Team:Sydney Australia/Notebook

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Notebook

We've been working hard inside and outside of the lab for almost five months now, and whilst it has been challenging we've had a lot of fun! Read more about it below.

23/5/19 - Team meeting

First ever team meeting - the project begins! So exciting!

31/5/19 - Team meeting

Team roles decided.

7/6/19 - Wet lab

Safety induction for the team - learning how to be safe in the lab

7/6/19 - Team meeting

Planning wet lab work, outreach measures, and human practices initiatives

14/6/19 - Team meeting

Discussed current lab work, further planning on outreach, discussed the need for Human Ethics approval for us to conduct the desired interviews for HP, discussed logo.

19/6/19 - Wet lab

Fahad prepared an overnight culture of E. coli containing pk18, pET28 and PUS250 plasmids.

24/6/19 - Wet lab

Fahad transformated pET28 and pk18 into TOP10 E. coli

25/06/19 - Wet lab

Fahad and Benj made a culture of pET28, pk18 and PUS 250. pET28 and pk18 were pelleted and stored in the -20C freezer. PUS250 had not reached a high enough concentration so was left to incubate overnight.

Merrie and Isobel made an overnight culture of Top10 E. coli on LB agar in incubator at 37 degrees overnight for preparation of competent cells.

26/6/19- Wet lab

Merrie made up a 10mM Tris PH8 from 1M stock solution and sent for autoclaving. Merrie also pelleted the overnight culture of PUS250. The 1:10 dilution had OD600 of 0.502.

Merrie completed plasmid DNA extraction of pET28, pk18 and PUS250 plasmids according to the Coleman lab protocol. The only variation to the protocol was that the pellets were resuspended in 1XTE pH8 instead of 10mM Tris as there was no sterile Tris. All plasmids were stored in the -20 freezer.

Isobel and Fahad performed a restriction digest of Pet28 PUS250 & PK18 with HindIII and EcoRI. The restriction digest was conducted according to manufacturer's protocol for ThermoScientific FastDigest restriction enzymes, and products were run on a 1% agarose gel in 1xTAE for 30 minutes at 200 V. The gel was run to quantitate and identify the prepared plasmids.

pK18 and pET28c(+) were grown in a 50 mL overnight culture of LB, at 37°C with shaking at 200 rpm. 5 x 10 mL of each culture was decanted into separate falcon tubes, then spun at 2500 rcf for 15 minutes. Supernatant was discarded and 500 uL of P1 resuspension buffer was added to each tube. After resuspending the pellet by vortex, 2 x 250 uL of each tube was added to separate microcentrifuge tubes, and miniprep proceeded according to manufacturer's protocol (ISOLATE II Plasmid Mini Kit, Bioline).

10 x 50 uL of purified plasmid was obtained for pK18 and pET28c(+), and samples for respective plasmids were combined into single tubes. Plasmid concentration was determined by spectrophotometer (ThermoFisher NanoDrop 2000c), and were found to be 281.6 ng/μL for pK18 and 38.1 ng/μL for pET28c(+).

To confirm, a restriction digest was conducted according to manufacturer's protocol for ThermoScientific FastDigest restriction enzyme, and products were run on a 1% agarose gel in 1xTAE for 30 minutes at 200 V. The results were consistent with the nanodrop.

27/6/19 - Wet lab

Merrie performed a digest of the Vivid G blocks. Each reaction (WT, CH1, CH2, CH3, CH4) was incubated for 15min at 37ºC then run through spin column to remove enzymes and eluted in EB buffer (15μL).

Merrie performed a digest of the PET28 plasmid. The reaction was incubated for 15min at 37ºC then run through spin column to remove enzymes and eluted in EB buffer (45μL).

Merrie performed a ligation reaction of the PK18 with each of the Vivid GBlocks (WT, CH1, CH2, CH3, CH4). The reactions were incubated at room temperature for 15 minutes and stored in -30 freezer overnight.

Isobel continued with process of making TOP10 competent cells by taking 2x 3mL cells from overnight incubation into and putting them into 100mL LB broth.

Initial OD600 ~0.1. Incubated in 37ºC shaking 200rpm until OD600 ~0.5 reached. The cells were pelleted at OD600 ~0.45 as per protocol - the pellet was hard to resuspend. Cells were divided into 70 220μL aliquots stored in -80ºC freezer. 28/06/19-Wet lab-Isobel, Benj, and Merrie performed a heat shock transformation of TOP10 Ecoli with pk18 Vivid ligation. Positive and negative controls were generated. Transformed cells were spread-plated onto LB-Km-Xgal plates and incubated at 37C overnight.

Fahad performed a PET28 purification using spin column using Coleman lab protocol and eluted in 20μL EB buffer. Final concentration was 38.1ng/μL.

Fahad also made up a stock of LB-Xgal-Km plates.

29/6/19 - Wet lab

Fahad performed a plate observation for the transformed Vivid cells. No successful transformations were observed.

2/7/19 - Wet lab

Isobel and Benj performed a Vivid gene digestion and ligation into PK18. The plasmids and G-blocks were digested using EcoRI and HindIII for one hour at 37C. The enzymes were then heat killed at 80C for 20mins. 10uL plasmid digest and 10uL Vivid digest were combined with 10uL digest buffer and 1uL T4 ligase and incubated for one hour at room temp.

Isobel and Benj then performed a heat shock transformation of ligated PK18 and Vivid G-blocks. Positive, negative, ligation, and digestion controls were generated. 100uL of the transformed cells were spread-plated onto LB-Km-Xgal plates. The plates were incubated at 37C overnight.

3/7/19 - Wet lab

Checked plates. Very few colonies observed, almost all were blue. White colonies did not fluoresce under UV light so presumed false positives. Decided to test a new ligation protocol the next day. 4/7/19-Wet lab/outreach prep-Isobel conducted a plasmid preparation for the WMBB activity. Plasmids B29, PUS116 & PK18 to be used for fruit genetics experiment. Each plasmid was grown inside TOP10 E. coli with the appropriate antibiotic in 1mL LB until it reached OD600 1.5. Each culture was then transferred to a 50mL LB broth to grow at 37C until it reached OD600 1.0. Each culture was then mini-prepped according to Coleman lab protocol.

WMBB write up.

4/7/19 - Wet lab

Merrie and Benj performed digest and ligation testing of PK18 to troubleshoot the Vivid transformations. Learned to decrease plasmid concentrations for future cloning. Confirmed that ligation was working as intended.

5/7/19 - Wet lab

Benj performed another digestion and ligation control test. Plasmid digested with various enzymes was run on a gel to ensure that the plasmid was being cut as expected. The band sizes and numbers were consistent with the digestions working correctly. Plates were prepared with cells transformed with either digested or digested+ligated plasmids. Significant differences were seen in between the colony counts of the digested and ligated plates, indicating a significant improvement in digestion when the concentration of plasmid was reduced for digestion.

5/7/19 - Wet lab/outreach prep

Nathan continued preparing for the WMBB outreach activity by mixing the watermelon supernatant with the plasmid mixtures. For further details please see our WMBB write up.

5/7/19 - Team meeting

Discussed current Vivid progress, finalised logo, ironed out details for WMBB outreach event, discussed contacting Usona - major psilocybin producer for clinical trials.

6/7/19 - Wet lab/Outreach prep

Isobel and Nathan continued to prepare for the WMBB outreach event by performing a restriction digest on B29, PK18, and PUS116 and running a gel with the Kiwi DNA and the plasmids. For further details please see our WMBB write up.

8/7/19 - Wet lab/Outreach prep

Isobel ran a gel with the three different fruit DNAs and the three different plasmids. She found that as the strawberry + pk18 produced the clearest band pattern that would be the designated ‘sick person’ sample for the activity.

She also decided that for the students all their reagents would be replaced with water and then the gel replaced so the students would experience the experimental procedure without the risk of not getting easily interpretable results. See our WMBB write up. for more details

8/7/19 - Wet lab

Benj formulated a revised cloning protocol for his vivid genes. This created Vivid Cloning Protocol Version 1. See our protocols section for the full protocol. He performed this new protocol on his Vivid genes.

9/7/19 - WMBB outreach event

Nathan and Isobel ran a short molecular biology class with Indigenous youth called ‘Fruit Forensics’ where students had to use molecular biology to find out which piece of fruit had given their patient food poisoning. See our full WMBB write up. for more details!

9/7/19 - Wet lab

Benj had great success today! Finally saw some positive colonies on his CH2 and CH3 plates. However, unfortunately there were still no positive colonies for WT, CH1, and CH4. These vivid gene blocks needed to be re-done using Vivid Protocol Version 2.

10/7/19 - Wet lab

Benj performed Vivid Protocol Version 2 (see our protocol section for full protocol - link) on the WT, CH1, and CH4 gene blocks. Due to a plate mix-up the newly transformed cells were not plated onto differential media so there was some delay in seeing results.

11/7/19-Wet lab

Benj had even more success! He got some positive patches on his plate. There was some contaminating blue colonies among the white patches so these were further purified by streak plating.

12/7/19 - Wet lab

Nathan performed some Psi gene cloning of PsiM/D/K into PET28. He got positive colonies for all three genes.

15/7/19 - Wet lab

Benj and Isobel worked on purifying the Vivid colonies. Unfortunately several blue colonies remained on the plates. Benj consulted Nick (supervisor) who suggested that the constitutive expression of PK18 may be stressing the cells. New patch plates were incubated at 30C to reduce the stress on the cells.

Isobel was able to streak out 2x CH3 white colonies from CH3-1 and CH3-2 plates from 11/7. She then streaked CH2 from patch plate square 3 from 12/7. Lastly she made a patch plate of CH1 colonies from 10/7 pellet plate with 3x controls and 6x white colonies.

Benj then patch plated WT, CH4 from spread plates made on 10/7. CH2 was patch plated from spread plates from 8/7. CH1 was streak plated from CH1 patch plates from 12/7 using squares 1, 2, 4 and 5. All the plates that were made up were incubated at 30°C to reduce stress on the cells.

Lastly, Benj and Isobel made 19 LB-KM-XGAL plates (400mL LB agar, 500μL Kanamycin, 1mL X-Gal) and 19 (LB-KM plates 400mL LB agar, 500μL Kanamycin).

16/7/19 - Wet lab

Benj continued the purification of the Vivid transformations. He made streak plates from the patch plates made on 15/7 of WT and CH4 onto LB-Km-Xgal plates.

17/7/19 - Wet lab

Benj found that the plates grown at 30°C showed distinct and isolated white colonies on all Vivid streak plates.

He re-streaked the white colonies onto LB-km-Xgal plates and KM-charcoal plates. The charcoal plates reduce fluorescence of LB and therefore background fluorescence, good for testing the fluorescence of the different harmonisations. Benj also performed repurification on KM-Xgal plates of CH1, CH2, and CH3, then made some more KM-LB-Xgal plates.

Benj and Nathan performed colony PCR on the Psi D, K, and M plates. Four positive colonies were obtained for PsiM, three positive colonies for PsiK, and seven positive colonies were found for PsiD. However, whilst bands were present some of the dye had migrated off the gel resulting in poor visualisation of the ladder. Benj and Nathan decided to run a gel again using three positives for each Psi gene to get better visualisation of the ladder and bands for each gene. 18/7/19-Wet lab-Benj and Nathan ran a gel using three positive colonies per Psi gene from yesterday. This gel confirmed their results. These positive colonies were streak-plated on LB-Km plates and inoculated into LB-Km broths, all cultures were incubated at 36C overnight.

19/7/19 - Wet lab

Benj and Nathan removed the Psi gene cultures from the incubator. The plates were stored in the 4C fridge. The cultures were transferred to 5mL falcon tubes and the cells pelleted. Excess supernatant was removed and the tubes were placed in the -30C freezer for 24hours.

Benj found that the CH1 AND CH3 cultures have stable incorporation of the Vivid/pk18 plasmid but CH2 and CH4 and WT appear to be rejecting the plasmid this may be due to the high copy number of the plasmid and strong expression of the VIVID protein. To test this theory he grew new plates of CH1 and CH3 and incubated them at 30C and 37C to test whether the E. coli would reject the plasmid at different temperatures.

20/7/19-Wet lab

Benj and Nathan removed the frozen tubes from the freezer and thawed them. Any remaining supernatant was removed with careful pipetting. The cells in each tube were resuspended in 1mL EB buffer before being transferred to individual 1.5mL eppendorf tubes. Plasmid was then extracted from tubes using the 'Plasmid miniprep protocol (5 ml culture) – Spin Column Method' protocol, except the tubes were left to dry at room temperature on a clean Kimwipe for 10mins instead of at 60C to evaporate residual ethanol. All plasmid samples, eluted into 30uL of 10mM Tris-HCl pH8, were then placed in a fridge at 4oC for 48 hours.

Benj purified and moved the CH1 and CH3 plates from 19/7/19 to the cool room.

22/7/19 - Wet lab

Benj checked in on his Vivid colonies. Found that the CH1 plates were stable at 37C and 30C whilst the CH3 plates were beginning to turn blue at 37C but still stable at 30C.

Both the CH3 30C and 37C plates were moved to 30C to confirm it is the speed of growth that is resulting in the reversion to blue colonies.

WT, CH4, and CH2 were re-streaked on Km-Xgal and grown at 30C.

23/7/19 - Wet lab

Nathan analysed the PET28-PsiK/M/D plasmids prepared on 20/7/19 for purity and concentration using a nanodrop spectrophotometer. The least contaminated tube for each Psi gene was selected for restriction digest preparation.

A single restriction digest using EcoR1 and a double restriction digest using EcoR1 and HindIII was then set up for each plasmid sample as well as an empty pET28 vector, all tubes were incubated for one hour at 36C. 15uL of each sample was then mixed with 2uL of 6X purple loading dye before being pipetted into individual wells on an Agarose gel for gel electrophoresis.

Nathan found that the results indicated that the PsiK and PsiM colonies selected were suitable for protein purification study. Unfortunately, PsiD showed double banding - indicating the presence of both an empty pET28 and the PsiD construct within the cells. Using these cells for protein purification study would result in suboptimal expression of the PsiD gene. Nathan decided to test the other tubes using restriction digest before performing protein purification steps for PsiD. New individual 5mL of LB/Kan broths were inoculated with the tested PsiK and PsiM colonies.

Benj cultured Vivid CH1 and CH3 in 5mL Km-LB overnight to prepare for mini-prep. More colonies from the WT-patch plate were streaked out and incubated at 30C. The CH2 and CH4 plates from 22/7 were re-streaked and incubated at 30C. 24/7/19-Wet lab-Benj plasmid purified the CH1 and CH3 cultures according to the Coleman labs nanodrop protocol. CH1 had a concentration of 434.6ng/uL and CH3 had a concentration of 30.7ng/uL.

25/7/19 - Wet lab

Nathan performed another restriction digest analysis on the other two tubes of PsiD cells. Results revealed no double banding in the single digest column for tubes 2 and 3, with a distinct size difference when compared to the empty pET28 plasmid. This meant that tubes 2 and 3 were suitable for protein purification work.

Nathan also performed PCR on the PsiH gene block to add BamH1 & Xba1 restriction sites to the construct to make it suitable for insertion into the PCW plasmid.

Benj performed another mini-prep on the CH1 and CH3 cells according to Coleman lab protocols. The concentrations were similar to 24/7: CH1 had a concentration of 431.3 ng/uL, CH3 had a concentration of 28ng/uL. He then performed a restriction digest on both of them and ran similar amounts of plasmid on a gel along with pk18 cut and uncut plasmid. No double banding was observed. It does appear that the CH3 plasmid is significantly lower in concentration than the CH1 plasmid in E. coli Top10 cells, possibly due to the constitutive expression stressing the cells.

Benj decided to try transforming the vivid genes into PUS250 - an inducible plasmid. Benj used the leftover G-blocks for CH2 and CH3. For CH1 and CH3, the positive colonies from prior experiments were mini-prepped and that DNA was digested then used to transform into PUS250.

26/7/19 - Wet lab

Benj grew the CH2 AND CH3 spread plates for 3 days at 37C. White colonies were patch plated on KM-Xgal plates and KM-Cumate.

The LB plates were used to determine which colonies were truly fluorescent. Cumate (the inducer) plates should fluoresce strongly if Vivid has been successfully transformed into the pus250 plasmid and into the E. coli Benj also patch plated white colonies from the digestion and ligation plates to compare expression of CH1 and CH3 to the white colonies.

26/7/19 - Team meeting

Discussed current Vivid progress, discussed Psi-gene successes, made a plan for putting PsiH in PCW plasmid. Discussed going to the SBA Conference, discussed Dusseldorf postcard collaboration.

30/7/19 - Wet lab

Isobel performed a nanodrop on the PCW plasmid stocks in the lab. Found that the stock had a concentration of 825ng/uL. Diluted the stock 1 in 4. Performed two double digests and two single digests using NdeI and SalI. Unfortunately, the gel had no discernable bands - possible due to the plasmid being degraded in the cool room, enzymes being heat killed, or the mung bean endonuclease that was added to blunt the ends not being in the appropriate concentration.

Fahad prepared an overnight culture of the PUS250 plasmid using two colonies from 100uL heat shock transformed plate were isolated and to incubate 2x 5mL vials of LB-Km. They were incubated in the shaking 37C incubator overnight.

31/7/19 - Wet lab

Isobel and Merrie transformed some TOP10 E. coli with the remaining PCW (as per Coleman lab protocol) as very little stock remained after the failed digestion. Grew 100uL aliquots of the supernatant and pellet of the transformed cells on Ampicillin plates, plus a TOP10 only negative control.

Merrie used 1 sample of the overnight PUS250 culture for miniprep of plasmid DNA. Miniprep of 5mL sample as per Coleman lab protocol with no changes. Nanodrop showed 18ng/uL DNA.

1/8/19 - Wet lab

Isobel and Merrie found that the supernatant and pellet plates from 31/7 had good growth and there was no growth on the negative control plate. A big loop-full of cells taken from pellet plate, vortexed in 1mL broth, then inoculated into 50mL LB with Ampicillin. Grown in 37C shaker for 24hrs.

2/8/19 - Wet lab

Merrie miniprepped the PCW cells from 1/8 and tested the concentration using the nanodrop. The concentration was 113ng/uL. A restriction digest was then performed on the PCW plasmid DNA to remove the p450 from the plasmid using Sal1 and Nde1.

The double digest, single digest, and uncut PCW plasmids were run on a gel. The use of LAB buffer in the gel resulted in poor resolution in the ladder making it hard to determine size. However, the double digest had a double band, indicating the removal of p450, which made the cut PCW plasmids suitable for end-blunting and ligation. The ends were blunted using mung-bean nuclease and the DNA purified using a spin-column. The yield was low as spin columns do not work well on DNA over 5kb. The ends were ligated together using T4 ligase.

Benj found that the transformation of of CH2 and CH3 in PUS250 into TOP10 appeared to have failed. Fluorescence did not vary between transformed and untransformed colonies. The CH2 & CH3 patch plated on LB-Km-Xgal and LB-Km-Cumate had similar fluorescence and growth to digestion and ligation controls. Benj also performed a restriction digest on the various CH1-PK18 cultures from previous weeks.

2/8/19-Team meeting

In depth human practices meeting, got updated on the process of our Human Ethics approval - still waiting. Decided our main focuses are ethics, medical, and economics. Also discussed our outreach theme and potential for iGEM to be integrated into the high school curriculum.

5/8/19 - Wet lab

Isobel tested the concentration of the digested and ligated PCW plasmid using the nanodrop. The concentration in one replicate was 569.9ng/uL, and in the second replicate 712.5ng/uL. She then ran the plasmids on a gel to check for a band of the correct size. The gel made it apparent how much yield had been lost from the column purification, a phenol chloroform purification would have been better.

6/8/19 - Wet lab

Nick performed error prone PCR done using primers NVC15b and 16b to amplify the VVD-36-CH4-C37A gene (CH4 codon variant) out of pK18 plasmid backbone, from a colony of E. coli TOP10 carrying pK18-VVD-CH4 plasmid. PCR as normal using Taq polymerase, except 0.15 mM MnCl2 added to make it error-prone. Band looks good on gel.

7/8/19 - Wet lab

Isobel ran PCR on the PCW plasmid to test whether the digested plasmid still contains p450. The primers were designed to span the region where p450 used to be. If the p450 was cut out, end up with 700bp product. If p450 still in, end up with approx 2000bp product.

7/8/19 to 8/8/19 - JAMS Science Week Booth Outreach Event

Isobel, Merrie, Nathan, Emma, and Benj volunteered to help JAMS Sydney with their Science Week booth at the Australian Museum. They helped kids with activities such as looking at pond water under a microscope, viewing their own skin or hair using a handheld microscope, and viewing plates containing different microbes. See our full JAMS write up for more details!

8/8/19 - Wet lab

Isobel ran a gel of the PCR product from 7/8. Found that the PCR product band was around 700bp, indicating that our PCW plasmid had its p450 successfully removed - ready to be replaced by PsiH!

9/8/19 - Wet lab

Nick purified the error-prone PCR product from 6/8 on column, digested with EcoRI+HindIII, the mixture was heat-killed, then ligated to pK18 that had been digested with same enzymes overnight at 4C.

9/8/19 - Team meeting and meeting with Alex Kelly

Talked to Alex Kelly, biotechnology entrepreneur and iGEM Sydney alumni, about the considerations we must think of regarding scaling up our project for industry.

10/8/19 - Wet lab

Isobel performed a PCR on the PCW plasmid with p450 inside to double check that the PCW plasmid tested on 8/8 had its p450 removed and it wasn’t a PCR error. She ran the whole-PCW PCR product on the same gel as the digested-PCW PCR product and found that the band sizes were as expected.

Nick transformed the error-prone PCR ligation mix into TOP10 cells, which were spread on several LB-Km-Xgal plates at different dilutions, then incubated 30C for 48 h.

11/8/19 - Wet lab

Nick examined the error-prone PCR plates under white light and with long-wave UV lamp. Under white light, approx. 50% appear recombinant (white colonies), while rest are blue (vector only). Under UV lamp, a mix of non-fluorescent and weakly fluorescent colonies seen, but also a few colonies showing obviously brighter fluorescence – these were picked and restreaked for later analysis (total 12 colonies).

12/8/19 - Wet lab

Isobel performed a colony PCR on colonies transformed with the digested-PCW plasmid to ensure that the colonies used for mini-prep and insertion of PsiH were p450-free plasmids. The PCR-product from the colony PCR was run on a gel. Found that all seven colonies tested had the correct PCW plasmid in them. Isobel selected one colony to grow up for mini-prepping.

12/8/19 - Human Practices

Emma finished writing up the literature review on the History of Psilocybin in Society. Read it on our Human Practices page!

13/8/19 - Wet lab

Emma, Nathan, and Fahad performed a miniprep of the PsiK and PsiD PET28 plasmids to prepare for sequencing.

The mini-prep was performed according to protocol with minor alterations: all samples were spun down into two columns instead of four, and the samples were eluted with only 12uL EB to increase the concentration of the final yield. The final concentration of PsiD was 119.3ng/uL. The final concentration of PsiK was 71.7ng/uL.

Merrie performed a PCR to add Bam1 and XBa1 restriction sites to PsiH. However, unfortunately this PCR was unsuccessful.

14/8/19 - Wet lab

Isobel performed a miniprep of pCW-no-p450 and then digested with BamH1 HF and Xba1. In the plasmid with the p450 still in, there's two restriction sites and the fragments would be approx 7900 and 800bp. In the plasmid with no p450, there's only 1 restriction site for BamH1 and fragment would be 7000bp.

The restricted pCW-no-p450 on the gel, there was only single bands in the BamH1 digest lane, so could presume that p450 had been removed.

Nathan grew more E. coli TOP10 containing PET28 PsiD/K in 50mL LB/Km, 50ug/mL, 37C overnight.

Fahad made more protein extraction buffer C made according to Coleman lab protocol. pH buffered to 8.5.

Benj prepared a 5ml culture of Vivid wild-type in Km-LB and ch1-5 to miniprep for sequencing.

Merrie re-tried the PCR to add Bam1 and XBa1 restriction sites to PsiH. The PCR product was run on a gel. There were nice bands between 2kB and 3kB as expected. The bands were cut out, weighed, and moved to the cool room for purification later.

14/8/19 - Team meeting

Outreach focused meeting: discussed the upcoming innovation games outreach event. Made a shopping list of supplies, plan for activities and posters, and decided who would be coming on the day.

15/8/19 - Wet lab

Nathan pelleted and froze the PsiD/K PET28 cells were cells at -30C with supernatant removed.

Benj performed a miniprep on his WT, CH1, and CH3 cells to prepare for sequencing. He tested the concentration of DNA using the Nanodrop. Final concentrations for WT, CH1, and CH3 (tubes 1 and 2) were 21ng/uL, 357ng/uL, 64.2ng/uL and 51.4ng/uL respectively.

Benj also performed some further purifications on Vivid CH1, and streak plated more colonies of CH4.

Merrie performed a gel DNA extraction on the PsiH from 14/8. The final concentration according to the nanodrop was 8.3ng/uL - not ideal!

15/8/19 - Human Practices

Our human ethics approval came through!

16/8/19 - Wet lab

Nathan performed a PsiD/K-PET28 plasmid purification using 800uL P1 added to defrosted cell pellets. This was split between 2x 4 epindorff tubes (200uL in each) before the 5mL Coleman purification protocol was followed.

Benj performed a 5mL miniprep on two CH4 colonies, CH2, and CH3.

16/8/19 - Human Practices

Emma completed her literature review “The What and How of Psilocybin”. Read it on our Human Practices page!

18/8/19 - Innovation games outreach event

Went to Sydney Olympic Park for the outreach event. Isobel, Merrie, Nathan, Benj, and Emma were there on the day. Fahad and Nathan made posters. Nathan and Benj ran a fluorescent protein activity where children could look at E. coli art. Merrie ran a strawberry DNA extraction demonstration. Emma ran a DNA modelling activity using paper and lollies. Isobel ran a creative activity where children could either draw a scientist or draw a hybrid animals using randomised ‘genes’. See our full Innovation Games write upfor more details!

20/8/19 - Human Practices

First round of emails to experts inviting them to be interviewed was sent out by Nick Coleman to the experts on Emma’s list.

20/8/19 - Wet lab

Merrie and Isobel attempted to perform a gel purification on the PsiH PCR product to prepare for PUS381 ligation. The first attempt had low yield (8.3ng/uL), the second attempt had a much better yield (24ng/uL).

21/8/19 - Wet lab

Merrie performed the PsiH/PUS381 ligation. Transformed TOP10 E. coli with the ligation product (as well as a positive control of PUS381 and a negative control of no DNA) using the Heat Shock protocol.

21/8/19 - Team Meeting

Discussed current Vivid optimisation work versus error-prone PCR work. Discussed Psi progress. Told Emma to stop being addicted to making her Human Practices lit reviews. Celebrated that we finally have Human Ethics approval and can contact our HP experts and organise interviews! Discussed USYD Open day outreach event, who would be there, and what we will do. Booked our flights to Boston!

21/8/19 - Human Practices

First two interview subjects responded positively to being interviewed. Emma sent emails to arrange one phone interview and one email interview.

22/8/19 - Wet lab

Merrie did patch plating and colony junction PCR of 24 colonies. Isobel ran the PCR products on 2x 1% TAE (50mL) with 7.5uL GelGreen. 100V, 40min. Loaded 5uL PCR product and ladder. Only one positive colony was found. Isobel streaked out a plate from that colony and inoculated a 50mL broth for use in a miniprep. The PsiH+PUS381 plasmid was named PUS382.

23/8/19 - Human Practices

Emma completed her Safety of Psilocybin literature review. Read it on our Human Practices page!

23/8/19 - Wet lab

Nathan received sequencing results back for PsiD/K/M. Decided PsiD needs resequencing close to the centre of the gene as there were many apparent deletions and low confidence regions. The PsiM construct was sequenced well with no deletions or substitutions. The PsiM-Pet28 construct can now be renamed PUS383.

PsiK had a few areas of low confidence sequence that need to be resequenced.

24/8/19 - Wet lab

Benj prepared patch plates of the different Vivid harmonisations.

26/8/19 - Wet lab

Nathan attempted to elute protein extracts of PsiD/K/M from TOP10 cells according to the protein extraction protocol. He ran these extracts on a gel and found no protein bands on his SDS-PAGE gel.

Benj streaked out more vivid colonies from patch plates made on 24/8 onto individual streak plates for the different Vivid harmonisations.

Nick amplified the inserts from his more-fluorescent error-prone-PCR colonies with primers NVC15b /16b, the PCR products were then purified with column, and sent for Sanger sequencing with primer 15b.

27/8/19 - Wet lab

Merrie performed a restriction digest of PUS381 using HindIII, ScaI, EcoRV, and Xba. Ran restriction products on 0.8% agarose TAE 100V 45 mins with 5uL gel green. Fahad performed a restriction digest on PUS382 using HindIII, ScaI, and Xba. Ran on a 0.8% 1xTAE agarose gel 50mL at 100V 60 min.

28/8/19 - Wet lab

Isobel redid the restriction digests of PUS381 that Merrie performed the day before as the resulting bands were too diffuse. Found that the bands were the correct size.

Nathan redid the PsiD/K/M protein extraction according to the protein purification protocol from the TOP10 cells containing the respective plasmids. Again, he got no product on his gel. He discovered that TOP10 cells were not appropriate for making the His-tag purification proteins. He checked the original workflow and found that this work was meant to be performed in BL21 cells.

Benj restreaked individual colonies from plates prepared on 26/8 to further purify.

Merrie set up an indole assay using PCW as a positive control and PUS382 as a test. Loops full of each plate’s colonies were transferred to 10mL LB-Carb and incubated, shaking, till the OD increased by 0.5. IPTG and Alanine was added to both cultures and left shaking overnight.

28/8/19 - Team meeting

Discussed progress of the codon harmonisation program. Benj was directed to prepare for the Vivid fluorescence assays, Nathan discussed his difficulties with protein purification, Merrie discussed her indole assay progress. Proposed that the next meeting needs to be a wiki working bee to get more of our writing on track.

29/8/19 - Wet lab

Nathan transformed TOP10 cells with PUS381 using the heat shock protocol. Incubated at 37C overnight on LB-Amp plates with a negative control.

Benj prepared 5mL individual cultures of each of the Vivid harmonisations to prepare for mini-prep. The vivid harmonisations were also plated onto charcoal plates to evaluate fluorescence.

The results of the indole assay from 28/8 were observed. A strong rust/purple colour change was observed in both. Decided to repeat the experiment with negative controls to be sure of results.

30/8/19-Wet lab

Isobel inoculated 2x 50mL LB-Carb broths with large loopfuls of PUS381 transformed cells from the supernatant plate Nathan prepared the day before.

Benj mini-prepped the 5mL cultures he made the day before.

Emma prepared some negative controls for the indole assay conducted on 28/8/19. PUS381 (no p450 or PsiH) induced/uninduced and PUS382 uninduced. Loops full of each plate’s colonies were transferred to 10mL LB-Carb and incubated, shaking, till the OD increased by 0.5. IPTG was added to one of the PUS381 cultures, alanine was added to all, then all were left shaking overnight.

31/8/19 - Wet lab

Benj noted the results of the negative controls indole assay - rust/purple colour change was seen in all cultures. This was unexpected and it was decided that the experiment needed to be repeated again. P.S. Little did they know, this experiment would continue to trip them up for many weeks to come!

1/9/19 - Wet lab

Benj performed a plasmid digest on several of his Vivid constructs. Some of the cells containing the tested constructs were incubated overnight in 5mL cultures to get enough DNA to confirm the band size, while other tested cells were streaked out on solid media for further tests.

Nick analysed the error-prone PCR sequences from 26/8 by aligning them to original VVD-CH4 sequence in Snapgene. 10/12 sequences gave good reads over the whole plasmid insert. many point mutations were seen in these clones, but all 10 mutants that had complete sequences shared a mutation at methionine-130, either M-->I or M-->V or M-->L. A new Gblock of VVD-CH4 was designed and ordered, which contained only the mutation M130I, to test if this was solely responsible for the increased fluorescence. This part would henceforth be referred to as VVD-Met-130, after the position in the DNA sequence that was mutated to generate the extra-fluorescent phenotype.

2/9/19 - Wet lab

Isobel miniprepped PUS381 then performed restriction digests on PCW, PUS382, and PUS381 to ensure that the plasmids used in the indole assay were correct. All plasmids’ digests were looking distinct from one another.

3/9/19 - Human Practices

Received interview response from Dr Francisco Moreno. Read our ‘Psilocybin Talks’ here!

3/9/19 - Wet lab

Merrie prepared PsiD and K for sequencing. She also prepared the PUS382 plasmid for sequencing.

Nathan performed PCR on the PsiD plasmid to add the BSA1 restriction site for our Golden Gate Cloning procedure. When the products were ran on gel there was some background banding, so gel purification was required.

Nathan also prepared overnight cultures of PsiD/K/M in both non-inducing and auto-inducing media. 50mL media in sterile 250mL bottles with two loopfuls of transformed BL21 cells.

Merrie and Emma transformed TOP10 cells with PCW, PUS382, and PUS381. The freshly transformed cells were transferred straight into the 10mL LB-Carb cultures. Cultures failed to grow.

4/9/19 - Wet lab

Benj set up a PCR of his tested Vivid recombinant constructs.

Nathan began protein extraction on the cells that were grown overnight. Extraction was performed using the Protein Extraction protocol. Protein concentration was estimated using the Nanodrop spectrophotometer. ~40ug each was run on an SDS page gel. Finished gel was stored in the cool room.

Nathan got sequencing results back for PsiD which revealed three deletions that would render the protein non-functional. He went into the cool room and found older plates of PsiD cultures from 17/7 and sent the other PsiD+ colonies off for sequencing after performing a junction PCR to amplify the region.

Merrie and Emma re-attempted the direct transformation to indole assay. Once again, the cultures failed to reach an appropriate OD. This was due to insufficient concentrations of cells - turns out there’s a reason why you plate your transformed cells first!

4/9/19 - Team meeting

Wiki working bee! Discussed the wiki progress at length and assigned wiki writing tasks. Had a lot of fun!

5/9/19-Wet lab

Isobel performed spanning and junction PCRs of PCW, PUS382, and PUS381 to confirm the results of her restriction digest from 2/9.

Benj performed more PCR on his Vivid constructs to amplify the region for sequencing.

Merrie transformed more TOP10 cells with PCW, PUS381, and PUS382. Once complete, they were plated on LB-Amp plates and incubated overnight.

8/9/19 - Wet lab

Isobel freshly transformed some TOP10 cells with PCW, PUS382, and PUS381. Benj streaked out some PUS381 to prepare for a colony PCR. Spanning PCR gave normal results, PsiH specific PCR gave weird results - possible contamination of our PsiH primer.

9/9/19 - Wet lab

Benj performed a colony PCR on the pure Vivid isolates using 2x Mango mix. Used three cultures per harmonisation type, plus one PK18 control and one no template control. He also restreaked several of the Vivid isolates onto Km only plates as the Xgal appeared to be interfering with the fluorescence of colonies. He also prepared some 5mL cultures of several Vivid isolates to incubate overnight.

Nathan performed a colony PCR on the PsiD colonies picked from the 17/7 plate.

Isobel performed a colony PCR on six different PUS381 colonies from the plates Benj prepared on 8/9.

10/9/19 - Wet lab

500mL cultures of PsiM and PsiK in BL21 cells were incubated, shaking, at 37C until they reached an OD~0.5. They were then induced with IPTG and moved to the room temperature shaker overnight.

Benj miniprepped the 5mL Vivid cultures he prepared the day before. He then sent the DNA off for sequencing.

Nathan prepared the PsiD colony PCR products for sequencing.

11/9/19 - Wet lab

Merrie streaked out colony 5 from Isobel’s colony PCR as it was the only colony to not give a band with the PUS382 primer.

Fahad pelleted the induced cultures of PsiM and PsiK from 10/9. The pellets were then stored in the cool room overnight. Fahad also made up some LB dry mix for the lab.

Benj miniprepped a 5mL wild type Vivid culture.

11/9/19 - Human Practices

Emma and Nathan conducted an interview with Dr Prashanth Puspanathan. Read our ‘Psilocybin Talks’ here!

11/9/19 - Team meeting

A bit of a difficulties troubleshooting meeting! Nathan revealed that it looks like PsiD has three deletions resulting in a nonsense mutation, made plans to do more protein ID work using larger cultures he made the day before. Isobel discussed that PUS381 has potential contamination so we need to take the colony PCR positive colony and use that to rebuild our plasmid stocks. Benj realised that the Vivid proteins on Xgal are still able to cleave the Xgal, making a product that will interfere with the fluorescence assay, will begin to replate out onto normal LB-Km plates.

However, great progress has been made on the website by Benj. Our drop down menus are coming along nicely and he aims to finish the HP page by the end of the week.

Discussed the HP interview progress - an interview with an MP is planned for later in the month, we need to interview some scalability and legal people.

Discussed who will be talking at the upcoming outreach presentations - Nathan and Emma at JAMS, Fahad and Isobel at the UNSW Symposium.

12/9/19 - Wet lab

Isobel decided to redo the restriction digests of the PUS381 stocks to see if any were better than those currently used to make colonies. No good results were found.

Nathan performed protein extraction on the pellets Fahad prepared on 11/9. The proteins were then run on an SDS-PAGE gel. However, it appeared that cell extract was still present in the elution columns which means that the samples require further purification.

13/9/19 - Wet lab

Nathan prepared two 50mL cultures each of PsiK and PsiM in BL21. Again, the cultures were shaken at 37C until they reached an OD of ~0.5. The cultures were then induced with IPTG and returned to the 37C shaker overnight.

Using the sequencing results generated from 10/9 colony PCR products, Nathan selected two colonies that did not have any deletions inside the PsiD gene to grow up in 50mL cultures for plasmid prep, and those plasmids used for BL21 transformation.

14/9/19-Wet lab

Nathan attempted an Ni+ column extraction of half of the cultures prepared the day before. Several different fractions were collected for running on an SDS-PAGE gel. The other two cultures were pelleted and left in the cool room.

Benj prepared another indole assay, incubating all cultures until they reached OD 0.5, adding Ala to all cultures, and inducing one culture per plasmid type (PCW, PUS381, PUS382). They were incubated at 37C overnight.

Benj also prepared one 50mL culture of PUS381 from 11/9 to use for DNA extraction to be grown overnight.

15/9/19 - Wet lab

Isobel miniprepped the PUS381 colony incubated the day before and performed a restriction digest on that DNA. Still no good looking results.

Isobel then reviewed the results of the indole assay and found that all cultures had turned red/brown. This is when we started to suspect that it was the Ala that was causing the colour change, not the IPTG.

Isobel also miniprepped the positive PsiD colonies from Nathan’s colony PCR on 9/9.

16/9/19 - Wet lab

Isobel picked a different freshly transformed colony of PUS381 to grow up, miniprep, and restriction digest. She also ran PCW as a control as she was beginning to suspect the enzymes had been contaminated. The results continued to look suspect, so a restriction digest with different stocks of the same enzymes was planned for 18/9.

17/9/19 - Wet lab

Benj perfomed PCR on two CH1 Vivid variants and one wild type variant. All Vivid variants remaining on Km-Xgal plates were restreaked onto Km only plates.

Merrie performed a heat shock transformation of PK18 into Top 10 cells and plated them onto LB-Kan plates. She also prepared 5mL cultures of each of the Vivid variants and one culture of Top 10 with PK18. These were shaken at 37C until they reached an OD of 0.5. Each culture was then diluted to an OD of 0.02 and transferred to a 96 well plate to prepare for a fluorescence assay.

Merrie prepared the fluorescence and particle standard curves for the assay using an adapted version of the iGEM fluorescence protocol using the TECAN plate reader.

PsiD and PUS382 PCR products were sent off for sequencing.

18/9/19 - Wet lab

Fahad performed a comparative restriction digest using the stocks Isobel had used for her previous restriction digests on PUS381 and some fresh enzyme stocks. The results clearly showed that those Bam and Xba stocks had been contaminated with HindIII. Those stocks were disposed of and future experiments would be carried out using fresh, uncontaminated stocks.

Nathan transformed BL21 cells with Psi D, K, and M.

19/9/19 - Wet lab

Nathan checked the DNA concentrations of the plasmid stocks and reattempted the transformations done the day before. However, the transformations failed due to bad LB from an autoclaving issue.

Merrie began to prepare Golden Gate Constructs of PsiD/K/M in PUS250.

19/9/19 - Team meeting

Discussed the progress with the fluorescence assay, Golden gate cloning, plans for a collaboration with Macquarie, the conference banner, and Integrated Human Practices.

20/9/19 - Wet lab

Merrie digested, ligated, and transformed Nick’s Vivid mutants into pk18, and then into TOP10 cells to prepare for a fluorescence assay.

Nathan retransformed TOP10 cells with Psi D, K, and M. Also performed a PCR across the insert region for each of the Pet28/Psi Constructs.

Merrie also transformed TOP10 cells with the golden gate products prepared the day before.

Nathan and Isobel also began some protein work to investigate the solubility of PsiM. Initial SDS PAGE gels showed no differences in banding between the induced and uninduced samples for both the insoluble and soluble lanes.

21/9/19 - Wet lab

Benj patch plated 20 clearly fluorescent Vivid mutants from the transformation performed 20/9/19.

Nathan discovered that all his transformants from yesterday grew.

23/9/19 - Wet lab

Isobel performed colony PCR on 30 white golden gate colonies. One purple colony was picked also as a negative control.

Nathan ran some PsiK and PsiM PCR products on a gel to observe PCR results.

Isobel performed gel purification on PsiM and PsiK DNA from Nathan’s gel.

Isobel also then transformed PsiM, K, and D into new BL21 cells containing pGro7 (a plasmid coding for a protein folding chaperone).

Isobel made more golden gate transformations of TOP10 cells as no positive colony PCR results were found.

Isobel performed gel purification on PsiM and PsiK DNA to prepare for sequencing.

Merrie grew up cultures of the Vivid clones in 5mL LB broth to prepare for the fluorescence assay. After all colonies reached an OD600 of ~1, she diluted the cells to an OD of 0.02, as per the iGEM measurement protocol. Samples were loaded into a 96-well plate with eight replicates of each culture, including one Pet28-cell control, and an LB only control.

23/9/19 - Human Practices

Emma and Isobel conducted an interview with Alan Davis at John Hopkins. Read our ‘Psilocybin Talks’ here!

24/9/19 - Wet lab

Merrie cloned PsiD/K/M into PUS250 using golden gate cloning again.

24/9/19 - Outreach

Presented at JAMS Sydney - an amazing opportunity to showcase our project to other microbiologists and microbiology fans! Read more about the evening here!

25/9/19 - Wet lab

Isobel performed colony PCR on 30 golden gate colonies from 23/9. No positive colonies were found.

25/9/19 - Human Practices

Emma and Isobel conducted an interview with Margaret Ross at St Vincents. Read our ‘Psilocybin Talks’ here!

25/9/19 - Team meeting
26/9/19 - Wet lab

Merrie performed another colony PCR on the golden gate clones using 21 colonies. Emma patch plated the colonies ready for isolation.

Merrie also transformed TOP10 cells with the golden gate product from 24/9.

Nathan grew up cultures of PsiD/K/M to try to purify out the enzymes. Cultures were induced ~OD600 0.5. Fahad pelletted the cells and saved the pellets for protein purification.

Fahad also transformed DH5-alpha cells with pGro. Also tried to transform some DH5-alpha cells with pGro and the following: PCW, PUS381, PUS382.

Benj set up 5mL cultures of Vivid WT, CH2, CH3, CH4, and three of Nick’s error prone PCR mutants to prepare for a fluorescence assay excitation and emission spectra. Assay was performed as per the iGEM measurement protocol.

26/9/19 - Human Practices

Conducted an interview with Sam Bannister from the Lambert Initiative. Read our ‘Psilocybin Talks’ here!

26/9/19 - Outreach and Collaboration

Presented at the UNSW symposium. Helping UNSW with their outreach by educating UNSW students to both the wonder of Synthetic Biology and the sustainability considerations was an awesome opportunity.

27/9/19 - Wet lab

Fahad performed bead-beater protein purification on the PsiD/K/M samples and prepared them for SDS-PAGE.

Benj miniprepped the Vivid WT, CH2, CH3, CH4, and error prone PCR mutants that were used in the fluorescence assay.

28/9/19 - Wet lab

Nathan ran Psi D, K, and M protein extracts on an SDS-PAGE gel after separating based on solubility. Protein was observed, but only in the insoluble fractions.

Isobel checked the transformations of DH5-alpha cells with pGro. The pGro only transformation and the pGro/PCW transformation worked but the others did not. She decided that the pGro-transformed cells needed to be made competent again. She began step one of that process.

She inoculated 3mL of the 5mL overnight cultures of the DH5-alpha-pGRO cells and top10 cells into 100mL LB and performed the chemical competency protocol on the cells. 100uL aliquots were plated onto plates of the appropriate agar and incubated overnight.

29/9/19 - Wet lab

Isobel performed the transformed various plasmids for the Macquarie collab into DH5-alpha cells.

She also tested the competent cells made on 28/9 by transforming them with PCW. These plates were then incubated overnight.

30/9/19 - Wet lab

Fahad prepared cultures of PsiD/K/M for induction. Cultures were induced when they reached an OD600 of ~0.5.

Merrie miniprepped cultures of the two positive golden gate colonies. The DNA was stored in the cold room for PCR the next day.

Merrie also prepared overnight liquid cultures of all the Macquarie collaboration constructs.

1/9/19 - Wet lab

Fahad decanted the overnight cultures of PSID/K/M (+/-) into 15ml falcon tubes and centrifuged 3900 for 15 min to pellet. The cells were resuspended in 5ml of wash buffer A by vortexing and were again pelleted by centrifugation 3900 for 15 min.

Nathan set up PsiD/K/M cultures at 37C shaking until they reached an OD600 of ~0.5. The cultures were then induced and incubated at 37C, 30C, and room temp after induction. After five hours all cells were pelleted, and the pellets were stored in the cold room.

Merrie performed spanning PCR on the golden gate constructs from 30/9. Both colonies were positive.

Merrie moved the Macquarie collaboration cultures to the cold room, prepared 0.1mM IPTG for induction, and prepared the LB-antibiotic mastermixes. The samples were put into the TECAN for the fluorescence assay, but it was aborted after ten hours as the cultures maxed out the machine.

2/10/19 - Wet lab

Merrie did PCR on codon-optimised PsiH to add the restriction sites to move it into PUS381.

Nathan resuspended the cell pellets from 1/10 in Buffer A, then centrifuged, then resuspended in TE. Soluble extracts were obtained by beadbeating and nanodropped. Cell extracts were then stored in the cold room overnight for SDS-PAGE.

Merrie also ran a gel on the PCR products from 1/9 to perform gel purification.

2/10/19 - Team meeting

We discussed the attributions page, collaborations page, and the Human Practices progress. We feel as though we have these aspects of the progress very much under control. We discussed where to characterise our WT-VVD and VVD-Met130 mutant parts in terms of the medal criteria. We also decided on a lab work cut off, October 16 for most wet lab work, October 23rd for characterisation wet lab work for our presentation and poster.

3/10/19 - Wet lab

Isobel digested codon-optimised PsiH (CO-PsiH) and PUS381 with Bam and Xba. Isobel then ligated CO-PsiH and PUS381. This mixture was then transformed into DH5-alpha/pGRO cells.

Nathan heated the SDS page gel of protein extracts from 2/10/19 3 minutes at 95°C before loading onto SDS page gel 10μg of protein per lane plus SDS buffer and water to make up 20μL.

Merrie performed gel purification on the gel ran yesterday. The concentration was not high enough after purification so another PCR and gel purification was performed. This second gel purification resulted in yields suitable for sequencing. The samples were set up and sent off for sequencing.

4/10/19 - Wet lab

Isobel performed colony PCR on 28 CO-PsiH-DH5alpha/pGRO colonies. 5 positive colonies were found. Decided to call these codon-optimised PsiH/PUS381 constructs PUS383 from now on.

Nathan ran 15°C soluble cell extracts on SDS page again heated for 3 minutes at 95°C before loading onto SDS page gel 10μg of protein per lane plus SDS buffer and water to make up 20μL.

Emma performed heat shock transformation of the golden gate constructs (14 and 16) in PUS250 - henceforth referred to as PUS387-14 and PUS387-16 - into DH5-alpha-pGRO as per the lab protocol. PUS250 was also transformed as a positive control. The transformations were plated on LB-Km-Cm plates and incubated overnight.

Merrie started new overnight cultures of the Macquarie constructs for a new fluorescence assay.

4/10/19 - Human Practices

Fahad met with Adamn Bandt, the Federal Greens spokesman for science, in Melbourne to discuss the responsible and democratic use of GM technology, especially in reference to our project. This chat was part of our Psilocybin talks series on our Human Practices page!

5/10/19-Wet lab

Isobel began making the indole assay pre-induction media. Inoculated 3mL bottles of the media with each of PUS381, PCW, PUS382, and three different PUS383 colonies that had positive PCR results the day before.

Isobel also performed a spanning PCR on the PUS383 colonies to determine which colony will be used for the indole assay. All three colonies had an insert of the correct size.

Merrie spun down the 30°C and 37°C induced and unindexed samples from PSIK and PSIM and replaced the TE with SDS loading buffer. They were then transferred to bead beating tubes and beat for 30s, iced for 2 min, and spun at max speed for two minutes. They were then transferred to new tubes on ice.

Nathan made 3x 500ml of LB Km/Cm with added arabinose to 2mg/ml. 50ml cultures of PSID, PSIK, and PSIM were used to seed the 500ml conical flasks. OD was measured periodically until it reached approx OD600 0.5. They were then induced with IPTG and returned to the 15C shaker.

Merrie made up 10mL liquid cultures of the PUS387 transformations from 4/10/19. Aliquots were taken to use to assess psilocybin production.

Merrie set up another fluorescence assay for Macquarie according to their protocol but with some minor changes. The cultures were pipetted uninduced, then the IPTG was added. She also changed the excitation to 480, the emission to 525, and the gain to 75, to better prevent maxing out the TECAN. The machine was set to perform 140 cycles, 30 min apart, shaking at 150rpm, 37 degrees celsius.

Merrie also made glycerol stocks of the error-prone PCR Vivid mutants to put in the -80C freezer.

6/10/19 - Wet lab

Isobel started the indole assay. To TB+additives bases, 0.5mL of each pre-induction culture was added: PCW, PUS381, PUS382, and PUS383.

Isobel also prepared a 50mL LB culture of PUS383 to prepare for a mini-prep.

Fahad prepared a 400mg/mL arabinose stock to induce the indole assay cultures. He then induced the cultures in the evening with arabinose, IPTG, and alanine.

Isobel streaked PSIDKM in Dh5a pGro7 onto kan/cm plate from bottle of culture in cold room put in 37 degree C incubator.

Fahad removed PSIK/D/M cultures from shaking incubator (15°C). The cultures were then decanted into 11x falcon tubes 50ml each and centrifuged 3900 for 15 min to pellet. The supernatant was poured off and cells resuspended in 5ml wash buffer A then decanted into single falcon tubes for each plasmid and centrifuged at 3900 for 15 minutes. Again, the supernatant was poured off and cells resuspended in 5ml 1x TE supplemented with 3ul of 10x protease inhibitor then frozen at -30C.

8/10/19 - Wet lab

Merrie miniprepped the PUS383 50mL LB culture.

Merrie also discovered that the PCR and sequencing of PUS387-14 and PUS387-16 failed due to the use of an incorrect primer. She decided to redo the PCR using the correct primers and a PUS250 and no template control.

Isobel checked the results of the indole assay. PCW changed colour very drastically, but no colour change was observed in any of the other cultures. One set of cultures was set aside to be used for protein purification. Protein purification was done according to the protocol for insoluble proteins, and the resulting protein extract was run on an SDS-PAGE gel. Extra protein extract was frozen in TE before SDS-loading buffer was added. The gel was not the best.

Merrie performed a heat shock transformation according to Coleman lab protocol colony 14 PUS387 into DH5a pGro7, she also prepared a NTC and PET28 positive control.

She also performed a PCR on PUS387-14 and 16, with PUS250 and a no template control.

9/10/19 - Wet lab

Isobel transformed DH5-alpha cells with PUS381/2/3 to prepare for a hydroxytryptamine assay. She also co-transformed DH5-alpha with PUS387(14/16) and PUS382/3.

She then also prepared PUS383 for sequencing.

Merrie took the golden gate transformation plates from 8/10 out of the incubator and used them to inoculate 2x 10mL cultures of TB for a single plasmid psilocybin assay. She realised that she had transformed the wrong control (pet28 instead of PUS250) and would have to repeat with PUS250 later. They were incubated for several hours, but overshot their required OD. They were diluted and returned to the incubator until they reached the required OD of 0.5. They were then induced with 10ul of 100mM cumate and placed in 15 degree shaker in 50ml falcons. After 7 hours incubating they were spun down and resuspended in phosphate-glucose buffer and 4-hydroxytryptamine was added. One tube of each plasmid was placed in the 37C shaker, and one in the 15C shaker.

Merrie also ran the results of yesterday’s PCR on a gel.

Nathan ran Fahad’s protein samples from 6/10 on an SDS page gel to visualise protein expression and solubility at 15C. Gels were stained with Coomassie blue.

He also performed dialysis of the his-tagged purified proteins using the cell pellets Fahad prepared according to protocol.

Isobel put Nathan’s gels in the high and low destain.

9/10/19 - Team meeting

Discussed the results of the indole assay, the upcoming scale-up experiment, the upcoming phosphate assays, and the need to send all our protein gels for LCMS. Fahad revealed that the Codonator would soon be up on the wiki - very exciting! Emma begged for help transcribing the recent interviews. Isobel offered to transcribe Margaret Ross's interview, Fahad his interview with Adam Bandt, and Nathan offered to transcribe the Dale interview. Merrie said that she has fully finished the work required for the Macquarie collab, she has sent them our data and we have received their gel. Benj taught us how to upload images for the wiki. We discussed the upcoming deadlines, Fahad offered to take the lead on the making and populating of the parts pages.

10/10/19 - Wet lab

Fahad retrieved the extra PUS381/2/3 protein extract from the freezer and performed another SDS page on the samples to get a better image.

Emma inoculated the PUS381/2/3 and PUS387-PUS382/3 cultures transformed yesterday into antibiotic-glucose-LB media. They were put into the 37C shaker overnight. Emma also prepared the TB for the next step of the hydroxytryptamine and psilocybin assays and sent it for autoclaving.

Merrie decided to repeat yesterday’s single-plasmid psilocybin assay with the correct controls and no shaking. She also spun down the tubes from the day before and found that PUS387-14 and 16 had black pigment to their pellets. The cells were stored in glycerol in the -20C freezer.

She also performed a miniprep of PUS387-14 and 16 to prepare for sequencing. The samples were prepared with primers and sent off for sequencing.

Nathan realised he left the PsiM fractionated samples out overnight (big sad). He ran all three on gels regardless. Nathan decided with the help of our supervisor, Nick, to abandon the in-vitro enzyme assay of PsiM due to it being likely degraded and didn’t purify well. He dialysed PsiD and PsiK again at 4C overnight, stirring.

Nathan and Benj performed a scale-up exercise in collaboration with the chemical engineering department. Nathan inoculated a 1.5L TB culture with DH5-alpha PUS250 cells. He took OD600 every 20mins.

Benj did the same but using the 1.5L incubator in the engineering department and measuring every 30mins.

11/10/19 - Wet lab

Merrie transferred the PUS381/2/3 and PUS387-382/3 cultures from the LB-glu media into the TB and added the additives from the indole assay. All cultures were incubated at room temp until induction.

Merrie induced the PUS381/2/3 cultures after six hours with IPTG and arabinose and left them shaking at room temperature, then induced the PUS387-PUS382/3 cultures with IPTG, arabinose, and cumate and moved them to the 15C shaker.

Fahad thawed more of the PsiD cultures frozen on 6/10. 10mL aliquots of these were decanted into 50mL falcon tubes, washed in PBS 5ml, and then after the supernatant was poured off. Cells were then resuspended in 5ml fresh phosphate buffer with 1mM tryptophan. Took 1ml supernatant and froze. Remainder spun down and returned to shaker.

Nathan performed some transformations of PUS250, PUS250/PUS381, PUS381, and PUS381/Pet28-PsiM, with a no template control into DH5-alpha-pGRO cells. They were plated and put in the incubator overnight.

In the evening (six hours after induction), Isobel pelleted the cells from the PUS381/2/3 assays and the PUS387-PUS382/3 assays and resuspended them in phosphate-glucose buffer. All cultures had their required intermediates added. Isobel also took time point zero samples. These were stored in the freezer to prepare for LCMS.

12/9/19 - Wet lab

Isobel performed a PCR to check 381/382/383 samples are correct due to suspicious OD600 results yesterday. The results showed that the cultures were in fact correct.

Merrie performed a restriction digest according to lab protocol of PCW, and PUS381/2/3 with Bam & XbaI to get a nice gel image for the wiki. However the gel showed incomplete digestion so would have to be repeated.

Nathan removed the dialysis tubes from the fridge and extracted 1mL of PsiD and PsiK into separate 1mL eppendorfs to prepare for the in vitro assay. In one tube there was 200uL enzyme alone, in another tube there was 200uL enzyme with the substrate. For PsiK, 5mM ATP MgCl2 was added to both tubes. Reactions were left to proceed for two hours before being moved to the freezer to wait for LCMS.

Emma span down the PUS381/2/3 and PUS387-PUS382/3 cells and took 1mL samples of the supernatant as our t=24 samples, these were stored in the freezer to send off for LCMS.

13/10/19 - Wet lab

Merrie performed another restriction digest on PCW and PUS381/2/3 according to protocol, but was still having troubles with incomplete digestion.

Merrie removed the PUS387 single plasmid psilocybin assays from the 15C shaker. The supernatant was removed and placed in the freezer until it could be sent off for LCMS.

13/10/19 - Outreach

Benj, Emma, Isobel, and Nathan set off for Brisbane to participate in the Synthetic Biology Australia (SBA) conference!

14/10/19 - Wet lab

Merrie prepared the protein samples for PsiD/K/M for LCMS. She also sent our enzyme assay supernatants off for LCMS.

Merrie also began work on the bSFP by reconstituting the G-block to clone it into pk18. The ligation mixture was transformed into TOP10 cells and plated onto LB-Km-Xgal plates.

14/10/19 - Outreach

Day one of the SBA conference! Nathan, Isobel, Benj, Emma, and Fahad attended.

15/10/19 - Wet lab

Merrie made 5ml cultures of bsfp, WT vivid, CH1, CH2, CH3, Met130-10 and put them in the 37 shaker overnight to prepare for the characterisation fluorescence assay. She was concerned by the small number of white colonies observed on the bSFP plates so she planned another transformation as a back up.

15/10/19 - Outreach

Day two of the SBA conference! Nathan, Isobel, Benj, Emma, and Fahad attended.

16/10/19 - Wet lab

Merrie performed another transformation of bSFP into TOP10 cells. She also set up the fluorescence assay with the overnight cultures from the day before as per the iGEM measurement protocol.

Merrie also performed a digest of PCW，PUS381/2/3 and ran the products on a gel to make a nice gel for the Wiki. However the gel was not so nice, so it needed to be performed again.

16/10/19 - Outreach

Day three of the SBA conference! Emma and Isobel presented our progress so far. The presentation went fantastically and they fielded questions well.

18/10/19 - Wet lab

Merrie received the results from the bSFP fluorescence assay. The levels were surprisingly low so she decided to redo the digestion and ligation of the G-block into pk18. The new ligation mix was then transformed into TOP10 cells and plated on LB-Km-Xgal plates.

19/10/19 - Wet lab

Isobel and Merrie performed colony PCR on bsfp using NVC15b and 16b plus patch plate and 5ml cultures of 16 white colonies.

They also set up 5ml cultures of CH1 and WT Vivid, PK18 and Met-130. Then they did another transformation of TOP10 cells with the ligation product from yesterday.

Nathan digested each pET28-psi construct using HindIII and EcoRI to make some nice gels for the wiki.

20/10/10 - Wet lab

Nathan performed a double restriction digestion of pUS250 and pUS387 clones (14 and 16) using NEB's HindIII-HF and EcoRI-HF enzymes.

Merrie attempted to make more cultures of bsfGFP but they were not growing. They were abandoned as our comparative protein for our VVD_Met130 experiment.

21/10/19 - Wet lab

Merrie Prepared cell lysates of MET130I and sfGFP for thermal stability assay and performed assay with StepOnePlus Real time PCR system. She also measured the excitation and emission spectrum of the MET130I fluorescent protein using the Tecan Spark plate reader.

21/10/19 - Team meeting

The team met to finalise details on the wiki and parts registry. Results from the melt curve assays were added to the wiki and registry.

And with that, all we needed to do was polish off our poster and presentation, and get ready to head off to the Jamboree! If you want to learn more about what we did in our final days of the project, check out our social media.