Experiments

Experiment Pre-Prep

Preparation of the LB Broth

LB broth was created using Invitrogen LB broth base (No. 12780-052).

• 1 litre of distilled water was added to 20 g of Invitrogen powder as per the company’s protocol.
• The solution was stirred with a magnetic rod until fully dissolved.
• Transferred 50ml into a small glass vial using a pipette and repeated 40 times; the 40 vials were placed in two racks
• The two racks of vials were placed in an autoclave at 121°C for 15 minutes; vial caps were unscrewed loosely to avoid bursting in the autoclave under pressure

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Antibiotic Dilution

Spectinomycin sulfate (CAS [64058-48-6], Batch No. 5016-01)

• 5g of weighed spectinomycin sulfate was dissolved in distilled water to make up 10ml solution
• 1ml of solution was transferred to each of the 9 eppendorf tubes and each tube was labelled with ‘spec’ before being placed in the freezer

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Agar preparation and plating

• 6.4g agar powder was weighed on a scale and added to 200ml distilled water in glass jar with a cap; repeated five times so 32g / L distilled water was used
• Five jars placed in autoclave at 121°C for 15 minutes with loosened caps
• The bench was wiped with antibacterial spray
• LB agar jars were placed in the microwave until fully dissolved (as agar had solidified)
• A specific antibiotic was transferred into each LB agar jar and mixed gently– each jar contained either ampicillin, xanamycin, spectinomycin, or chloramphenicol
• A layer of approx. 25ml of LB agar containing an antibiotic was poured into each agar plate – 8 plates were used per jar of 200ml solution
• Plates were left to set before being flipped over to prevent condensation interfering with agar and each plate was labelled accordingly with the antibiotic used
• Plates were stored in the fridge stacked and wrapped in plastic film
• This process was repeated four times for each antibiotic

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Final Plate Summary

• 8 plates – amp
• 8 plates – kanamycin
• 8 plates – spectinomycin
• 8 plates – chloramphenicol

Toehold switch assembly and testing:

JUMP Cloning

Transformation of XL1-Blu E.Coli with LacZ, mRFP and pJUMP29A1 plasmid backbone

• mRFP and the LacZ were taken from the iGEM 2025 Distribution Kit (wells O3 and E23 respectively)
• 10 uL of dH2O were added to each well, mixed and then left to rest for 5 minutes
• Then, 1uL of the plasmid was transformed into competent E.Coli
• pJUMP29A1 was taken from another source

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Transformation of E.Coli with pJUMP29A1, LacZ and mRFP

• Take 50uL of competent E.COli cells and rest on ice for 5 mins
• Add 1uL of plasmid DNA to the competent E.Coli and incubate on ice for 5 min
• Heat shock E.Coli at 42°C for 1 minute
• Put on ice for 5 mins
• Add 250 uL of SOC at room temperature
• Incubate for 1hr at 37°C in a shaking incubator at 250rpm
• Plate on a labeled agar petri dish with an appropriate antibiotic
• Incubate overnight at 37°C

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Spin column plasmid purification<

• Kit used - QIAprep Spin Miniprep Kit
• We have resuspended our cells in 250uL of Buffer P1 and transferred the mix into a 1.5ml microcentrifuge tube
• Then, we added 250 uL of Buffer P2 and mixed the solution
• After that, we added 350uL of Buffer N3 and mixed
• Then the mix was centrifuged at 13,000g for 10 minutes at room temperature
• After which, the supernatant was collected and added to a spin column that was provided in the kit. The column was placed into another 2ml collection tube beforehand
• Then, the column was centrifuged for 1 minute at 10,000g and the flowthrough discarded
• 500 uL of PB buffer were added to the colum
• Then, the column was centrifuged for 1 minute at 10,000g and the flowthrough discarded
• After that, 75- uL of PE buffer was added to the column
• Then, the column was centrifuged for 1 minute at 10,000g and the flowthrough discarded
• The column was then placed back into the same tube, and centrifuged at 10,000g for 1 min again
• After that, the column was placed into a clean microcentrifuge tube
• 30 uL of dH2O were added, and the column was left to rest for 1 min
• Then, the column was centrifuged for 1 min at 10,000g
• After that, the column was discarded, and the flowthrough was the purified plasmid DNA
• Following that, the concentration of the plasmid DNA was measured using a NanoDrop
• Purified plasmid DNA was then stored at -20°C until needed.

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JUMPcloning of our composite parts + reporter gene

• First, all our sequences that we have ordered were diluted to 10 fmol/uL (total volume of 20 uL)
• Then, we have labeled tubes CP1. CP2, CP3, CP4, CP5luc, CP6luc, CP7luc, CP8luc, CP5mRFP, CP6mRFP, CP7mRFP and CP8mRFP
• Then the following mixes were created

After mixing tubes were then placed in a PCR machine on this cycle

• Phase 1 – 37C for 10 mins
• Phase 2 – 15 cycles of [37C for 2 mins, 16C for 2 mins] then 75C for 15 mins
• Phase 3 – 37C for 15 mins

After cloning, some DNA was sent to be sequenced for verification purposes and some was used in a PCR to test for the length of the cloned fragments

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Cell free system assembly

NEBExpress system assembly and testing

Storage: NEBExpress® Cell-free E. coli Protein Synthesis System was stored in a -80°C freezer until needed.

Assembly of the NEBExpress® Cell-free E. coli Protein Synthesis System

1. We thawed the components of the kit on ice for 10 mins.
2. We then gently vortexed the NEBExpress® S30 Synthesis Extract and Protein Synthesis Buffer to mix.
3. Then, we combined reagents in a 1.5 ml microcentrifuge tube on ice as follows:

Component name	- Control	+ Control	hsa-miR-7850-5p miRNA + TH1	hsa-miR-1306-5p miRNA + TH2	TH3*	hsa-miR-6529-5p miRNA + TH4
NEBExpress® S30 Synthesis Extract	12 μl	12 μl	12 μl	12 μl	12 μl	12 μl
Protein Synthesis Buffer (2X)	25 μl	25 μl	25 μl	25 μl	25 μl	25 μl
T7 RNA Polymerase	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl
RNase Inhibitor, Murine	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl
Control DHFR-His template (125 ng/μl)	---	2 μl	---	---	---	---
Toehold	---	---	6 μl	5 μl	8 μl	6 μl
miRNA	---	---	5 μl	6 μl	---	5 μl
Water	11 μl	9 μl	---	---	3 μl	---

*Due to the miRNA containing plasmid failing to express after multiple attempts, no miRNA was inserted into the TH3 system, with the TH3 system serving as a second negative control.

After mixing all the components, tubes were placed on an incubator at 37°C with shaking at 225 rpm for 3 hours.

After the incubation, samples were transported on a 96-well plate for further analysis.

Assays:

mRFP Assay

• Samples were pipetted onto a 96 well plate, with wells A1 and A2 being negative and positive control respectively, wells A3-6 were toehold systems CP1/5, CP2/6, CP7 and CP4/8 respectively.
• The 96 well plate was then transported into the FLUOstart omega spectrophotometer and the absorbance reading was taken
• When results came with no mRFP expression we incubated the cell free system for 1 more hour and repeated the measurements - no change was observed.

PCR + gel electrophoresis

2% gel was prepared using 4g agar and 200ml TAE buffer

• 12.5ul PCR Master Mix was added to the DNA template with 2.5ul forward primer, 2.5ul reverse primer and 2.5ul water to a test tube
• Invert the tube multiple times and centrifuge
• Amplification was carried out using the following parameters:
• Phase 1 cycle: 37°C for 10 minutes
• Phase 2 cycle: [37°C for 2 minutes and 16°C for 2 minutes] for 15 cycles and then 75°C for 15 minutes
• Phase 3 cycle: 37°C for 15 minutes
• Agarose gel electrophoresis was then performed:
• Diluted TAE buffer was poured into the electrophoresis chamber to cover the agarose gel.
• 100 base pair DNA ladder was added to the first 10ul well.
• Each cell component was added to the following cells in this order:

Well	Sample
1	DNA ladder (100bp)
2	CP1
3	CP2
4	CP3
5	CP4
6	CP5 luc
7	CP6 luc
8	CP7 luc
9	CP8 luc
10	CP5 mRFP
11	CP6 mRFP
12	CP7 mRFP
13	CP8 mRFP
14	FR6 ladder (1kbp)

• The last well contained 10ul of the 6754A DNA ladder
• The lid was then placed, the wires connected and the cycle started; gel electrophoresis was run for one hour at 120V.

Sequences:

TH1+mRFP+T1

TH2+mRFP+T1

TH3+mRFP+T1

TH4+mRFP+T1