We also considered adding signal peptides to the sequence of both enzymes. Thereby PsiH and CPR would be transported to the cell membrane rather than floating around in the cytosol. Potentially, one might be able to preserve the native form in that way and thus greater activity, because P450 monooxygenases are often still partially misfolded by N-terminal modification - either as a result of the new N-terminal or simply because the protein is used to be closely associated with a membrane which is shown in the indelible hydrophobic domains.[12] However, this was an idea that came up late in the project and as we at some point plan to move the entire system to *Rhodovulum sulfidophilum*, which probably would require another signal peptide to work compared to *E. coli*, we never actually tested this, but it might be interesting in the future. In addition to this, it would also be interesting to investigate whether the use of a peptide linker between PsiH and CPR would optimize the production of psilocybin in terms of N-terminal modification. Hypothetically, this might have an impact by simply placing the two enzymes close to each other thereby making the transfer of electrons from CPR to the reaction catalyzed by PsiH more probable compared with the alternative scenario in which N-terminal modification that makes the enzymes more soluble also creates distance in between making the interaction more stochastic. Furthermore, one could improve tryptophan production in *E. coli* thereby obtaining a higher yield in psilocybin without adding tryptophan to the medium from start.[22]

## References

References
[1] Fricke J, Blei F, Hoffmeister D. Enzymatic Synthesis of Psilocybin. Angewandte Chemie In-ternational Edition [Internet]. 2017 [cited 22 October 2021];56(40):12352-12355. Available from: https://pubmed.ncbi.nlm.nih.gov/28763571/
[2] Adams A, Kaplan N, Wei Z, Brinton J, Monnier C, Enacopol A et al. In vivo production of psilocybin in *E. coli*. Metabolic Engineering [Internet]. 2019 [cited 22 October 2021];56:111-119. Available from: https://pubmed.ncbi.nlm.nih.gov/31550507/
[3] Team:Sydney Australia - 2019.igem.org [Internet]. 2019.igem.org. 2021 [cited 22 October 2021]. Available from: https://2019.igem.org/Team:Sydney\_Australia
[4] Hoefgen S, Lin J, Fricke J, Stroe M, Mattern D, Kufs J et al. Facile assembly and fluores-cence-based screening method for heterologous expression of biosynthetic pathways in fun-gi. Metabolic Engineering [Internet]. 2018 [cited 22 October 2021];48:44-51. Available from: https://pubmed.ncbi.nlm.nih.gov/29842926/
[5] Milne N, Thomsen P, Mølgaard Knudsen N, Rubaszka P, Kristensen M, Borodina I. Metabol-ic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and re-lated tryptamine derivatives. Metabolic Engineering [Internet]. 2020 [cited 22 October 2021];60:25-36. Available from: https://pubmed.ncbi.nlm.nih.gov/32224264/
[6] Hausjell J, Halbwirth H, Spadiut O. Recombinant production of eukaryotic cytochrome P450s in microbial cell factories. Bioscience Reports [Internet]. 2018 [cited 22 October 2021];38(2). Available from: https://pubmed.ncbi.nlm.nih.gov/29436484/
[7] Ichinose H, Wariishi H. High-level heterologous expression of fungal cytochrome P450s in Escherichia coli. Biochemical and Biophysical Research Communications [Internet]. 2013 [cit-ed 22 October 2021];438(2):289-294. Available from: https://pubmed.ncbi.nlm.nih.gov/23886957/
[8] Baek M, DiMaio F, Anishchenko I, Dauparas J, Ovchinnikov S, Lee G et al. Accurate prediction of protein structures and interactions using a three-track neural network. Science [Internet]. 2021 [cited 22 October 2021];373(6557):871-876. Available from: https://pubmed.ncbi.nlm.nih.gov/34282049/
[9] Mbaye M, Hou Q, Basu S, Teheux F, Pucci F, Rooman M. A comprehensive computational study of amino acid interactions in membrane proteins. Scientific Reports [Internet]. 2019;9(1). Available from: https://www.nature.com/articles/s41598-019-48541-2
[10] Hausjell J, Halbwirth H, Spadiut O. Recombinant production of eukaryotic cytochrome P450s in microbial cell factories. Bioscience Reports [Internet]. 2018 [cited 22 October 2021];38(2). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835717/
[11] Feyereisen R. Insect CYP Genes and P450 Enzymes. Reference Module in Life Sciences [Internet]. 2019 [cited 22 October 2021];. Available from: https://www.sciencedirect.com/science/article/pii/B9780128096338040401
[12] Ichinose H, Hatakeyama M, Yamauchi Y. Sequence modifications and heterologous ex-pression of eukaryotic cytochromes P450 in Escherichia coli. Journal of Bioscience and Bio-engineering [Internet]. 2015 [cited 22 October 2021];120(3):268-274. Available from: https://pubmed.ncbi.nlm.nih.gov/25732206/
[13] Kallunki, Barisic, Jäättelä, Liu. How to Choose the Right Inducible Gene Expression Sys-tem for Mammalian Studies?. Cells [Internet]. 2019 [cited 22 October 2021];8(8):796. Availa-ble from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721553/
[14] Brautaset T, Lale R, Valla S. Positively regulated bacterial expression systems. Microbial Biotechnology [Internet]. 2008 [cited 22 October 2021];2(1):15-30. Available from: https://pubmed.ncbi.nlm.nih.gov/21261879/
[15] Salcedo-Sora J, Jindal S, O'Hagan S, Kell D. A palette of fluorophores that are differentially accumulated by wild-type and mutant strains of Escherichia coli: surrogate ligands for profil-ing bacterial membrane transporters. Microbiology [Internet]. 2021 [cited 22 October 2021];167(2). Available from: https://pubmed.ncbi.nlm.nih.gov/33406033/
[16] Kell D, Yang L, Malla S, inventors. Engineered cells for production of indole-derivatives. WO2020187739. 2020 Sep 24.
[17] Bhandol H, Alindogan J, de Guzman A, Lim R. Review: Structure and regulation of the Acr efflux pumps and their role in antibiotic resistance in Escherichia coli [Internet]. UJEMI. 2021 [cited 22 October 2021]. Available from: https://ojs.library.ubc.ca/index.php/UJEMI/article/view/193259
[18] Hirakawa H, Inazumi Y, Masaki T, Hirata T, Yamaguchi A. Indole induces the expression of multidrug exporter genes in Escherichia coli. Molecular Microbiology [Internet]. 2004 [cited 22 October 2021];55(4):1113-1126. Available from: https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2004.04449.x
[19] Kneen M, Farinas J, Li Y, Verkman A. Green Fluorescent Protein as a Noninvasive Intracel-lular pH Indicator. Biophysical Journal [Internet]. 1998 [cited 22 October 2021];74(3):1591-1599. Available from: https://pubmed.ncbi.nlm.nih.gov/9512054/
[20] Janczak M, Bukowski M, Górecki A, Dubin G, Dubin A, Wladyka B. A systematic investiga-tion of the stability of green fluorescent protein fusion proteins. Acta Biochimica Polonica [Internet]. 2015 [cited 22 October 2021];62(3):407-411. Available from: https://pubmed.ncbi.nlm.nih.gov/26192770/
[21] Saragliadis A, Trunk T, Leo J. Producing Gene Deletions in *Escherichia coli* by P1 Transduction with Excisable Antibiotic Resistance Cassettes. Journal of Visualized Experiments [Internet]. 2018 [cited 22 October 2021];(139). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235078/
[22] Niu H, Li R, Liang Q, Qi Q, Li Q, Gu P. Metabolic engineering for improving l-tryptophan production in Escherichia coli. Journal of Industrial Microbiology and Biotechnology [Inter-net]. 2019 [cited 22 October 2021];46(1):55-65. Available from: https://academic.oup.com/jimb/article/46/1/55/5996789?fbclid=IwAR2abxJn\_iTnnqpNv6of1kJKSrZhZVi3slzhLAb0k2RH8ZRGqzcveoa-85o

![](https://static.igem.org/mediawiki/2021/1/15/T--SDU-Denmark--frontpagemushroom.svg)

#### Results

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# Team:SDU-Denmark/Results

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# Results

![](...)

# Abstract

On this page, you can read about how we achieved a succesful production of psilocybin.

# Introduction

## PsiDKM

## Assembly of 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.

![](https://static.igem.org/mediawiki/2021/b/b4/T--SDU-Denmark--A_Gel_PsiDKM.svg )

*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

### 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.

![](https://static.igem.org/mediawiki/2021/1/15/T--SDU-Denmark--A_Gel_pSB1K3-LacI.svg )

*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.

![](https://static.igem.org/mediawiki/2021/2/26/T--SDU-Denmark--A_Gel_pSB1K3-LacI-T7lac-PsiDKM.svg)

*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.*

## Sequencing

### 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.

## Transcriptional and translational control

### 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.

![](https://static.igem.org/mediawiki/2021/f/fd/T--SDU-Denmark--QPCR_PsiDKM.svg)

*qPCR analysis of PsiDKM: Gene expression of PsiD, PsiK and PsiM and the T7-polymerase.*

## Detection for psilocybin

### 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.

![](https://static.igem.org/mediawiki/2021/0/0e/T--SDU-Denmark--Falkon.svg)

*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.

![](https://static.igem.org/mediawiki/2021/8/88/T--SDU-Denmark--LCMS_PsiDKM.svg)

*LC/MS of PsiDKM: Arbitrary areas of three different PsiDKM samples of fragtment ions m/z=204 and m/z=160.*

## PsiH

## Assembly of PsiH/CPR

### 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.

![](https://static.igem.org/mediawiki/2021/6/6e/T--SDU-Denmark--T7LacPsiH1.svg)

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.

![](https://static.igem.org/mediawiki/2021/c/c4/T--SDU-Denmark--T7LacPsiH2.svg)

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)

![](https://static.igem.org/mediawiki/2021/6/66/T--SDU-Denmark--CPR_Overhang_Int_PCR.svg)

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.

![](https://static.igem.org/mediawiki/2021/0/05/T--SDU-Denmark--CPR_Overhang_Int_PCR2.svg)

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.

![](https://static.igem.org/mediawiki/2021/b/bc/T--SDU-Denmark--CPR_Overhang_N.svg)

![](https://static.igem.org/mediawiki/2021/c/c4/T--SDU-Denmark--CPR_Overhang_N2.svg)

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).

![](https://static.igem.org/mediawiki/2021/c/c8/T--SDU-Denmark--PCR_PsiH_ST.svg)

![](https://static.igem.org/mediawiki/2021/d/d7/T--SDU-Denmark--PCR_CPR_ST2.png)

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.

![](https://static.igem.org/mediawiki/2021/e/e5/T--SDU-Denmark--N_PsiH_GyrA.png)

![](https://static.igem.org/mediawiki/2021/8/89/T--SDU-Denmark--N_PsiH_GyrA2.png)

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:

![](https://static.igem.org/mediawiki/2021/c/cf/T--SDU-Denmark--Overview_CPR_NT.png)

![](https://static.igem.org/mediawiki/2021/8/8a/T--SDU-Denmark--Overview_CPR_NT2.png)

### 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).

![](https://static.igem.org/mediawiki/2021/3/30/T--SDU-Denmark--CPR_Del_5_14.png)

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

### 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.

![](https://static.igem.org/mediawiki/2021/6/69/T--SDU-Denmark--figur22.png)

![](https://static.igem.org/mediawiki/2021/4/43/T--SDU-Denmark--SDSPAGE1.png)

## Production of Psilocybin

## 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.

![](https://static.igem.org/mediawiki/2021/c/cb/T--SDU-Denmark--Billede11.png)

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).

![](https://static.igem.org/mediawiki/2021/c/cb/T--SDU-Denmark--PTRP.png)

### 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:

![](https://static.igem.org/mediawiki/2021/4/4f/T--SDU-Denmark--FORMEL1.png)

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.

![](https://static.igem.org/mediawiki/2021/5/54/T--SDU-Denmark--figur15.png)
*Figure 15 illustrates the fragment ion located at m/z=204*

![](https://static.igem.org/mediawiki/2021/6/6d/T--SDU-Denmark--figur16.png)
*Figure 16 Illustrates the fragment ion located at m/z=160*

![](https://static.igem.org/mediawiki/2021/4/47/T--SDU-Denmark--figur17.png)
*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.*

![](https://static.igem.org/mediawiki/2021/8/8a/T--SDU-Denmark--figur18.png)
*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.

![](https://static.igem.org/mediawiki/2021/0/0e/T--SDU-Denmark--Table_1.png)
*Table 1 illustrates the different samples as well as the result of the analysis*

![](https://static.igem.org/mediawiki/2021/8/85/T--SDU-Denmark--figur19.png)

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

![](https://static.igem.org/mediawiki/2021/0/01/T--SDU-Denmark--Table_2.png)
*Table 2 illustrates the different samples as well as the result of the analysis*

![](https://static.igem.org/mediawiki/2021/0/0d/T--SDU-Denmark--figur21.png)

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

## 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.

![](https://static.igem.org/mediawiki/2021/e/e4/T--SDU-Denmark--Transport1.png)

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.

![](https://static.igem.org/mediawiki/2021/c/cd/T--SDU-Denmark--Transport2.png)

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.

![](https://static.igem.org/mediawiki/2021/1/1f/T--SDU-Denmark--Transport3.png)

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.

![](https://static.igem.org/mediawiki/2021/a/a6/T--SDU-Denmark--Transport4.png)

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.

![](https://static.igem.org/mediawiki/2021/3/36/T--SDU-Denmark--Transport5.png)

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:

![](https://static.igem.org/mediawiki/2021/9/98/T--SDU-Denmark--Tabel.png)

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.

![](https://static.igem.org/mediawiki/2021/c/c5/T--SDU-Denmark--Transport6.png)

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.

![](https://static.igem.org/mediawiki/2021/f/ff/T--SDU-Denmark--Transport7.png)

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.

![](https://static.igem.org/mediawiki/2021/f/f9/T--SDU-Denmark--Transport8.png)

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.

![](https://static.igem.org/mediawiki/2021/3/3f/T--SDU-Denmark--PsiT1_Fluor1.svg)

![](https://static.igem.org/mediawiki/2021/3/32/T--SDU-Denmark--PsiT1_Fluor2.svg)

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.

![](https://static.igem.org/mediawiki/2021/4/43/T--SDU-Denmark--PsiT1_Fluor3.svg)

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](https://2021.igem.org/Team%3ASDU-Denmark/Human_Practices) 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.

![](https://static.igem.org/mediawiki/2021/7/7e/T--SDU-Denmark--Transport12.png)

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.

![](https://static.igem.org/mediawiki/2021/e/e2/T--SDU-Denmark--Transport13.png)

Our actual western blot is shown below with the wells corresponding to the theoretical images.

![](https://static.igem.org/mediawiki/2021/b/b3/T--SDU-Denmark--Transport14.png)

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.

![](https://static.igem.org/mediawiki/2021/4/44/T--SDU-Denmark--Transport15.png)

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.

![](https://static.igem.org/mediawiki/2021/9/93/T--SDU-Denmark--Transport16.png)

### Transformation of PsiDKM into knockout strain

Next goal was a knockout mutant capable of producing psilocybin from 4-hydroxyindole.

![](https://static.igem.org/mediawiki/2021/5/53/T--SDU-Denmark--Transport17.png)

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.

![](https://static.igem.org/mediawiki/2021/e/e3/T--SDU-Denmark--Transport18.png)

![](https://static.igem.org/mediawiki/2021/1/15/T--SDU-Denmark--frontpagemushroom.svg)

#### Parts

![](https://static.igem.org/mediawiki/2021/1/1c/T--SDU-Denmark--Next_pil.svg)