Notebook

Purpose: grow individual constructs on agar plates for future cloning

Procedure:

From the iGEM distribution kit 2025 (estimated 1-2ng of DNA in each well)

• With a pipette tip, a hole was punched through the foil cover into the corresponding well of the part we want.
• 10μL of dH2O (distilled water) was pipetted into the well. This was pipetted up and down a few times and left to sit for 5 minutes to make sure the dned DNA is fully resuspended.
• 1μL of resuspended DNA was transformed into the desired cells.

XL1-Blu E.coli transformation:

• 50μL of prepared E.coli competent cells was put on ice for 5 minutes.
• 1μL of plasma DNA was added and then incubated on ice for 5 minutes.
• This was heat shocked for 1 minute at 42°C.
• This was put on ice for 5 minutes.
• 250μL of room temperature SOC buffer was added.
• The sample was incubated for 1 hour at 37°C while shaking at 2590 rpm.
• One LB agar petri dish per part (mRFP, LacZ and pJUMP29A1) with antibiotic was plated and then incubated at 37°C overnight.

Purpose: to extract a concentrated and purified plasmid for cloning.

Notes: cells in the P2 vial (LacZ) were dead when the experiment started. Reason for this is unknown

Procedure:

• Cells were resuspended in 250μL of P2 buffer and mixed.
• Added 350μL of N3 buffer was added and mixed.
• At room temp the sample was centrifuged at 13,000 rpm for 10 minutes.
• The supernatant was collected.
• The spin column was placed in the 2ml collection tube
• We applied the sample to the column.
• The sample was centrifuged for 1 minute, then the flowthrough was discarded and the column was placed back into the sample tube.
• 500μL of the PB buffer was added to the column to wash.
• The sample was centrifuged for 1 minute, then the flowthrough was discarded and the column was placed back into the sample tube.
• 750μL of the PE buffer was added to the column to wash.
• The sample was centrifuged for 1 minute, then the flowthrough was discarded and the column was placed back into the sample tube.
• The sample was centrifuged for an additional 1 minute, then placed in a clean 1.5mL microcentrifuge tube.
• 50μL of H2O was added and the column was left to stand for 1 minute for elution of DNA.
• The column was centrifuged for 1 minute.
• The purified samples were stored in the freezer at -20°C.
• We did this for agar plates A2, P1 and P2.
• We recorded the concentration of plasmid using a nanodrop, which was initially calibrated using water.

Results:

Final concentrations

• A2 (pJUMP29A1) - 21ng/uL, A260/A280 = 1.98
• P1 (mRFP) - 68.6ng/ul, A260/A280 = 1.87
• P2 (LacZ) - 5.1ng/uL, A260/A280 = 1.86

This concentration was too small to be useful, so we re-did this construct on day 3

Purpose: re-do the LacZ failed plasmid purification

Procedure:

• 50μL of prepared E.coli competent cells was put on ice for 5 minutes.
• 1μL of plasma DNA was added and then incubated on ice for 5 minutes.
• This was heat shocked for 1 minute at 42°C.
• This was put on ice for 5 minutes.
• 250μL of room temperature SOC buffer was added.
• The sample was incubated for 1 hour at 37°C while shaking at 2590 rpm.
• One LB agar petri dish per part (LacZ) with antibiotic was plated and then incubated at 37°C overnight.

Results:

The transformation of LacZ failed again - cultures grew a plate but when transformed into liquid media they died.

Conc from nanodrop- 11.4 and 11.8 ng/μL

Purpose: to assemble the constructs that were necessary for toehold testing

Procedure:

513372229 - CP1 250ng= 2142fmol
Dilution to 100fmol/μL
1. 10μL of dH2O was added to the CP1 tube (make 214.2 fmol/μL)
5.33μL of dH2O was added to 4.67mL of the diluted solution just made.
18μL of dH2O was added to 2μL of this solution (make up 20μL of 10fmol/μL).

513373230 - CP2 250ng= 2131fmol
Dilution to 100fmol/μL
2. 10μL of dH2O was added to the CP2 tube (make 213.1 fmol/μL)
5.31μL of dH2O was added to 4.69mL of the diluted solution just made.
18μL of dH2O was added to 2μL of this solution (make up 20μL of 10fmol/μL).

513373231 - CP3 250ng= 2131fmol
The concentration is the same as CP2s so repeat the steps as the one for CP2.

513373232 - CP4 250ng= 2131fmol
The concentration is the same as CP2s so repeat the steps as the one for CP2.

513373233 - CP5 250ng= 2213fmol
Dilution to 100fmol/μL
3. 10μL of dH2O was added to the CP5 tube (make 221.3 fmol/μL)
5.48μL of dH2O was added to 4.52mL of the diluted solution just made.
18μL of dH2O was added to 2μL of this solution (make up 20μL of 10fmol/μL).

513373234 - CP6 250ng= 2201fmol
Dilution to 100fmol/μL
4. 10μL of dH2O was added to the CP6 tube (make 220.1 fmol/μL)
5.46μL of dH2O was added to 4.54mL of the diluted solution just made.
18μL of dH2O was added to 2μL of this solution (make up 20μL of 10fmol/μL).

513373235 - CP7 250ng= 2201fmol
The concentration is the same as CP6s so repeat the steps as the one for CP6.

513373236 - CP8 250ng= 2201fmol
The concentration is the same as CP6s so repeat the steps as the one for CP6.

513373223 - T1 250ng= 2875fmol
Dilution to 100fmol/μL
5. 10μL of dH2O was added to the CP6 tube (make 287.5fmol/μL)
6.52μL of dH2O was added to 3.48mL of the diluted solution just made.
18μL of dH2O was added to 2μL of this solution (make up 20μL of 10fmol/μL).

513373237 - Nanoluc 500ng= 1288fmol
Dilution to 100fmol/μL
6. 10μL of dH2O was added to the Nanoluc tube (make 128.8fmol/μL)
2.24μL of dH2O was added to 7.76mL of the diluted solution just made.
18μL of dH2O was added to 2μL of this solution (make up 20μL of 10fmol/μL).

Each CPX tube was labelled= CPX and either 100 or 10 (depending on the dilution).

Cloning

We have labeled tubes CP1. CP2, CP3, CP4, CP5luc, CP6luc, CP7luc, CP8luc, CP5mRFP, CP6mRFP, CP7mRFP and CP8mRFP

Then the following mixes were created

CP1

Component name	Volume added (uL)
pJUMP29A1	2
CP1	2
T4 DNA ligase buffer	2
T4 DNA ligase	0.5
Restriction enzyme	1
Water	12.5

CP2

Component name	Volume added (uL)
pJUMP29A1	2
CP2	2
T4 DNA ligase buffer	2
T4 DNA ligase	0.5
Restriction enzyme	1
Water	12.5

CP3

Component name	Volume added (uL)
pJUMP29A1	2
CP3	2
T4 DNA ligase buffer	2
T4 DNA ligase	0.5
Restriction enzyme	1
Water	12.5

CP4

Component name	Volume added (uL)
pJUMP29A1	2
CP4	2
T4 DNA ligase buffer	2
T4 DNA ligase	0.5
Restriction enzyme	1
Water	12.5

CP5luc

Component name	Volume added (uL)
pJUMP29A1	2
CP5	2
T1	2
Luciferase	2
T4 DNA ligase buffer	2
T4 DNA ligase	1
Restriction enzyme	0.5
Water	8.5

CP6luc

Component name	Volume added (uL)
pJUMP29A1	2
CP6	2
T1	2
Luciferase	2
T4 DNA ligase buffer	2
T4 DNA ligase	1
Restriction enzyme	0.5
Water	8.5

CP7luc

Component name	Volume added (uL)
pJUMP29A1	2
CP7	2
T1	2
Luciferase	2
T4 DNA ligase buffer	2
T4 DNA ligase	1
Restriction enzyme	0.5
Water	8.5

CP8luc

Component name	Volume added (uL)
pJUMP29A1	2
CP8	2
T1	2
Luciferase	2
T4 DNA ligase buffer	2
T4 DNA ligase	1
Restriction enzyme	0.5
Water	8.5

CP5mRFP

Component name	Volume added (uL)
pJUMP29A1	2
CP5	2
T1	2
mRFP	2
T4 DNA ligase buffer	2
T4 DNA ligase	1
Restriction enzyme	0.5
Water	8.5

CP6mRFP

Component name	Volume added (uL)
pJUMP29A1	2
CP6	2
T1	2
mRFP	2
T4 DNA ligase buffer	2
T4 DNA ligase	1
Restriction enzyme	0.5
Water	8.5

CP7mRFP

Component name	Volume added (uL)
pJUMP29A1	2
CP7	2
T1	2
mRFP	2
T4 DNA ligase buffer	2
T4 DNA ligase	1
Restriction enzyme	0.5
Water	8.5

CP8mRFP

Component name	Volume added (uL)
pJUMP29A1	2
CP8	2
T1	2
mRFP	2
T4 DNA ligase buffer	2
T4 DNA ligase	1
Restriction enzyme	0.5
Water	8.5

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

Cloning went without any incidents

Purpose: to propagate the number of plasmids that we have

Procedure:

XL1-Blu E.Coli transformation

• 50μL of prepared E.coli competent cells was put on ice for 5 minutes.
• 1μL of plasma DNA was added and then incubated on ice for 5 minutes.
• This was heat shocked for 1 minute at 42°C.
• This was put on ice for 5 minutes.
• 250μL of room temperature SOC buffer was added.
• The sample was incubated for 1 hour at 37°C while shaking at 2590 rpm.
• One LB agar petri dish per part (CP1-4, CP5luc-8luc, CP5mRFP - 8mRFP) with antibiotic was plated and then incubated at 37°C overnight.

Purpose: to prepare constructs for spin column purification and to analyse the sequences via gel electrophoresis

No growth was observed on CP3 plate, so we decided not to test with that miRNA

E.Coli inoculation

1. To 100ml of LB broth 10ml of antibiotic (kanamycin) was added
2. A single colony from cloned plates was taken and placed into the tube with broth and kanamycin
   • 11 plates = 11 tubes
3. Tubes were then put into a shaking incubator at 37C, 225 rpm overnight

PCR

1. 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
2. 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

Gel electrophoresis

1. 2% gel was prepared using 4g agar and 200ml TAE buffer
2. Into a gel electrophoresis chamber TAE buffer was added until it was covering the gel fully
3. PCR products were transported into the gel in this arrangement
   • Well 1 = DNA ladder (100bp)
   • Well 2 = CP1
   • Well 3 = CP2
   • Well 4 = CP3
   • Well 5 = CP4
   • Well 6 = CP5 luc
   • Well 7 = CP6 luc
   • Well 8 = CP7 luc
   • Well 9 = CP8 luc
   • Well 10 = CP5 mRFP
   • Well 11 = CP6 mRFP
   • Well 12 = CP7 mRFP
   • Well 13 = CP8 mRFP
   • Well 14 = FR6 ladder (1kbp)
4. The lid was then placed and the gel was running for an hour at 120 V

Purpose: To analyse the lengths of the constructs for verification purposes

Plasmid Purification:

1. Inoculated cells were transferred into 15 ml tubes and labeled CP1-4, CP5luc - 8luc, CP5mRFP - CP*mRFP
2. For 5 minutes these samples were centrifuged at 3500 rpm.
3. The pellets were left at the bottom of the tube.
4. 250μL of P1 buffer was added to each tube, then mixed and transferred to labelled microcentrifuge tubes.
5. 250μL of P2 buffer was added to each tube, followed by the addition of N3 buffer.
6. The tube was centrifuged at 13000rpm for 10 minutes then the supernatant was collected.
7. The sample was applied to a 2mL collection tube with a column, then was centrifuged for 1 minute.
8. The flow-through was discarded.
9. Add 500μL of PB buffer to the column, centrifuged for 1 minute and discarded the flow-through.
10. Add 750μL of PB buffer to the column, centrifuged for 1 minute and discarded the flow-through.
11. We centrifuged for 1 extra minute and placed the column into a clean 1.5mL tube (labelled).
12. 30μL of H2O was added and left for 1 minute (to elute the DNA).
13. This was lastly centrifuged for 1 minute.

• It was stored at -20°C.

We recorded the concentration of all the sample using a Nano-drop.

Nano-drop results:

• CP1 - 46.4ng/uL A260/280 1.75
• CP2 - 33.3ng/ul A260/280 1.86
• CP4 - 47.7ng/ul A260/280 1.91
• CP5mRFP - 37.6 ng/ul A260/280 1.88
• CP6mRFP - 38.9 ng/ul A260/280 1.87
• CP7mRFP - 37.8 ng/ul A260/280 1.81
• CP8mRFP - 30.7 ng/ul A260/280 1.92
• CP5luc - 37.3 ng/ul A260/280 1.92
• CP6luc - 44 ng/ul A260/280 1.83
• CP7luc - 33.5 ng/ul A260/280 1.79
• CP8luc - 32.5 ng/ul A260/280 1.78

Purpose: to verify the length of our sequences

Procedure:

PCR reaction

The following components have been added to the PCR tubes

1. 12.5 uL PCR mastermix (2X)
2. 5 uL of DNA (CP1-8, T1 and NanoLuc)
3. 2.5 forward primer
4. 2.5 reverse primer
5. 2.5 ul dH2O

Amplification was carried out using the following parameters:

1. Phase 1 cycle: 37°C for 10 minutes
2. Phase 2 cycle: [37°C for 2 minutes and 16°C for 2 minutes] for 15 cycles and then 75°C for 15 minutes
3. Phase 3 cycle: 37°C for 15 minutes

Gel electrophoresis

1. 2% gel was prepared using 4g agar and 200ml TAE buffer
2. Into a gel electrophoresis chamber TAE buffer was added until it was covering the gel fully
3. PCR products were transported into the gel in this arrangement
   • Well 1 = DNA ladder (1kbp)
   • Well 2 = CP1
   • Well 3 = CP2
   • Well 4 = CP3
   • Well 5 = CP4
   • Well 6 = CP5
   • Well 7 = CP6
   • Well 8 = CP7
   • Well 9 = CP8
   • Well 10 = T1
   • Well 11 = NanoLuc
   • Well 12 = 100 bp DNA ladder
   • Well 13 = 1 kbp DNA ladder

The reason for adding 2 DNA ladders was that after adding one of them we realised that it was expired, so we decided to add another one just in case.

Purpose: To test for leaky expression when no miRNA is present

Procedure:

Four plates were labelled with: "CP5,6… mRFP BL21-de3 15.09.25 KCL iGEM"

For Plate 5:

• 4 sections were created (I, II, III and IV, each resembling CP5mRFP, CP6mRFP, CP7mrFP and CP8mRFP respectively)
• Nictrocellulose was placed onto the agar and colonies were streaked onto their corresponding sector
• We added 1μLof each plasmid into the 50μL e.coli tubes and let them incubate on ice for 5 minutes. [CP5 plasmid was added to the CP5 E-coli tubes so we restarted].
• The tubes were heat shocked for 1 minute at 42°C, then the tubes were put on ice for 5 minutes.
• Add 250μL of room temperature SOC buffer to each tube.
• They were incubated for 1 hour at 37°C at 250rpm.
• We then plated them and the nitrocellular paper was added to the plate.
• They were incubated overnight at 37°C.

Purpose: to test our toehold for sensitivity to correct miRNA and to test for leaky expression when no miRNA is present.

Procedure:

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 225rpm for 3 hours

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

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.