iGEM 2023 — Team warwick

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This page contains chronological notes of our labbook. Detailing our daily tasks and who participated, including pictures of our progress and containing all protocols used in our experiments. See our procedures [here](https://2023.igem.wiki/warwick/experiments) and our results [here](https://2023.igem.wiki/warwick/results).

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#### Introduction

When planning out and finalising our project idea before summer, everyone in the team had aimed to undergo multiple design cycles. We were aspiring to test many different approaches; including: pumps, feedback systems to shut off enzyme production, degraders to stop further production or even using enzymes to detoxify the products which could be converted back after extraction, just to name a few. By the end of the summer, we aspired to have an optimised and innovative solution for the production of perillyl alcohol in *E. coli* using information gathered from experiments and modelling.

However, with substantial delays in the delivery of our parts as well as some large changes to the experiment that had to be made part way through (which will be discussed later), we had a much tighter time frame to run it through numerous cycles than we had hoped. Despite this, taking a step back to optimise or redesign our experiments entirely was vital in reaching our desired end goal before the iGem deadlines.

![e-f1](https://static.igem.wiki/teams/4600/wiki/e-f1.jpg)

#### Changing pJBEI-6410+ Plasmid Assembly Method

When designing the feedback system of our project, pJBEI-6410 only contains a small ~50bp tet promoter sequence that will respond to the tet produced by pJBEI-6411 to downregulate the expression of downstream enzymes. Because it is such a short sequence, we intended to add the sequence onto the end of primers and clone it in by an overlap extension PCR instead of ordering a large gibson fragment that is mostly backbone.

We designed the complementary primers around specific restriction sites and, after they eventually arrived, we had early success in the production of A and B fragments, joining the A and B fragments together and cutting open the plasmid. Despite this, after weeks of repeats and slight alterations to the procedure, we still couldn’t produce cells containing the modified plasmid. Most of our samples didn’t grow cells on ampicillin plates, and the few colonies that did grow failed our colony PCR tests (an example gel is shown in Figure 2).

![e-f2](https://static.igem.wiki/teams/4600/wiki/e-f2.jpg)

#### Using HP20 Resin to Isolate Our Products From Liquid Culture

With perillyl alcohol and limonene not having a unique absorbance signal to identify them with, our chosen method of analysing their presence was using gas chromatography-mass spectrometry (GCMS). After growing a liquid culture and homogenising the cells, all of the leftover cellular components in the sample can create a lot of background noise on the GCMS trace and make our products harder to extract. Because of these reasons, and hearing the idea from a chemistry professor, we aimed to use HP20 resin during the extraction. It has been used to isolate terpenes of similar structure to PA and limonene in previous research so, with only one functional group atom difference, we believed that it could be equally very effective (Ignea et al., 2019).

![e-f3](https://static.igem.wiki/teams/4600/wiki/e-f3.jpg)

Regrettably, however, as shown in Figure 3, the addition of HP20 resin removes the PA peak at ~6 minutes entirely and isn’t especially effective at removing impurities either. Because of this, we obviously cannot use the resin during extraction. During our second run of GCMS (being carried out post wiki freeze deadline) we are however using an internal standard that, despite not removing any impurities, should make it possible to more accurately assess the amount of PA/limonene produced. Any updates to our progress using the internal standard will be shown on the [results page.](https://2023.igem.wiki/warwick/results)

#### Modelling

During our project the modelling team helped us to plan some of our experiments, including the IPTG induction assays that allowed us to express the plasmid at different levels to see the effect of our pathway and its products on growth. The act of balancing production of PA/limonene against the growth of our cell is something that we were considering a lot in the creation of our project. More info on the [modelling page.](https://2023.igem.wiki/warwick/model)

References

Figure 1 created in <https://www.biorender.com/>

Ignea, Codruta, et al. “Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate.” Nature Communications, vol. 10, no. 1, 2019, https://doi.org/10.1038/s41467-019-11290-x

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**Making chemically competent cells (for heat-shock transformation):**

* Requirements

+ Sterile LB
+ Overnight liquid culture containing desired cells
+ 10% glycerol
+ 25 ml, 10 ml, 5 ml pipettes and PIPETBOY
+ Ice and dry ice
+ 1ml cuvettes
+ Sterile Eppendorf tubes
+ Sterile conical flasks
+ Spectrophotometer
+ 4°C centrifuge
+ Gloves

* Make a 1/10 dilution of the overnight culture and measure the OD600 (100 µl liquid culture in 900 µl sterile LB in 1 ml cuvette)
* In two sterile conical flasks, combine LB and overnight culture to get a 50 ml solution with an OD600 of roughly 0.15 (using OD600 previously obtained)
* Shaking incubate the two flasks at 37°C and regularly check OD600 until it reaches 0.4 and then put on ice OD600 of 0.4, transfer the 50 ml cultures into two sterile 50 ml falcon tubes
* Centrifuge the two balanced tubes at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 50 ml of 10% glycerol
* Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 50 ml of 10% glycerol.
* Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 25 ml of 10% glycerol.
* Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 5 ml of 10% glycerol.
* Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 1 ml of 10% glycerol.
* Prepare 50 µl aliquots into sterile Eppendorf tubes on dry ice and transfer into a -80°C freezer

**Making electro-competent cells (for transformation via electroporation):**

* Requirements

+ Sterile LB
+ Overnight liquid culture containing desired cells
+ 10% glycerol
+ 25 ml, 10 ml, 5 ml pipettes and PIPETBOY
+ Ice and dry ice
+ 1ml cuvettes
+ Sterile Eppendorf tubes
+ Sterile conical flasks
+ Spectrophotometer
+ 4°C centrifuge
+ Gloves

* Make a 1/10 dilution of the overnight culture and measure the OD600 (100 µl liquid culture in 900 µl sterile LB in 1 ml cuvette)
* In two sterile conical flasks, combine LB and overnight culture to get a 50 ml solution with an OD600 of roughly 0.15 (using OD600 previously obtained)
* Shaking incubate the two flasks at 37°C and regularly check OD600 until it reaches 0.4 and then put on ice OD600 of 0.4, transfer the 50 ml cultures into two sterile 50 ml falcon tubes
* Centrifuge the two balanced tubes at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 50 ml of 10% glycerol
* Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 50 ml of 10% glycerol.
* Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 25 ml of 10% glycerol.
* Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 5 ml of 10% glycerol.
* Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 1 ml of 10% glycerol.
* Prepare 50 µl aliquots into sterile Eppendorf tubes on dry ice and transfer into a -80°C freezer

**Gel Electrophoresis:**

* Requirements

+ Agarose
+ 1% TAE/TBE
+ Gel tray and comb
+ DNA ladder (1kb plus)
+ 6X loading dye
+ GelRed® stain
+ Microwavable flask
+ Precise weight scales
+ Microwave
+ Electrophoresis tank and power pack
+ Gel imager
+ Gloves

* Mix 0.5 g of agarose with 50 ml of TAE in a microwavable flask
* Microwave for 1-2 minutes until the agarose powder is dissolved
* Let it cool before adding 3 µl of GelRed and pour into the gel tray containing the comb. Leave to set at room temp for 20-30 minutes.
* Add 6X purple loading dye to samples and 3 µl of DNA ladder to the first well (e.g. 2 µl 6X purple loading dye to a 10 µl sample)
* Run at 100V for 20 minutes or until the bands are as desired

**Restriction Digest**

* Requirements

+ CutSmart buffer
+ Relevant restriction enzyme
+ Relevant plasmid
+ Water
+ Gel electrophoresis equipment
+ Gel imager
+ Gloves

* Add 1 µl of CutSmart buffer, 0.5 µl total of restriction enzyme (or 0.25 µl of each enzyme), and 7.5 µl of water to a 1.5 ml tube
* Add 1 µl of plasmid and mix well throughout
* Incubate at 37°C for 1 hour in a static incubator
* Afterward, add 2 µl of 6X loading dye to each sample and mix well
* Load onto gel with DNA ladder and run gel electrophoresis

**Heatshock**

* Requirements:

+ 2/3 µl desired plasmid/ligation mix
+ 50 µL of chemically competent cells
+ Sterile SOC
+ 42°C water bath
+ Sterile Eppendorf tubes
+ Selective media plate (with relevant antibiotics)
+ Bunsen burner
+ Spreaders
+ Gloves

* Procedure:
* Take out competent cells from -80°C freezer and thaw on ice for 15 minutes
* After thawed, add 2 µl of plasmid (or 3 µl of ligation mix) into 50 µl of chemically competent cells and mix by flicking the tube
* Incubate on ice for 30 minutes
* Heat shock in 42°C water bath for 45 seconds
* Place the cells back on ice for 2 minutes
* Add 500 µl of SOC and shake incubate at 37°C for 45 minutes
* Plate 50 µl of mixture onto one plate and 300 µl onto another (don’t forget to use plates that have the desired antibiotic)
* Incubate plates overnight at 37°C

**Electroporation:**

* Requirements:

+ 1/2 µl desired plasmid/ligation mix
+ 50 µL of electro-competent cells
+ Sterile SOC
+ 2mm blue electroporation cuvettes
+ Electroporation machine
+ Sterile Eppendorf tubes
+ Selective media plate (with relevant antibiotics)
+ Bunsen burner
+ Spreaders
+ Gloves

* Procedure:
* Take out competent cells from -80°C freezer and thaw on ice for 15 minutes
* After thawed, add 2 µl of plasmid (or 3 µl of ligation mix) into 50 µl of electro-competent cells and mix by flicking the tube (keep on ice)
* Transfer all of the mix into a 2mm blue electroporation cuvette, on ice
* Dry the slides so no moisture is on the metal plates and ensure there are no bubbles present in the cuvette
* Shock at 25 µF and 2.5 volts, hold down buttons until it beeps
* Immediately add 500 µl of 37°C SOC and incubate at 37°C for 1 hour
* Plate 300 µl of mixture onto one plate and 50 µl onto another
* Incubate plates overnight at 37°C

**Gel Extraction**

* Requirements:

+ 96% ethanol
+ Gel containing desired bands
+ Gel imager
+ QIAquick®reg; Gel Extraction Kit
+ 50°C water bath
+ 13,000rpm tabletop centrifuge
+ Gloves

* Procedure:
* First ensure that buffer PE contains the added ethanol
* After looking at the gel bands, using a clean scalpel, cut out the bands as precisely as possible (keep checking on the machine)
* Weigh the gel slice in a colorless 1.5 ml Eppendorf tube and add 100 µl of QG buffer for every 100 mg of gel slice.
* Incubate at 50°C for 10 min and vortex occasionally (or until the gel slice has completely dissolved).
* Transfer the solution into a QIAquick spin column and centrifuge for 1 minute at 13,000 rpm, discard the flow-through
* Add 500 µl buffer QG, centrifuge for 1 minute, discard flow-through
* Add 750 µl buffer PE, centrifuge for 1 minute, discard flow-through.
* Centrifuge for a further minute to remove residual wash buffer
* Place the QIAquick column into a clean 1.5 ml microcentrifuge tube and elute by adding buffer EB (20-50 µl depending on desired concentration). To increase concentration, incubate it for 4 mins before spinning.

**Plasmid Purification:**

* Requirements:

+ RNase
+ 96% ethanol
+ Overnight culture of cells containing plasmid
+ QIAprep®reg; Spin Miniprep Kit
+ 13,000rpm tabletop centrifuge
+ Gloves

* Procedure:
* Start by adding RNase to buffer P1 and ethanol to buffer PE
* Add 1.5 ml of overnight liquid culture to an Eppendorf tube and centrifuge it at 13,000 rpm for 3 mins at room temperature (15-25°C).
* Remove the supernatant and leave just the pellet.
* Resuspend the pellet in 250 µl of buffer P1
* Add 250 µl buffer P2 and mix by inverting, the reaction will turn the solution blue. Don’t let the reaction occur for longer than 5 mins
* Add 350 µl N3 and mix by inverting, the solution will turn colorless
* Centrifuge for 10 mins at 13,000 rpm
* Apply 800 µl of supernatant to a QIAprep 2.0 spin column and centrifuge at 13,000 rpm for 60 seconds, discard the flow-through
* Wash the spin column with 750 µl of buffer PE and centrifuge for 60 seconds, discard flow-through. Centrifuge for another minute to remove the residual wash buffer.
* Place the QIAprep 2.0 spin column into a 1.5 ml Eppendorf tube and add buffer EB (20-50 µl), let stand for 1 minute then centrifuge.

**Overlap Extension PCR:**

* Materials:

+ All relevant 10 µM primers (1-4)
+ 5X Q5 reaction buffer
+ Q5 polymerase
+ 2 mM dNTPs
+ Purified template DNA
+ Nuclease-free water
+ Nanodrop device
+ Phosphatase
+ T4 ligase
+ T4 Ligase buffer
+ Gel electrophoresis materials
+ Gel extraction materials
+ Restriction enzymes
+ Thermocycler
+ PCR tubes
+ Selective media plate (with relevant antibiotics)
+ Heatshock/electroporation materials
+ Bunsen burner
+ Spreaders
+ Gloves

* Procedure:
* Using four pre-designed primers (primers 1-4), determine the annealing temperature and extension time for each.
* Prepare a reaction mixture on ice with 10 µl of 5X Q5 reaction buffer, 5 µl of 2 mM dNTPs, 2.5 µl of 10 µM forward primer, 2.5 µl of 10 µM reverse primer, 1 µl of purified template DNA, and 0.5 µl of Q5 polymerase. Make up to 50 µl with nuclease-free water (28.5 µl).
* Repeat the above step for primer pairs 1:3 (2.3 kb) and 2:4 (0.4 kb).
* Put the reaction mixtures in a thermocycler for the following conditions:

+ Initial denaturation: 98°C for 30 seconds
+ Denaturation: 98°C for 10 seconds
+ Annealing: 67°C for 20 seconds
+ Extension: 72°C for 85 seconds
+ Final extension: 72°C for 120 seconds
+ Hold: 4-10°C for 10 mins

* Run the PCR products on a gel and cut out the bands containing the amplified region.
* Use a QIAquick gel extraction kit to purify DNA, resulting in fragment A (from 1:3 primer amplification) and fragment B (from 2:4).
* Prepare another reaction mix on ice with 10 µl of 5X Q5 reaction buffer, 5 µl of 2 mM dNTPs, 2.5 µl of 10 µM forward primer 1, 2.5 µl of 10 µM reverse primer 4, 5 µl of fragment A, 5 µl of fragment B, and 0.5 µl of Q5 polymerase. Make up to 50 µl with nuclease-free water (23.5 µl).
* Put the reaction mixture in a thermocycler for the following conditions:

+ Initial denaturation: 98°C for 30 seconds
+ Denaturation: 98°C for 10 seconds
+ Annealing: 50-72°C for 20 seconds
+ Extension: 72°C for 25 sec/kb
+ Final extension: 72°C for 120 seconds
+ Hold: 4-10°C for 10 mins

* Run the PCR products on a gel and extract the 2.7 kb band with the joined AB fragment.
* Nanodrop the fragment to determine the DNA concentration (ng/µl).
* Using 1000 ng of AB fragment, add 1 µl of each restriction enzyme (EcoRI and SacI), 10 µl of CutSmart buffer, and make up to 100 µl with water.
* Meanwhile, make up another solution containing 5 µl CutSmart buffer, 2 µl of enzyme (or 1 µl of both), 1000 ng of original plasmid DNA, and make up to 50 µl with water.
* Incubate both solutions for 1 hour at 37°C, then add phosphatase and incubate for a further 5 minutes.
* Run both solutions on a gel and extract the digested AB fragment (2.7 kb) and the digested plasmid (12.5 kb) with sticky ends made by enzymes.
* Prepare a solution of 1 µl T4 ligase, 2 µl T4 ligase buffer, 12 µl of cut plasmid, and the calculated volume of fragment (in 1:3 ratio). Make up to 20 µl with water. Do a control without any fragments.
* Incubate the ligation mix at room temperature for 1 hour and then heatshock the formed plasmid into cells and plate onto an antibiotic plate.

**Colony PCR**

* Requirements:

+ 2X DreamTaq MasterMix
+ 10 µM forward and reverse primer
+ Thermocycler
+ PCR tubes
+ Gel electrophoresis materials
+ Gloves

* Procedure:
* Set up a MasterMix containing 12.5 µl 2X DreamTaq, 1 µl of forward primer, 1 µl of reverse primer, 0.5 µl of plasmid (or ½ of a bacterial colony), and make up to 25 µl with 10.5 µl of water.
* Gently mix the samples with a pipette.
* Put the samples in a thermocycler for the following conditions:

+ Initial denaturation: 95°C for 10 minutes
+ Denaturation: 95°C for 30 seconds
+ Annealing: Tm-5 (~50-55°C) for 30 seconds
+ Extension: 72°C for 60sec/kb
+ Final extension: 72°C for 5 minutes
+ Hold: 4-10°C for 10 mins
+ 35 cycles

* After the PCR, run it on a gel, and if the insert is present, you should see an additional band.
* At this point, make a liquid culture of the ½ colony or plate out the liquid culture.

**Gisbon Assembly**

* Requirements:

+ CutSmart® Buffer
+ Relevant enzyme
+ Relevant plasmid
+ Phosphatase
+ Thermocycler (heat denaturation and 50°C incubation)
+ QIAprep® Spin Miniprep Kit (if not heat denaturation eligible)
+ Gibson Assembly (GA) 2X MasterMix
+ Heatshock/Electroporation materials
+ Bunsen burner
+ Spreaders
+ Gloves

* Procedure:
* Begin the digestion with 2 µl CutSmart buffer, 2 µl of enzyme, 10 µl of plasmid, and 6 µl of water up to 20 µl. Incubate the tube for 2 hours at 37°C.
* After 2 hours, add 2 µl of phosphatase and incubate for a further hour.
* If eligible, heat denature the enzymes. If not, then add 22 µl of QG buffer and continue with column purification into 20 µl of elution buffer.
* Use nanodrop to measure the concentration of the purified plasmid.
* With 100 ng of plasmid, calculate the amount of plasmid in pmols and triple it (0.03 pmols) to find how much of the 10 µM fragments to use.
* Add 10 µl of GA 2x MasterMix to 100 ng of plasmid and all the fragments and make up to 20 µl with water.
* Incubate the mixture at 50°C for 1 hour in a thermocycler.
* Add 2 µl of the mixture to 50 µl of competent cells (thawed on ice).
* Transform the Gibson sealed fragment into cells using electroporation or heatshock and add 500 µl of SOC.
* Shaking incubate at 37°C 220 rpm for 1 hour and plate 300 µl of the mixture onto one plate and 50 µl onto another.

**Creating Growth Media for Producing Perillyl Alcohol**

* Materials:

+ Sterile LB
+ 1% glucose stock
+ Riboflavin
+ Arabinose
+ IPTG
+ Antibiotics (Kanamycin and Ampicillin)
+ 10 ml Falcon tube
+ Bunsen burner
+ Gloves

* Procedure:
* Begin by adding 5.4 ml of LB to a Falcon tube.
* Then add 600 µl of 10% glucose stock (resulting in a 1% final concentration).
* Dilute 5000x riboflavin (0.1 g in 1000 µl of water) and then add 1.25 µl to the final 6 ml media (resulting in 1x concentration in the final solution).
* Add 0.12 g of arabinose to the 6 ml stock as well (to get 0.2% concentration).
* Dilute 1000 mM IPTG stock to 1 mM using water. Next, add 150 µl to the media to achieve a 25 µM final concentration.
* Finally, add any antibiotics needed for plasmid survival.

**Using HP-20 Resin to Isolate Terpenes**

* Requirements:

+ HP20 resin
+ Ethyl acetate (EtOAc)
+ Growth media
+ Clean autoclaved bottle
+ Funnel
+ 4°C centrifuge
+ Fume hood
+ Gloves

* Procedure:
* Cover 5 g of HP20 resin with 15 ml of EtOAc in a clean autoclaved bottle and shake for 10 minutes.
* Filter the resin using a funnel and discard EtOAc in flammable waste.
* Add HP20 resin back into the used bottle and cover it with 15 ml of EtOAc.
* Add 2.5 g of 5% HP20 resin into 50 ml media and autoclave it.
* Mix 2.5 ml of bacterial culture with the 50 ml of media (containing 5% HP20), shaking incubate for 1 hour.
* Centrifuge at 4000 rpm at 4°C for 10 mins and discard the supernatant.
* Freeze the cell pellet/HP20 resin overnight at -80°C and dry the pellet.
* Transfer the dry pellet/resin into a clean flask and add 20 ml EtOAc, shake overnight at 100 rpm.
* Add another 20 ml of filtered EtOAc and shake overnight again.
* Evaporate EtOAc under nitrogen and record the extract weight.

**Homogenising Liquid Cell Cultures for GCMS (Without HP20 Resin)**

* Requirements:

+ Liquid culture of cells to homogenize
+ Ethyl acetate (EtOAc) or dichloromethane (DCM)
+ Sodium sulfate (Na2SO4)
+ 13,000 rpm centrifuge
+ GCMS-grade vial
+ Sterile Eppendorf tube
+ Fume hood
+ Gloves

* Procedure:
* Extract the POH from the sample by adding EtOAc in a 1:1 ratio to the media in an Eppendorf tube under a fume hood.
* Centrifuge to pellet down particles at 13,000 rpm for 1 minute.
* Transfer the supernatant to another sterile Eppendorf tube.
* Add Na2SO4 to extract extra water remaining from the EtOAc extract.
* Filter the supernatant to remove the solids and transfer the liquid to another sterile Eppendorf tube.
* Centrifuge the filtrate again for 10 minutes at 13,000 rpm.
* Transfer the supernatant to a GC/MS vial.

**Preparing Perillyl Alcohol Sample from HP-20 Resin for GC/MS**

* Requirements:

+ Liquid culture containing HP-20 resin to homogenize
+ Ethyl acetate (EtOAc) or dichloromethane (DCM)
+ Sodium sulfate (Na2SO4)
+ 13,000 rpm centrifuge
+ GCMS-grade vial
+ Sterile Falcon tube
+ Filter paper
+ Scintillation vial
+ Rotary evaporator
+ Fume hood
+ Gloves

* Procedure:
* Transfer all the liquid culture into a sterile Falcon tube.
* Centrifuge for 10 minutes at 4 degrees and 5000 rpm.
* Remove the Falcon tube from the centrifuge and discard the supernatant.
* Transfer the pellet with HP20 resin into a sterile conical flask and add 20 ml EtOAc/DCM, shake overnight at 100 rpm for extraction.
* After overnight shaking, add Na2SO4 to the sample to remove residual water and filter the solid out into a pre-weighted 20 ml scintillation vial with filter paper.
* Attach it to a rotary evaporator without heating (depending on time and speed), remove the EtOAc in a vacuum, record the exact weight.
* The sample is then reconstituted into 1 ml of EtOAc and further diluted in a 1:50 ratio (10 uL in 490 uL EtOAc) for quantitative accuracy and comparison with the media extract.
* Transfer the supernatant to a GC/MS vial.

**Variable IPTG Experiment**

* Requirements:

+ Overnight liquid culture
+ IPTG dilutions
+ Growth media
+ Sterile conical flasks
+ Sterile 96-well plate
+ 37°C shaking absorbance plate reader
+ Bunsen burner
+ Gloves

* Procedure:
* Grow overnight culture of all cell types that will be tested (including any additional antibiotics).
* Dilute the culture 1/10 with LB and measure the OD600. Using these values, make 10 ml dilutions of the cultures with an OD600 of roughly 0.15 in a sterile conical flask.
* Incubate these flasks at 37°C and check frequently until they reach OD600=0.6. At which point, keep it on ice to prevent further growth.
* In a sterile environment, set up 10 µL IPTG dilutions in each well at different concentrations and add a 20 µL of ‘growth media’ (comprising of 10% glucose, riboflavin, and arabinose).
* Next, add 60 µL of OD600=0.4 liquid culture for each cell type to individual wells (excluding controls) and immediately after adding the culture, place in a 37°C shaking incubating absorbance plate reader.

**Interlab Experiment 1** [PDF Here](https://static.igem.org/websites/technology/interlab/2023/protocols/interlab-2023-exp1-v3.pdf)

**Find where we used the protocols in [results.](https://2023.igem.wiki/warwick/results)**

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#### Initial testing

The basis of our project revolves around counteracting the toxic effects of the mevalonate pathway products. As such, we first performed a growth assay to better quantify the effects of PA on cell survival. We found the most resilient of the strains we had access to (DH5α, BL21, DH10b, C41 and C43). Figure 1. shows that all cells have no growth under 1024 µg/ml perillyl alcohol (PA) concentration but at all of the lower concentrations they all grow. BL21 had the highest growth and DH5α having the lowest growth, shown with the final readings of each graph having little to no error bar overlap. We chose to use DH5α cells to perform our initial cloning experiments (due to its higher insert stability) and then once we confirmed the plasmid are correct, we transform it into the more PA resistant and faster growing BL21 cells.

![r-f1](https://static.igem.wiki/teams/4600/wiki/r-f1.jpg)

With our project revolving around mevalonate pathway plasmids that produce limonene and PA purchased from Addgene (more info on the [contributions page.](https://2023.igem.wiki/warwick/contribution)), the next step was to transform them into our cells (Alonso-Gutiérrez et al., 2013). After determining the cell types we want to use, we next confirmed the plasmids were as expected. To do this, we performed a restriction digest using enzymes EcoRI and SacI (as shown in Figure 2). For the most part, the digest was very successful however we did see an unexpected band that persisted after numerous digests with different enzymes. It did not affect the plasmids function, however we contacted the Addgene who provided it to us for more information (see more information on the [contributions page.](https://2023.igem.wiki/warwick/contribution) We then transformed the plasmids into homemade chemically competent *E. coli* BL21 and DH5α through heat-shock.

![r-f2](https://static.igem.wiki/teams/4600/wiki/r-f2.jpg)

Using these cells, we next tested to visualise the burden of inducing our pathway on cell growth and survival. As you can see in Figure 3, more notably in the case of BL21 cells, the more IPTG present (and the more the plasmid is induced) the lower the cell growth. This is because the cell is spending more resources to induce the plasmid, which increases the amount of toxic products being produced by the mevalonate pathway enzymes (also lowering growth). As the IPTG concentration decreases, the BL21+ cells spend fewer resources on the plasmids and due to lower induction, less toxic products will be present to slow growth - because of both these reasons the BL21+ cells grow more. At 0µM IPTG, the BL21+ cells containing mevalonate pathway plasmids have very similar growth to the normal BL21 cells and the error bars overlap, this is expected as it doesn’t have any induction of the plasmid. DH5α on the other hand shows significantly less growth with and without the plasmid, and even with no IPTG the DH5α cells with plasmid grow less than those without. Confirming that for our final tests, we should use BL21 cells to host our plasmids and synthesise our product.

![r-f3](https://static.igem.wiki/teams/4600/wiki/r-f3.jpg)

Meanwhile, we also saw fit to test for the production of PA. Without a distinct absorbance wavelength, the method we used for analysing the presence of perillyl alcohol was gas chromatography-mass spectrometry (GCMS). Using the literature to see the most common fragmentation patterns of limonene and PA we could find the retention time in each sample by looking at the chromatograms (Figure 4). Limonene has a retention time of 4.849 minutes within our runs with m/z 57, 69 and 98 peaks being the identifiable fragments showing in the chromatograms. Looking at the standard, PA has a retention time of 6.028 minutes within our runs and its most notable fragments showing in the chromatograms are m/z 67, 79 and 91. With the intensity on the Y axis, it is clear that the BL21+ sample has a higher PA concentration than DH5α+ and PA is produced in large amounts compared to limonene.

![r-f4](https://static.igem.wiki/teams/4600/wiki/r-f4.jpg)

#### Testing our idea

After laying the foundation for our project with the previous tests, the next step was to edit our plasmids and test our modifications. Using primers designed on Benchling and ordered from IDT/Twist, our goal was to add a pump and tet protein sequence to the pJBEI-6411 plasmid and add a short Ptet promoter sequence to the pJBEI-6410 plasmid (to introduce a pump that removes the toxic products alongside a feedback system that downregulates enzymes in response to stress that upregulates tet production). After the Gibson assembly reaction was performed, the reaction was transformed into chemically competent DH5α cells and grown on an antibiotic selective plate. After this, the plasmid was checked for the presence of the insert using colony PCR followed by plasmid purification, restriction digest and full-plasmid sequencing.

After checking over 50 colonies for potential pJBEI-6411\* (containing modified plasmid insert) with predesigned primers, we chose 3 colonies (18, 54, 55) that had a band at 709bp (shown in Figure 5). The purified plasmids were then digested using AvrII and EcoRI enzymes to further confirm that they are correct (shown in Figure 5).

![r-f5](https://static.igem.wiki/teams/4600/wiki/r-f5.jpg)

With the modified pJBEI-6411 plasmid (6411\*) complete much earlier than 6410\*, we wanted to assess the effectiveness of our pump without any input from the feedback system that reduces enzyme expression. Similar to before, we performed an assay to test the growth of cells in different concentrations of IPTG (which induces the plasmid). With more IPTG, the plasmid will be induced at higher levels and as such more enzymes will be made to contribute to the production of limonene and PA. As you can see in Figure 6, at very large IPTG concentrations (100uM), expression of the pump is very effective at increasing cell survival compared to without but is still lower than the cells without plasmid. As the IPTG concentration is lowered, the cells containing plasmid are induced less and grow more (closing the gap between growth of the three BL21 types).

![r-f6](https://static.igem.wiki/teams/4600/wiki/r-f6.jpg)

Eventually, after changing the cloning method from overlap extension PCR to Gibson assembly (see Engineering success for more detail), we obtained 6410\* plasmid (containing the Ptet insert). This was first confirmed by doing colony PCR on ~50 colonies with an expected band for 6410\* being 768bp using a predesigned primer. The first colony PCR (Figure 7) had a primer annealing temperature too low so results are inconclusive, though promising colonies were still kept for a restriction digest test (8, 11, 14 and 16). In the second gel things (Figure 7) were clearer and we thought that colony 38 looked the most accurate, we also took colonies 39, 40 and 48 that we could test later.

![r-f7](https://static.igem.wiki/teams/4600/wiki/r-f7.jpg)

Using these 8 colonies, we then set up a restriction digest of the purified plasmids using enzymes EcoRI and ApaI that will give a 6410\* band at 1061bp and the unmodified pJBEI-6410 will give a band at 1007bp. As seen in Figure 8 (on the left gel), despite the pJBEI-6410 band not showing up, the colony 11 and 14 bands appeared higher than the others so we decided to repeat the digest using a higher concentration of control plasmid. We also included colony 39. On the second gel, all three of the colony bands appear above the unmodified pJBEI-6410 control so we were confident that they had the insert. Nevertheless, using a primer that binds in the insert region, we sent the plasmids off for Sanger sequencing to do one final check and results were positive. Using this data, after the wiki freeze on the 12th we plan to test out the construct effectiveness by doing another IPTG assay and another GCMS experiment (if possible).

![r-f8](https://static.igem.wiki/teams/4600/wiki/r-f8.jpg)

Overall, after running out of time to put more work into our project, there are still plenty of experiments that we would have liked to have done. In particular, testing each stage of the system individually using fluorescence to monitor membrane stress, production of our membrane pumps and production of tet would be very helpful to confirm that our system is working. Doing even more repeats of both growth curves and GCMS would also be helpful to ensure our results are all valid and accurately quantify production. Having more data for our models would also give us many more insights.

#### Conclusion

We began the summer with the goal of increasing production of perillyl alcohol (PA) and limonene by mitigating the toxic effects of said products. We initially planned to modify both mevalonate plasmids to contain not only a pump system but also a potential feedback system that would downregulate enzymes. Despite not performing as many tests as we had hoped, we still laid a strong foundation for our project to work off by showing the limitations of producing PA/limonene intracellularly. At the end of the project, we successfully modified both plasmids and, with quite promising preliminary results, showed that our pump system in particular resulted in higher cell growth. While our feedback system wasn’t tested (before the wiki deadline) and we couldn’t accurately quantify the PA produced with and without each component of our project, we are pleased with our progress and can say without a doubt that we enjoyed our time in the lab and learning just a fraction of what synthetic biology is capable of.

References

1. Alonso-Gutiérrez, J., Chan, R., Batth, T. S., Adams, P. D., Keasling, J. D., Kim, Y., & Lee, T. S. (2013). Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metabolic Engineering, 19, 33–41.

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