iGEM 2022 — Team lambert-ga

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{% block title %}Notebook{% endblock %}
{% block image %}https://static.igem.wiki/teams/4245/wiki/notebook-header.jpg{% endblock %}
{% block lead %}The Day-to-Day Operations{% endblock %}
{% set list = [["RCA", "div-rca"],["RCT", "div-rct"],["Characterization", "div-char"]] %}
{% set footerlist = [["RCA", "Wetlab", "wetlab", "https://2022.igem.wiki/lambert-ga/rca"], ["Characterization",
"Wetlab", "wetlab", "https://2022.igem.wiki/lambert-ga/characterization"]] %}
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![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/experiments-rca-icon.svg)

## Rolling Circle Amplification (RCA)

Figure 1. RCA Lab Notebook

![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/rct-icon.svg)

## Rolling Circle Transcription (RCT)

Figure 2. RCT lab notebook

![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/poc-sample-testing.svg)

## Characterization

Figure 3. Characterization lab notebook

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block image %}https://static.igem.wiki/teams/4245/wiki/engineering-success-header.jpg{% endblock %}
{% block lead %}Design ⋅ Build ⋅ Test ⋅ Learn{% endblock %}
{% set footerlist = [["RCA", "Wetlab", "wetlab", "https://2022.igem.wiki/lambert-ga/rca"], ["Part Collections", "Wetlab", "wetlab", "https://2022.igem.wiki/lambert-ga/part-collection"]] %}
{% block page\_content %}{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block image %}https://static.igem.wiki/teams/4245/wiki/experimentation-header.png{% endblock %}
{% block lead %}All Protocols in One Place{% endblock %}
{% set list = [["Cloning Protocols", "div-cloning"],["Rolling Circle Amplification", "div-rca"],["Rolling Circle
Transcription", "div-rct"],["Proof of Concept", "div-poc"]] %}
{% set footerlist = [["Results", "Wetlab", "wetlab", "https://2022.igem.wiki/lambert-ga/results"], ["Notebook",
"Wetlab", "wetlab", "https://2022.igem.wiki/lambert-ga/notebook"]] %}
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![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/experiments-icon-cloning.svg)

## Cloning Protocols

### Restriction Digest

| Reagent | Amount |
| --- | --- |
| Restriction Enzyme 1 | 1 µL |
| Restriction Enzyme 2 | 1 µL |
| 10x Cutsmart Buffer | 5 µL |
| Insert or Vector DNA | 1 µg |
| Nuclease Free Water | To 50 µL |

1. Shave ice in a styrofoam container.
2. Label PCR tubes.
3. Add all reagents to the tube.
4. Incubate for 30 minutes at 37°C. If both enzymes are Time-Saver Qualified, only incubate for 5-15 minutes.
5. Incubate for 20 minutes at 65°C.

### Ligation

| Reagent | Amount |
| --- | --- |
| T4 Ligase Buffer | 2 µL |
| T4 DNA Ligase | 1 µL |
| Vector DNA | 50 ng |
| Insert DNA | 150 ng |
| Nuclease Free Water | To 20 µL |

1. Shave ice in a styrofoam container.
2. Label PCR tubes.
3. Add all reagents to the tube.
4. Incubate for 10 min at room temperature.
5. Incubate for 10 min at 65°C.

### Transformation

1. Label tubes with initials, date, and what is being transformed.
2. Place all tubes on ice.
3. Put SOC/LB and antibiotic plates in the incubator to warm at 37°C.
4. Pipette 1 µL of the desired plasmid(s) and 1 µL of PUC19 into the corresponding labeled transformation
   tubes.
5. Thaw competent cells on ice for 10 minutes or just until the cells inside are moving when you flick the
   tube.
6. Pipette 50 µL of competent cells into each labeled transformation tube on ice.
7. Incubate tubes on ice for 30 minutes.
8. Heat shock tubes at 42°C for 45 seconds.
9. Place on ice for 2 minutes.
10. Add 950 µL of SOC/LB media into each tube with cells.
11. Place tubes shaking in the incubator for 60 minutes at 37°C.
12. Pipette 120-150 µL of cells with SOC/LB media onto warm plates with the correct antibiotics.
13. Place antibiotic plates in the incubator at 37°C for 24 hours to let cells grow.

### Inoculation for Culture

1. Prepare liquid LB.
2. Add LB to a tube or flask.
3. Add 1 µL of antibiotic for every 1 mL of LB (typically use 5 µL : 5 mL).
4. Antibiotics should correspond to the cell’s antibiotic resistance gene.
5. Using a sterile pipette tip, pick up a single colony from your LB agar plate.
6. Drop the tip into liquid LB and antibiotic, then swirl to release cells.
7. Cover culture with sterile aluminum foil or cap that is not airtight.
8. Incubate culture at 37℃ for 12-18 hours in a shaking incubator.
9. After incubation, check for growth.
10. For long-term storage, continue with creating a glycerol stock.

### Miniprep

1. Grow 1-5 mL culture overnight in a 10 mL - 20 mL culture tube.
2. Centrifuge at 2500 xg for 10 minutes at room temperature. Decant or aspirate and discard culture media.
3. Add 250 µL of solution 1 mixed with RNase. Vortex to mix, then transfer suspension into a new 1.5 mL microcentrifuge tube.
4. Add 250 µL of solution 2. Invert until there is a clear lysate.
5. Add 350 µL to solution 3. Invert until white precipitate forms. Centrifuge at 13,000 xg for 10 minutes.
6. Insert mini column into a 2 mL collection tube.
7. Transfer the clear supernatant into a mini-column using a micropipette. Centrifuge at 13,000 xg for 60
   seconds. Discard filtrate and reuse the collection tube.
8. Add 500 µL of HBC Wash Buffer diluted in ethanol. Centrifuge at 13,000 xg for 60 seconds. Discard filtrate
   and reuse the collection tube.
9. Add 700 µL of the DNA Wash Buffer diluted in ethanol. Centrifuge at 13,000 xg for 30 seconds. Discard
   filtrate, and reuse collection tube.
10. Centrifuge empty mini-column at 13,0000 xg for 2 minutes to remove ethanol.
11. Transfer mini-column to a nuclease-free 1.5 mL microcentrifuge tube.
12. Add 50 µL of Elution Buffer. Let it sit at room temperature for 5 minutes. Centrifuge at 13,000 xg for 60
    seconds.
13. Store eluted DNA at -20℃.

### Gel Electrophoresis

#### Making Gels

1. Mix 0.5 g of agarose with 50 mL of 1x TAE buffer in a 100 - 250 mL Erlenmeyer flask.
2. Heat up the solution until it turns clear.
3. Cool solution to 65°C.
4. Add 6 µL of SYBR™ Safe and gently swirl the flask to mix the solution.
5. Pour the solution into a gel chamber and put in the gel comb.
6. Wait for the gel to solidify before loading.

#### Running the Gel

1. Once solidified, fix the gel’s position so that the wells of the gel are at the end of the chamber and DNA
   runs to red.
2. Pour used 1x TAE buffer into the gel chamber evenly to completely cover the gel.
3. On a piece of parafilm, mix 5 µL of DNA and 1 µL of purple loading dye using a micropipette.
4. Add the mixed 6 µL solution into each well.
5. Add 6 µL of a ladder into an empty well.
6. Close the gel chamber and connect electrodes to the power supply.
7. Set power supply with voltage and time.
8. Turn on the power supply and make sure bubbles are rising on the sides of the gel chamber.
9. Once gel electrophoresis is completed, put the gel under UV (ultraviolet) light to compare the bands of DNA
   to the bands of the ladder.

### PCR

| Reagent | Amount |
| --- | --- |
| Q5 High Fidelity Master Mix | 12.5 µL |
| 10 μM diluted forward primer | 1.25 µL |
| 10 μM diluted reverse primer | 1.25 µL |
| Template DNA | 1 µL |
| Sterile Water | To 25 µL |

1. Label PCR tubes.
2. Add reagents to PCR tube.
3. Set thermocycler with temperatures and times (annealing temperature and elongation time can vary depending
   on part).
4. Run thermocycler.

### Colony PCR

| Reagent | Amount |
| --- | --- |
| Q5 High Fidelity Master Mix | 12.5 µL |
| 10 μM diluted forward primer | 1.25 µL |
| 10 μM diluted reverse primer | 1.25 µL |
| Diluted Colony | 1 µL |
| Sterile Water | To 25 µL |

1. Dilute colony from LB agar plate into 40 µL of water.
2. Use 1 µL for colony PCR reaction.
3. Label PCR tube.
4. Add reagents to PCR tube.
5. Run in Thermocycler.

### Glycerol Stock

1. Grow 1-5 mL culture overnight in a 10 mL - 20 mL culture tube.
2. Centrifuge at 2500 xg for 10 minutes at room temperature. Decant or aspirate and discard culture media.
3. Remove all but 500 µL of growth media.
4. Add 500 µL of glycerol stock and resuspend.
5. Transfer solution to 1.5 mL microcentrifuge tube and store in -80°C.

![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/experiments-rca-icon.svg)

## Rolling Circle Amplification (RCA)

### SYBR™ Safe Experimentation

#### 1. miRNA - DNA hybridization and Ligation Reaction

1. Make sure all the PCR tubes are RNAse free. Label the PCR tubes.
2. Label three PCR tubes A1. Label three PCR tubes A2. Label three PCR Tubes A3. Label three PCR Tubes A4.
   Label
   three PCR Tubes A5. Label three PCR Tubes A6. Label three PCR Tubes A7. Label three PCR Tubes A8. Label
   three
   PCR Tubes A9. Label three PCR Tubes A10. Pipette 1 µL of 10 x Ligation Buffer (from NEB) into all of the PCR
   tubes.
3. Add 0.5 µL of 100 nM padlock probe. Add .1 µL of 1000 pM of the miRNA-1. Add 6.9 µL of water.
4. Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes through
   the
   use
   of a thermocycler.
5. Then, add 20 U or 0.5 µL of RNase inhibitors.
6. Add 1 µL of Splint R Ligase.
7. Incubate the reaction for 2 hours at 37°C.
8. Bring reaction up to 65°C for 10 minutes to terminate. The reaction was brought up based on gradient
   63-68°C.
   In
   addition, to determine the ideal situations where the miRNA and the padlock do not detach, the reaction was
   brought
   up to the gradient temperatures for 10 minutes as well as 20 minutes to determine the ideal temperature.
   * Each of the A1- A5 tubes are on the gradient for 10 minutes. Each of the A6- A10 tubes are on the
     gradient
     for
     20 minutes.

#### 2. RCA Reaction (Reaction volume will be 25 µL so add 0.5 µL of water)

1. Pipette 2.5 µL of 10x phi29 buffer (from NEB) into previous ligation reaction mixture.
2. Add 12.5 units or 1.25 µL of phi29 DNA polymerase.
3. Add 6.75 ul of 10 mM dNTPs.
4. Incubate the reactions for 8 hours at 37°C.
5. Bring up the reactions to 65°C for 10 minutes to inactivate the DNA polymerase.

#### 3. Readout

1. The readout reaction utilizes Sybr Safe; use a 5:1 ratio of reaction volume: SYBR™ Safe.
2. Run all the reactions through the plate reader to obtain fluorescence values.

### Gel Experimentation1. miRNA - DNA hybridization and Ligation Reaction 1. Pipette 1 μL of NEB 10 x Ligation Buffer into pcr tubes. 2. Add 2.5 μL of 20 nM padlock probe . For miRNA, do a 1:10 dilution using a 1000 pM stock and add 1 μL of diluted miRNA into reaction. 3. Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes. 4. Add 1 μL of Splint R Ligase and 0.5 μL of RNAse inhibitor into sample. 5. Incubate the reaction for 2 hours at 37°C. 6. Bring reaction up to 65°C for 20 minutes to terminate. 2. RCA Reaction 1. Pipette 2.5 μL of NEB 10x phi29 buffer into previous ligation reaction mixture. 2. Add 12.5 units or 1.25 μL of phi29 DNA polymerase. Add 1 μL of aluminum. 3. Add 6.76 μL of 10 mM dNTPs. 4. Incubate the reactions for 8 hours at 37°C. 5. Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase. 3. Readout 1. Add 10 μL of reaction + 2 μL of loading dye into 1% agarose gel with SYBR™ Safe. Detection Limit Experimentation The following concentrations of miRNA were used to determine the lower detection limit: 1. Add 5 µL of 1000 pM of the miRNA-1. * Final Concentration: 204 pM 2. Add 1 µL of 1000 pM of the miRNA-1. * Final Concentration: 40.8 pM 3. Add 0.5 µL of 1000 pM of the miRNA-1. * Final Concentration: 20.4 pM 4. Add 1 µL of 100 pM of the miRNA-1. * Final Concentration: 4.08 pM 5. Add 0.8 µL of 100 pM of the miRNA-1 * Final Concentration: 3.27 pM 6. Add 0.5 µL of 100 pM of miRNA-1 * Final Concentration: 2.04 pM 7. Add 0.3 µL of 100 pM of miRNA-1. * Final Concentration: 1.22 pM 8. Add 1 µL of 10 pM of miRNA-1. * Final Concentration: 0.408 pM 9. Add 0.5 µL of 10 pM of miRNA-1. * Final Concentration: 0.204 pM 10. Add 3 µL of 1 pM of miRNA-1. * Final Concentration: 0.122 pM 11. Add 1 µL of 1 pM of miRNA-1. * Final Concentration: 0.0408 pM Lettuce with Complement 1. In nuclease-free PCR tubes add the following triplicates: | | | --- | | Dye Control (Background) | * 25 μL water* 2.5 μL phi29 DNA polymerase buffer* 1 μL SplintR Ligase buffer || Lettuce Left + Lettuce Right + Dye | * 19 μL RNase-free water* 1 μL 10X Splint R Ligase Buffer* 2.5 μL 10X phi29 Reaction Buffer* 3 μL Split Lettuce Left Sequence (100 uM stock)* 3 μL Split Lettuce Right Sequence (100 uM stock) | | Complement + Lettuce Left + Lettuce Right + Dye | * 17.5 μL RNase-free water* 3 μL Split Lettuce Left Sequence (100 uM stock)* 3 μL Split Lettuce Right Sequence (100 uM stock)* 1.5 μL complement* 1 μL 10X Splint R Ligase Buffer* 2.5 μL 10X phi29 Reaction Buffer | - Centrifuge the reagents- On the sides of the centrifuged PCR tubes, carefully add 1.5 μL of DFHBI-1T (100 uM stock)- For controls without the dye, repeat steps 1-3 were repeated with 1.5 μL RNase-free water being added in place of the dye.- Vortex and centrifuge reagents.- Incubate the reaction at 70°C for 5 minutes, then cool to and incubate at 25°C for 1 hour.- Read fluorescence on the plate reader. The emission spectra is 528 nm, while the excitation spectra is 480 nm. Lettuce with RCP 1. In nuclease-free PCR tubes add the following triplicates: | | | | --- | --- | | Dye Control (Background) | * 25 μL water* 2.5 μL phi29 DNA polymerase buffer* 1 μL SplintR Ligase buffer | | Lettuce Left + Lettuce Right + Dye | * 19 μL RNase-free water* 1 μL 10X Splint R Ligase Buffer* 2.5 μL 10X phi29 Reaction Buffer* 3 μL Split Lettuce Left Sequence (100 uM stock)* 3 μL Split Lettuce Right Sequence (100 uM stock) | | Complement + Lettuce Left + Lettuce Right + Dye | * 17.5 μL RNase-free water* 3 μL Split Lettuce Left Sequence (100 uM stock)* 3 μL Split Lettuce Right Sequence (100 uM stock)* 1.5 μL complement* 1 μL 10X Splint R Ligase Buffer* 2.5 μL 10X phi29 Reaction Buffer | 2. Centrifuge the reagents 3. On the sides of the centrifuged PCR tubes, carefully add 1.5 μL of DFHBI-1T (100 uM stock) * For controls without the dye, repeat steps 1-3 were repeated with 1.5 μL RNase-free water being added in place of the dye. 4. Vortex and centrifuge reagents. 5. Incubate the reaction at 70°C for 5 minutes, then cool to and incubate at 25°C for 1 hour. 6. Read fluorescence on the plate reader. The emission spectra is 528 nm, while the excitation spectra is 480 nm.Linear DNA Probes with Complement 1. Add the following to an amber microcentrifuge tube to make FAM-Probe Mastermix: | | | | --- | --- | | FAM Dye Tagged Linear DNA Probe Mastermix | * Volume of FAM Dye Tagged Linear DNA Probe: (# of reactions) x (1.6 µL FAM Dye Tagged Linear DNA Probe) x 1.1 * Volume of TE: (# of reactions) x (29 µL TE) x 1.1 | 2. Add the following to a PCR tube: | | | | --- | --- | | Tris-EDTA Buffer (Background) | * 34 µL TE Buffer | | FAM Dye Tagged Linear DNA Probe + TE | * 1.6 µL BHQ-1 Quencher Tagged Probe * 32.4 µL TE Buffer | | BHQ-1 Quencher Tagged Linear DNA Probe + TE | * 1.6 µL BHQ-1 Quencher Tagged Probe * 32.4 µL TE Buffer | | FAM Dye Tagged Linear DNA Probe + BHQ-1 Quencher Tagged Linear DNA Probe + Linear Probe Complement + TE | * 29.4 µL Mastermix * 1.6 µL BHQ-1 Quencher Tagged Probe * 3 µL Linear Probe Complement | 3. Vortex several seconds and spin down tubes. 4. Place tubes in thermocycler at 41°C for 1 minute. 5. Place tubes in thermocycler 37°C for 1 minute. 6. Pipette all 32 μL of solution into a 384 well plate to measure fluorescence at excitation wavelength of 480 nm and emission intensity at 518 nm using a plate reader.Linear DNA Probes with RCP 1. Add the following to an amber microcentrifuge tube to make FAM-Probe Mastermix: | | | --- | | FAM Dye Tagged Linear DNA Probe Mastermix | * Volume of FAM Dye Tagged Linear DNA Probe: (# of reactions) x (0.4 µL FAM Dye Tagged Linear DNA Probe) x 1.1* Volume of TE: (# of reactions) x (29 µL TE) x 1.1 | | | | | --- | --- | | Tris-EDTA Buffer (Background) | * 34 µL TE Buffer | | FAM Dye Tagged Linear DNA Probe + TE | * 29.4 µL Mastermix* 4.6 µL TE Buffer | | BHQ-1 Quencher Tagged Linear DNA Probe + TE | * 1.6 µL BHQ-1 Quencher Tagged Probe* 32.4 µL TE Buffer | | FAM Dye Tagged Linear DNA Probe + BHQ-1 Quencher Tagged Linear DNA Probe + Padlock Probe + miRNA | * 29.4 µL Mastermix* 1.6 µL BHQ-1 Quencher Tagged Probe* 3 µL Padlock + miRNA Solution | | FAM Dye Tagged Linear DNA Probe + BHQ-1 Quencher Tagged Line0ar DNA Probe + Rolling Circle Product + TE | * 29.4 µL Mastermix* 1.6 µL BHQ-1 Quencher Tagged Probe* 3 µL Linear Probe Rolling Circle Product | - Vortex several seconds and spin down tubes.- Place tubes in thermocycler at 41°C for 1 minute.- Place tubes in thermocycler 37°C for 1 minute.- Pipette all 32 μL of solution into a 384 well plate to measure fluorescence at excitation wavelength of 480 nm and emission intensity at 518 nm using a plate reader. ![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/rct-icon.svg) Rolling Circle Transcription (RCT) Overview of Steps 1. Ligation Reaction 2. RCT Reaction 3. Aptamer Reaction1. Ligation Reaction 1. Pipette 6.5 μL of RNase-free water into RNase-free PCR tube 2. Add 1 μL of 10x Ligation Buffer 3. Add 0.5 μL of cDNA (padlock) stock and 1.0 μL of miR-1 stock (Stock: 1,000 pM diluted ten-fold) 4. Incubate the mixture at 65°C for 3 minutes 5. Cool the mixture to 25°C over 10 minutes 6. Add 25U (1.0 μL) of Splint R ligase 7. Incubate the mixture at 37°C for 2 hours 8. Incubate the mixture at 65°C for 20 minutes to inactivate the Splint R ligase2. RCT Reaction 1. Pipette 16.3 μL of RNase-free water into ligation product 2. Pipette 2 μL of 10x transcription buffer 3. Add the following to the ligation product: * 40 U (0.8 μL) T7 RNA polymerase (from Stock: 50,000 units/mL) * 20 U (0.5 μL) RNase inhibitor (from Stock: 40,000 units/mL) * 0.4 μL NTPs (Stock: 25 mM) 4. Incubate the mixture at 37°C for 8 hours 5. Inactivate T7 RNA polymerase at 70°C for 10 minutes3. Aptamer Reaction 1. Directly add 40 μM of DFHBI-1T to the reaction mixture 2. Read fluorescence values using a plate reader. The emission spectra is 528 nm. The excitation spectra is 480 nm. ![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/experiments-icon-characterization.svg) Characterization Aptamer Characterization Protocol 1. Grow biosensor cells in liquid culture tubes with 5 mL LB and 5 μL antibiotic for 24 hours shaking at 170 RPM at 37°C. 2. Add 5 μL of culture cells into Erlenmeyer flasks with 50 mL LB and 50 µL antibiotic and grow for an additional 24 hours shaking at 170 RPM at 37°C. 3. Ensure that the cells are at an OD600 value between 0.4 and 0.8 using a spectrophotometer. * If the OD600 value is less than 0.4, grow cells for longer, checking the value every 20-30 minutes. * If cells have grown too much, dilute using LB until the concentration matches the desired OD600 value. 4. Add 3.5 mL of the cell culture into 15 mL liquid culture tubes. 5. Add 500 µL of diluted IPTG solution (0, 1, 10, 100 µM) . 6. Grow biosensor cells for an additional 2-4 hours shaking at 170 RPM at 37°C. 7. Add DFHBI to a final concentration of 200 µM. 8. Pipette 150 µL of the grown biosensor cells into a well plate to measure cell density (OD600) and fluorescence at excitation wavelength of 492 nm and emission intensity at 501 nm using a plate reader.Linear DNA Probes Characterization Protocol 1. Add the following to an amber microcentrifuge tube to make FAM-Probe Mastermix: * # of reactions x 1.6 μL= μL Volume of 1 mM FAM Probe * # of reactions x 29 μL = μL Volume of TE Buffer (pH 7.5 - 8) 2. Add the following to a PCR tube: * 29.4 μL Mastermix * 1.6 μL of 1 mM BHQ-1 Probe * 1 μL of diluted complement DNA solution (0, 0.1, 1, 10, 100 μM) 3. Vortex several seconds and spin down tubes. 4. Place tubes in thermocycler at 41°C for 1 minute. 5. Place tubes in thermocycler at 37°C for 1 minute. 6. Pipette all 32 μL of solution into a 384 well plate to measure fluorescence at excitation wavelength of 480 nm and emission intensity at 518 nm using a plate reader. ![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/experiments-icon-proof-of-concept.svg) Proof of Concept Overview of Steps 1. Serum Spiking 2. miRNA - DNA Hybridization and Ligation 3. RCA 4. Readout1. Serum Spiking 1. Add the following in a PCR tube: * 17.6 μL of pooled human serum * 0.4 μL RNase inhibitor * 2 μL of 10nM miRNA2. miRNA - DNA Hybridization and Ligation 1. Pipette 1 μL of NEB 10 x Ligation Buffer into new PCR tubes. 2. Add 2.5 μL of 20 nM padlock probe. Add 1 uL of the serum. 3. Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes. 4. Add 1 μL of Splint R Ligase and 0.5 μL of RNAse inhibitor into the sample. 5. Incubate the reaction for 2 hours at 37°C. 6. Bring reaction up to 65°C for 20 minutes to terminate the reaction.3. RCA Reaction 1. Pipette 2.5 μL of NEB 10x phi29 buffer into previous ligation reaction mixture. 2. Add 12.5 units or 1.25 μL of phi29 DNA polymerase. Add 1 μL of aluminum. 3. Add 6.76 μL of 10 mM dNTPs. 4. Incubate the PCR tube for 8 hours at 37°C. 5. Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase.4. Readout 1. Add 10 μL of reaction + 2 μL of loading dye into prepared gel.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block image %}https://static.igem.wiki/teams/4245/wiki/results-header.png{% endblock %}
{% block lead %}All Results in One Place{% endblock %}
{% set list = [["RCA", "div-rca"],["RCT", "div-rct"],["Proof of Concept", "div-poc"],["Micro-Q", "div-mq"]] %}
{% set footerlist = [["Model", "Drylab", "model", "https://2022.igem.wiki/lambert-ga/model"], ["Human Practices", "Human
Practices", "hp", "https://2022.igem.wiki/lambert-ga/human-practices"]] %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/experiments-rca-icon.svg)

## Rolling Circle Amplification (RCA)

### SYBR™ Safe Experimentation

Our team ran the first round of experimentation to determine the success of our miRNA-1 biosensor in an in vitro reaction. Our goal was to determine the success of the ligation and rolling circle amplification. Instead of using a molecular beacon-based reporting mechanism, we utilized a SYBR™ Safe-based fluorescence readout to limit our reaction to the ligation and RCA steps.

Additionally, we ran three triplicates of controls: the first with the padlock probes, second with the miRNA, and the last with both the padlock and the miRNA. To analyze the results, we added SYBR™ Safe to all the samples and read the results in a plate reader.

We found one sample that showed some improvement over our controls. The plate reader’s readout from sample A6’s readout was 9899.64 which showed a significant increase from our third control which averaged 103.39. However, due to the lack of consistency, these results also conveyed the fact that either our padlock likely did not produce a successful RCP, or the SYBR safe detection mechanism did not succeed.

### Gel Experimentation

After our first round of experimentation, we could not successfully detect a long strand of DNA. Therefore, we decided to run our RCP (rolling circle product) on a gel in order to determine if our output is a very long DNA strand.

By analyzing the results on the gel, our team concluded that a very long strand of DNA, likely the RCP, was produced (see Fig. 1). The gel exhibited a fluorescent band of DNA very close to the well, which indicates that a long strand of DNA, greater than 1 kB, was produced due to our reaction. As a result, we can infer that the RCA reaction allowed the creation of a really long DNA stand — our RCP.

![](https://static.igem.wiki/teams/4245/wiki/updatedwetlab/screen-shot-2022-10-12-at-1-47-05-am.png)

Figure 1. Gel electrophoresis of RCA reactions, depicting bands greater than 1 kB.

### Lettuce with Complement

From these results, we saw an increase in fluorescence with the presence of simulated RCP and lettuce in the reaction as compared to the controls (see Fig. 2). This shows that the lettuce aptamer is actually binding to the RCP and binding the dye causing fluorescence. A significant decrease in fluorescence is also observed in the lettuce reaction tube compared to the dye background. This decrease is most likely due to the opacity of the lettuce DNA sequences. However, overall, the lettuce with complement reaction greatly increased in fluorescence compared to the controls.

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updated-figure-2.png )

Figure 2. Lettuce with Complement Results with SplintR buffer.

### Lettuce with Rolling Circle Product (RCP)

The results of the split Lettuce reaction in conjunction with RCP suggested that this reporter mechanism can successfully correlate the presence of a specific miRNA to an increase in fluorescence through RCA and the Lettuce aptamer (see Fig. 3).

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updated-figure-3.png)

Figure 3. Graph of fluorescence from left to right: 1) water + dye control, 2) split lettuce + dye control, 3) split lettuce + RCP + dye, 4) split lettuce + padlock&miRNA control + dye. The results display a significantly increased fluorescence of the reaction with RCP and split lettuce as compared to the controls, indicating that the split lettuce successfully binded the RCP and induced fluorescence of DFHBI-1T.

### Linear Probes with Complement

We initially tested linear probes with the complement of the middle sequence to ensure that linear probes were an effective and characterizable means of quantifying miRNA. Figure 4 displays a significant decrease in the fluorescence intensity of a triplicate with FAM Probe, BHQ Probe, and Linear Probe Complement as compared to a triplicate of just FAM tagged Probes. Therefore, we concluded that linear probes were an efficient means of reporting the output of our biosensor.

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updated-figure-4.png)

Figure 4. Linear Probe Complement Fluorescent Readout

In order to quantify the relationship between linear probe complement concentration and fluorescence, we further characterized these parts with varying linear probe complement concentrations. There is a negative logarithmic correlation between the complement concentrations ranging from 0.1-100 mM and the relative fluorescence units (RFU) (see Fig. 5). The 0 mM complement concentration outputs less RFU than 0.1 mM, which does not align with the model. However, the large error bars at 0 mM suggests that there was some degree of significant error. Thus, this data point is insignificant and further trials should be performed to achieve more accurate results. Moreover, the data from 0.1-100 mM closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 6) correlating complement concentration to RFU (see [Model](https://2022.igem.wiki/lambert-ga/model)).Therefore, the overall data collected depicts an accurate relationship between the complement concentration and RFU.

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updated-figure-5.png)

Figure 5. Characterization curve for parts BBa\_K4245130 and BBa\_K4245132 showing a negative logarithmic relationship between RFU and complement concentrations ranging from 0.1-100 μM. Note: 0-0.1 μM shows positive relationship, but large error bars at 0 μM suggest this was due to faulty pipetting.

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updated-figure-6.png)

Figure 6. Deterministic ODE Model Simulation of RFU output dependent on concentration of linear DNA probe complement concentration.

### Linear DNA Probes with RCP

We use linear probes as a means to quantify and report the miRNAs that we sensed through rolling circle amplification (RCA) reactions. We confirmed their efficiency through related experiments. As shown by Figure 7, there is statistically significant decrease in the fluorescent output of a triplicate with FAM Probe, BHQ Probe, and RCP as compared to a triplicate of just FAM tagged Probes. This confirms that we did produce our desired RCP in the RCA reaction for our miRNA-1-3p and miRNA-133a-3p sensors and that this mechanism was an effective reporting method for our sensor.

![this is an image]( https://static.igem.wiki/teams/4245/wiki/results-updated-figure-7.png)

Figure 7. Fluorescent Read of Rolling Circle Product for miRNA-133-3p and miRNA-1-3p

In order to quantify the relationship between miRNA concentration and fluorescence, we further characterized these parts with varying linear probe complement concentrations. There is a negative logarithmic correlation between the complement concentrations and the relative fluorescence units (RFU) (see Fig. 7. Moreover, the data shown above closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 8) correlating complement concentration to RFU (see [Model](https://2022.igem.wiki/lambert-ga/model)).Therefore, the overall data collected depicts an accurate relationship between the miRNA concentration and RFU, further validating that RCA coupled with linear probes are an effective and efficient means of quantifying miRNA concentrations.

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updated-figure-8.png)

Figure 8. Characterization curve for showing a negative logarithmic relationship between RFU from linear DNA probes and miRNA concentrations

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updted-figure-9.png)

Figure 9. Deterministic ODE Model Simulation of RFU output dependent on miRNA concentration.

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updated-figure-10.png)

Figure 10. Figure 10. Linear DNA Probe Fluoresence from Serum Extracted miRNA-1-3p Rolling Circle Amplification. Results show significant decrease in fluorescence, indicating a successful Proof of Concept.

As shown by Figure 10, there is statistically significant decrease in the fluorescent output of a triplicate with FAM Probe, BHQ Probe, and RCP as compared to a triplicate of just FAM tagged Probes. This confirms that we did produce our desired RCP in the RCA reaction performed on our miRNA-1-3p spiked serum. This further validates that biosensors utilizing RCA coupled with FAM/BHQ-1 linear DNA probes is an effective sensing and reporting mechanism for miR-1-3p.

![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/rct-icon.svg)

## Rolling Circle Transcription (RCT)

Lambert iGEM attempted Rolling Circle Transcription but was unsuccessful. The products of RCT did not appear on a 1% agarose gel, shown in Figure 11, as it did with the RCA reaction gels, Figure 1, for example. To test if the secondary structure of the RCT padlock probe from the Broccoli aptamer complement was problematic to the transcription process by T7 RNA polymerase, we ran RCA with the RCT padlock probe. The results of a gel electrophoresis showed that RCA was successful with the RCT padlock probe, revealing that the folding of the RCT padlock probe is likely not the major problem (see Fig. 12). Considering the possibility that the RNA products may have degraded while loading and running RCT products on a gel, Lambert iGEM decided to continue testing RCT with the DFHBI-1T dye.

![](https://static.igem.wiki/teams/4245/wiki/rca-updated-figure-6.png)

Figure 11. Picture of RCT products run on a 1% agarose gel. There were no visible bands that indicate the production of long RNA strands.

![](https://static.igem.wiki/teams/4245/wiki/rca-updated-figure-7.png)

Figure 12. Picture of RCA gel products run with the miR-1 RCT padlock probe. There were visible bands near the wells that indicate the production of long DNA strands.

To test the efficacy of the miRNA-Broccoli-spacer design incorporated into the RCT padlock probe, the sequence ([BBa\_K4245210](http://parts.igem.org/Part%3ABBa_K4245210)) was transformed, cloned, mini-prepped, and tested in a cell-free reaction. As seen in Figure 12, the results showed a significant increase in fluorescence in reactions containing the miRNA-Broccoli-spacer coding plasmid, revealing the potential of the use of fluorescent aptamers through RCT. However, when we added DFHBI-1T, the fluorophore that the Broccoli aptamer uses to induce fluorescence, to the RCT products, there was no significant increase in fluorescence observed as compared to the controls (see Fig. 13). Therefore, Lambert iGEM discontinued the experimentation with RCT and focused on improving and testing RCA.

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updated-figure-13.png)

Figure 13. Graph of fluorescence output of miR-Broccoli-spacer produced through cell-free Biobits. When we added the miR-Broccoli-spacer plasmid to the cell-free reaction, the fluorescence was significantly greater than without the plasmid.

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updated-figure-14.png)

Figure 14. Graph of fluorescence output before and after the addition of DFHBI-1T to RCT products and controls.

![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/poc-sample-testing.svg)

## Proof of Concept (POC)

![this is an image](https://static.igem.wiki/teams/4245/wiki/untitled-5.png)

Figure 15. Linear DNA Probe Fluoresence from Serum Extracted miRNA-1-3p Rolling Circle Amplification. Results show significant decrease in fluorescence, indicating a successful Proof of Concept.

As shown by Figure 15, there is statistically significant decrease in the fluorescent output of a triplicate with FAM Probe, BHQ Probe, and RCP as compared to a triplicate of just FAM tagged Probes. This confirms that we did produce our desired RCP in the RCA reaction performed on our miRNA-1-3p spiked serum. This further validates that biosensors utilizing RCA coupled with FAM/BHQ-1 linear DNA probes is an effective sensing and reporting mechanism for miR-1-3p.

![](https://static.igem.wiki/teams/4245/wiki/icons-for-wiki/saftey-hardware-software.svg)

## Micro-Q

Micro-Q was tested using fluorescein from concentrations 0 - 500 µM. In order to determine its efficacy compared to a commercial fluorometer, we measured triplicates of several concentrations with sample sizes of 100µL in a plate reader and in Micro-Q. Since Relative Fluorescence Units (RFU) are relative, we scaled the output of Micro-Q to match the scale of the output from the Plate Reader so that they can be compared (see Fig. 16). In order to have an accurate comparison of the data from the Plate Reader and Micro-Q, we added points at the origin to our data set, and calculated a slope from a linear regression for each measurement device, now assuming a y-intercept of 0. The slopes of the data are ~0.9473 and ~0.9194 from Micro-Q, respectively, achieving a percent error of -0.02952%.

![this is an image](https://static.igem.wiki/teams/4245/wiki/mq-processed-compiledvs.png)

Figure 16. Scatter plot showing measurements from Plate reader and Micro-Q at different concentrations of fluorescein.

Micro-Q was also compared against a Plate Reader in the quantification of [BBa\_J428112](http://parts.igem.org/Part%3ABBa_J428112) DH5-alpha and BL21 using the iGEM InterLab
Experiment 1 Protocol. The InterLab study instructs to quantify the sample at a 0 hour and 6 hour time mark. To
better align with members’ schedules, we only quantified fluorescence at a 0 hour time mark. As expected, BL21
exhibited statistically greater amounts of fluorescence than DH5-alpha (see Fig. 17). Moreover, to evaluate the
accuracy of our hardware component, we quantified the same samples in both plate reader and Micro-Q. The values
gathered from Micro-Q were scaled up by a factor of 104 to be comparable with those of the plate reader. The
fluorescence/OD600 values are consistent between MicroQ and plate reader at 6766.657 and 7405.837 for DH5-alpha, and
16916.56 and 21328.17 for BL21. When comparing fluorescence in each cell strain individually, the error bars for
MicroQ and plate reader overlap (see Fig. 17). Therefore, the outputs of MicroQ and plate reader are not
significantly different, validating the fluorescence measurement of our hardware device.

![this is an image](https://static.igem.wiki/teams/4245/wiki/results-updated-figure-17.png)

Figure 17. Graph depicting characterization of BBa\_J428112 in DH5-alpha and BL21 quantified in both Micro-Q and Plate Reader. The error bars between Micro-Q and Plate Reader do overlap, suggesting there is not a statistically significant difference.

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