iGEM 2019 — Team SASTRA_Thanjavur

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# Team:SASTRA Thanjavur/Notebook

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* Project

  [Description](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Description)
  [Design](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Design)
  [Parts](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Parts)
  [Judging](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Judging)
* Lab Work

  [Results](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Results)
  [Experiments](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Experiments)
  [Safety](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Safety)
  [Notebook](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Notebook)
* Modelling

  [Biomarker Panel Development](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Model#omics)
  [Toehold Design](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Model#toehold)
  [Reaction Modelling](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Model#mak)
* Software

  [ML Problem](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Software#ml)
  [Feature Engineering](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Software#fe)
  [Predictive Modelling](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Software#pfs)
* Human Practices

  [Silver](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Silver)
  [Engagement](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Public_Engagement)
  [Integrated Human Practices](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Integrated_Human_Practices)
  [Activities](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Activities)
* People

  [Team](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Team)
  [Collaborations](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Collaborations)
  [Attributions](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Attributions)

# Notebook

Throughout our struggles and hard work, people came and people left, but one thing stood by our side - Our notebook. Errors, mistakes and perfect results - Our notebook has seen it all, documenting our entire journey, every step of the way. Here it is - Hope you enjoy reading it as much as we enjoyed writing it.

| Date | Description |
| --- | --- |
| 02-July | Preservation of E.coli MG1655 Culture for plasmid transfection |
| 09-July | Plasmid isolation by alkaline lysis method |
| 13-August | Planning and protocol discussion for PCR experiments |
| 15-August | Analysis of DNA Distribution kit for parts characterization - to study the effect of pH on GFP expression on the selected plasmid- pSB1C3 |
| 22-August | High efficiency transformation of pSB1C3 using NEB C2987I - protocol design |
|  | Preparation of experimental requirements and its sterilization |
|  | Requirements - Distilled water for preparation of antibiotics, LB agar, eppendorf vials & tip boxes |
| 23-August | Implementation of DNA resuspension protocol |
| 24-August | Plasmid transformation into the competent cells provided |
| 26-August | Repetition of transformation experiment due to poor transformation efficiency. |
|  | 1st PCR experiment - Toehold for miR20a |
| 28-August | Preparation of experimental requirements and its sterilization |
|  | Requirements - Distilled water for preparation of antibiotics, LB broth, LB agar, eppendorf vials & tip boxes |
| 29-August | Subculturing of the formed colonies of plasmid transformed competent cells |
| 30-August | Repetition of transformation experiment - We noticed some precipitation in the SOC broth and decided to repeat the experiment using LB broth and decreasing the RPM to 150. |
| 07-September | Fluorescence analysis of DH5 Alpha and the transfected cells (Strain 8) after 48 hours of incubation |
| 09-September | Trial 2 - Inoculation of colonies from Strain 8 and DH5 Alpha into freshly prepared LB broth |
| 10-September | Fluorescence analysis of the samples inoculated on 09-Sept, after 24 hours of incubation |
| 11-September | Trial 3 - Inoculation of colonies from Strain 8 and DH5 Alpha into freshly prepared LB broth |
|  | Fluorescence analysis of the samples inoculated on 09-Sept, after 6 hours of incubation |
| 12-September | Performed OD normalization to set the number of cells for fluorescence measurement after 24 hours |
|  | Fluorescence analysis of the samples inoculated on 09-Sept, after 24 hours of incubation |
|  | 2nd PCR experiment - Toehold for miR 21, miR 29 |
|  | Inoculation of Strain 8 in different CAMP (chloramphenicol) concentrations |
| 14-September | Performed OD normalization at 595nm to normalize the number of cells for fluorescence measurement after 48 hours |
|  | Fluorescence analysis of the samples inoculated on 12-Sept, after 48 hours of incubation |
|  | Planning and protocol discussion for pH study |
| 15-September | Preparation of LB broth media in varying pH (5, 6, 7, 8, 9) and sterilized |
|  | Preparation of 20 µg/mL of CAMP stock |
| 16-September | Trial 1 - Inoculated Dh5 Alpha and Strain 8 in varying pH media (with CAMP for Strain 8) |
|  | Preperation of LB broth media in varying pH (5, 6, 7, 8, 9) and sterilized |
| 17-September | Trial 1 - Fluorescence study after 24 hours of inoculation |
|  | Trial 2 - Inoculated Dh5 Alpha and Strain 8 in varying pH media (with CAMP for Strain 8) |
|  | 3rd PCR experiment - Toehold for miR20a and miR 21 (sample 1 in 3 dilution) |
| 19-September | Trial 2 - Performed OD normalization at 595nm to set the number of cells for fluorescence measurement after 48 hours |
|  | Trial 2 - Fluorescence analysis of the samples inoculated on 17-Sept, after 48 hours of incubation |
| 21-September | Preparation of LB broth media in varying pH (5, 6, 7, 8, 9) and sterlized |
| 22-September | Trial 3 - Inoculation of colonies from Strain 8 and DH5 Alpha into freshly prepared LB broth |
| 23-September | Trial 3 - Performed OD normalization to set the number of cells for fluorescence measurement after 24 hours |
|  | Trial 3 - Fluorescence analysis of the samples inoculated on 22-Sept, after 24 hours of incubation with LB broth as reference |
| 24-September | Trial 3 - Performed OD normalization at 595nm to set the number of cells for fluorescence measurement after 48 hours |
|  | Trial 3 - Fluorescence analysis of the samples inoculated on 22-Sept, after 48 hours of incubation with LB broth as reference |
|  | 4th PCR experiment - Toehold for miR20a and miR21 (1 uL sample directly without dilution) |
| 26-September | 5th PCR experiment - Toehold for miR20a and miR21 (1 uL sample directly without dilution) |
| 03-October | Trial 1 - Fluorescein Standard graph plot for instrument calibration & GFP reference (Auto gain and gain set to 70) |
| 04-October | Trial 2 - Fluorescein Standard graph plot for instrument calibration & GFP reference (Gain set to 65, 70, 90, 100) |
| 09-October | Purification of PCR product |
|  | Characterization of toehold for miR-200a ( Trial run 1) |
| 10-October | Quantification of purified PCR product |
|  | Characterization of toehold for miR-21 ( Trial run 2) |
| 14-October | Characterization of toehold for miR-21 ( Concentration and specificity Study). Performed by the sensor team. |
| 16-October | Characterization of toehold for miR-20a ( Concentration and specificity Study) Performed by the sensor team. |

![First slide](https://static.igem.org/mediawiki/2019/5/5b/T--SASTRA_Thanjavur--NotebookS1.jpeg)

The "blueprint" of the House of Wetlab and Sensors can be accessed here for your reference.

[House of Wetlab Notebook](https://static.igem.org/mediawiki/2019/d/d3/T--SASTRA_Thanjavur--WetlabNotebook.pdf)

[House of Sensors Noteboook](https://static.igem.org/mediawiki/2019/4/48/T--SASTRA_Thanjavur--SensorsNotebook.pdf)

Our Sponsors

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# Team:SASTRA Thanjavur/Experiments

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* Project

  [Description](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Description)
  [Design](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Design)
  [Parts](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Parts)
  [Judging](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Judging)
* Lab Work

  [Results](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Results)
  [Experiments](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Experiments)
  [Safety](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Safety)
  [Notebook](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Notebook)
* Modelling

  [Biomarker Panel Development](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Model#omics)
  [Toehold Design](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Model#toehold)
  [Reaction Modelling](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Model#mak)
* Software

  [ML Problem](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Software#ml)
  [Feature Engineering](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Software#fe)
  [Predictive Modelling](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Software#pfs)
* Human Practices

  [Silver](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Silver)
  [Engagement](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Public_Engagement)
  [Integrated Human Practices](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Integrated_Human_Practices)
  [Activities](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Activities)
* People

  [Team](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Team)
  [Collaborations](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Collaborations)
  [Attributions](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Attributions)

PROTOCOLS

Index

[High Efficiency Transformation](#trans)

---

[DNA Resuspension](#dna)

---

[PCR Protocol](#pcr)

---

[Characterization of BBa\_K608011](#cob)

---

[Characterization of BBa\_K3154001](#twogen)

High Efficiency Transformation Protocol by NEB - modified (NEB#29871)

**Materials:**

1. NEB#C2987I
2. LB medium
3. Crushed Ice
4. Microcentrifuge tubes
5. Pipettes and tips

**Procedure:**

1. Thaw a tube of NEB 5-alpha competent E.coli cells on ice. Gently mix the tube.
2. Transfer 50 µL of cells into an microcentrifuge on ice.
3. Add 3 µL of Plasmid DNA to the cell mixture. Carefully flick the tube to mix the cells and the DNA. (DO NOT VORTEX)
4. Allow the solution to remain undisturbed on ice for 30 minutes.
5. Heat shock at exactly 42 ℃ for 30 seconds. Do not mix the tube.
6. Place the tube on ice for 5 minutes.
7. Pipette 950 µL of room temperature LB into the mixture.
8. Incubate the mixture for 60 minutes at 37 ℃. Shake the tube at 250 rpm.
9. Spin the cell suspension at a low rpm and remove the supernatant. Resuspend the cell pellet in LB medium and plate the cell suspension on LB agar with a concentration of 35 µg/mL Chloramphenicol.
10. Incubate the plates at 37 ℃ for 24 hours to obtain colonies of the transformed cells.

DNA Resuspension Protocol (For BioBrick Bba\_K608011)

**Materials:**

1. DNA distribution plates
2. Distilled water
3. Chloramphenicol
4. Pipettes and tips

**Procedure:**

1. Punch a hole in the well containing the desired biobrick, through the foil cover.
2. Pipette 10 µL of distilled water into the well.
3. Mix the contents in the well gently, with a pipette.
4. Leave the plate undisturbed for 5 minutes.
5. Resuspension of the DNA is indicated by the presence of a red color due to the crescent dye.
6. Use the resuspended DNA to transform the competent cells using the high efficiency transformation protocol

PCR Protocol

To amplify the toehold construct for cell free reactions (miRNA 21 and 20a)

1. Add TE buffer to resuspend the gblock to your final desired concentration
2. Vortex it briefly to suspend it
3. It can be stored at -80 ℃ upto 2 years
4. Freeze thaw the master mix before usage
5. Dilute the gblocks to the required concentration using nuclease free TE buffer
6. Keep all reaction components on ice and add them to a PCR tube  and transfer the reactions immediately to the thermocycler
7. The thermocycler should be preheated to the denaturation temperature and the components must be well mixed before transferring
8. Allow the thermocycler to run to the set cycle parameters.
9. Run the PCR product on a 1.5% agarose gel and check the amplicon size
10. The product can be purified by using a purification kit and sequenced to confirm the amplification of gblocks

| **COMPONENTS** | **25 µL reaction** | **15 µL reaction** |
| --- | --- | --- |
| Q5 High fidelity 2X master mix (NEB kit) | 12.5 µL | 7.5 µL |
| 10 uM FP (VF2) | 1.25 µL | 0.75 µL |
| 10 uM RP (VR) | 1.25 µL | 0.75 µL |
| Template DNA (1ng) | 1 µL - 3µL | 1-3 µL |
| Nuclease free water | 6-9 µL | 6-9 µL |

**GRADIENT PCR (Temperature set up and no. of cycles)**

|  |  |
| --- | --- |
| **98℃**- denaturation (initial) | **30 seconds** |
| **98℃** | **10 s** |
| **62/63℃ -** annealing temperature | **20s** |
| **72℃-** extension | **30s** |
| **72℃ -**final extension | **3 minutes** |
| **4℃** | **∞** |

Characterization of Biobrick Bba\_K608011 pH experiment Protocol

**Materials:**

1. LB Broth
2. Centrifuge Tubes
3. Chloramphenicol
4. 1M NaOH
5. 35% HCl
6. Transformed E.Coli, containing Biobrick Bba\_K608011
7. pH meter, fluorescence spectrophotometer
8. Pipettes and tips

**Procedure:**

1. Prepare 300 mL of LB broth in distilled water.
2. Separate the LB broth into 6 different 50 mL centrifuge tubes, each containing 40 mL of the prepared broth.
3. Prepare 1N NaOH and 35% HCl. Adjust the pH of the broth in 5 of the tubes to the desired values of 5,6,7.2 (pH of standard LB broth), 8 and 9.
4. Keep a separate tube with 40 mL of the broth (with unaltered pH) to use as a control.
5. Autoclave the Tarson tubes.
6. Add Chloramphenicol to all the Tarson tubes to obtain a final antibiotic concentration of 20 µg/mL. This was done by initially creating a stock of 40 mg/mL.
7. Leave the LB Broth undisturbed overnight to check for contamination.
8. Separate the 40 mL into 4 different Tarson tubes containing 10mL each- 3 of them to perform the experiment in triplicates and 1 tube, kept aside with LB for dilution to adjust Optical Density before fluorescence measurement.
9. Inoculate the triplicate tubes at pH 5, 6, 7.2, 8 and 9 with the transformed E.Coli cells containing the biobrick
10. At this stage, each Tarson tube at pH values of 5, 6, 7.2, 8 and 9 contain:
    * 10 mL LB broth at the particular pH
    * Chloramphenicol- concentration of 20 µg/mL
    * Inoculated cells
11. Place the tubes in the orbital shaker at 37º Celsius for 48 hours until turbidity is visible.  Measure the OD (595nm) of the cultures grown under different pH values and normalize the OD to a constant value by diluting the samples with plain LB medium kept aside at that pH. (We used an OD value of 0.35, which was the OD obtained at pH 8).
12. Measure the fluorescence intensity of the cell solutions (triplicates), keeping uninoculated LB broth as the control. Excitation at 485 nm and Emission at 520 nm.

Characterization of Biobrick Bba\_K3154001 miR-21 2nd generation toehold switch

**Materials:**

1. 96 - U bottom fluorescent well plate
2. Toehold DNA
3. miR & anti-miR
4. NEB (#E6800 Pure Synthesis Kit)
5. Micropipette
6. Fluorescent plate reader

**Concentration study:**

1. Freeze thaw miR, anti-miR in ice for 5 mins.
2. Quick spin miR and anti-miR @1000rpm (<1min)
3. Resuspend the miR & anti-miR with T10E0.1 to obtain 100 µM stock solution

   |  |  |  |  |
   | --- | --- | --- | --- |
   | miR type | Volume of TE buffer added (mL) | Anti-miR type | Volume of TE buffer added (mL) |
   | 21 | 3.144 | 21 | 2.569 |
   | 20a | 2.896 | 20a | 3.116 |
   | 200a | 2.401 | 200a | 2.863 |
   | 29a | 2.532 | 29a | 2.592 |
4. Dilute the respective miR and anti-miR to prepare the working stock of 100 nM
5. Mix 100nM of miR & anti-miR solution of required volume (50 µL each)
6. Incubate for 2hrs at room temperature to facilitate hybridization of miR and anti-miR
7. Freeze thaw:

1. Solution A & Solution B from NEB #E6800 cell free protein expression kit
2. Toehold DNA obtained after PCR & purification with required DNA concentration (125 ng/µL)

8. Prepare the master mix containing solution A (10 µL) and solution B (7.5 µL)
9. Prepare the varying concentrations (100pM, 1nM, 10nM, 100nM) of the miR – anti-miR (MA) complex
10. Mix the following contents:

1. Master Mix = 17.5 µL
2. Toehold DNA = 1 µL **(21)**; 3 µL (33ng/µL)-4 µL (26ng/µL) **(20a)**
3. MA = 2 µL
4. Nuclease free water = X µL
Total = 30 µL

11. For specificity study, perform the same steps but add MA at a fixed concentration (100 nM in our case) that does not correspond to the respective toehold DNA. Example – add MA of 29a to Toehold DNA for miR 21.
12. Incubate the reaction systems at 37 °C and perform fluorescence intensity measurements at 2nd and 4th hr with the following settings:

1. Excitation Wavelength = 395 nm
2. Emission Wavelength = 520 nm
3. System Manual Gain = 100

References

1. <https://international.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987>
2. [http://parts.igem.org/Help:2018\_DNA\_Distribution](http://parts.igem.org/Help%3A2018_DNA_Distribution)
3. <https://international.neb.com/protocols/0001/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273>

Our Sponsors

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# Team:SASTRA Thanjavur/Results

[![](https://static.igem.org/mediawiki/2019/4/43/T--SASTRA_Thanjavur--LogoInverted.png)](https://2019.igem.org/Team%3ASASTRA_Thanjavur)

* Project

  [Description](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Description)
  [Design](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Design)
  [Parts](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Parts)
  [Judging](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Judging)
* Lab Work

  [Results](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Results)
  [Experiments](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Experiments)
  [Safety](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Safety)
  [Notebook](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Notebook)
* Modelling

  [Biomarker Panel Development](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Model#omics)
  [Toehold Design](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Model#toehold)
  [Reaction Modelling](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Model#mak)
* Software

  [ML Problem](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Software#ml)
  [Feature Engineering](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Software#fe)
  [Predictive Modelling](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Software#pfs)
* Human Practices

  [Silver](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Silver)
  [Engagement](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Public_Engagement)
  [Integrated Human Practices](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Integrated_Human_Practices)
  [Activities](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Activities)
* People

  [Team](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Team)
  [Collaborations](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Collaborations)
  [Attributions](https://2019.igem.org/Team%3ASASTRA_Thanjavur/Attributions)

RESULTS

Index

[Transformation](#council)

---

[Effect of pH](#arch)

---

[PCR](#hands)

---

[Switch Characterization](#wetlab)

---

![](https://static.igem.org/mediawiki/2019/4/42/T--SASTRA_Thanjavur--ResultsTitle.jpeg)

The House of Toeholds was able to characterize a second generation toehold switch for detecting the presence of hsa-miR-21-5p and hsa-miR-20a-5p, the differentially expressed miRNA biomarkers for early-stage detection of cervical cancer. Briefly, toehold switches are translational regulators that modulate the translation of a reporter protein (in our case, GFP). In the absence of the trigger RNA, the toehold switch is stable in its secondary structure thereby representing the OFF state of the switch as the ribosomes do not garner access to the RBS. On the contrary, in the presence of the trigger RNA, the switch linearizes owing to the hybridization between the participating RNA structures thereby promoting the translation of the reporter protein and representing the ON state of the switch.

Hence, the biosensor involving toehold switches as the sensing element will provide a fluorescence output, which is a function of the miRNA levels in the sample. But, in our case, since the chosen miRNAs have a stop codon in the open reading frame of the toehold switch construct, the use of a hybrid trigger element consisting of the biomarker and anti-miRNA is necessitated to prevent the stop codon problem. Hence, it is not required to expect a linear correlation between miRNA levels and the obtained fluorescence, which is what we obtained through our studies involving the second generation toehold switches.

We performed a cell-free characterization of our computationally designed second generation toehold switches, which is our novel BioBrick contribution to iGEM 2019 and the synthetic biology community. The performance of our switch was rudimentary and gave us preliminary results, which could possibly indicate the presence of the chosen miRNA biomarkers. We strongly believe that our BioBrick could be further enhanced with regards to its design features and sensor characteristics to produce an effective, patient compliant screening mechanism for a panel of cervical cancer biomarkers through a simple blood test.

As a part of our Bronze medal criteria, we performed a varying pH study on E.coli DH5alpha containing biobrick Bba\_K608011, and were able to conclude that pH 7.2 was the optimum pH for the growth of our transformants, followed closely by pH 8 and then pH 6.

Transformation of DH5-alpha competent cells

E.coli DH5 alpha cells (C2987I, New England Biolabs) were transformed with the plasmid pSB1C3 containing Bba\_K608011 to study the effect of pH conditions on the activity of the medium strength promoter, which could be correlated to the fluorescence intensity of the Green Fluorescence Protein (GFP) encoded by the biobrick.

![...](https://static.igem.org/mediawiki/2019/b/b5/T--SASTRA_Thanjavur--2R.jpeg)

##### Take 1

Take 1:

For the first transformation experiment, we observed one transformant colony for the control sample (pUC19 plasmid DNA) and one for the test sample (Bba\_K608011). Due to the very low transformation efficiency and discrepancies such as higher heat shock incubation temperature period and clump formation in the SOC cell growth media during the transformation process, we decided to repeat the experiment.

Take 2:

Steps were taken to ensure accurate heat shock incubation temperature and time period. Cells were grown in SOC medium and LB medium. We still observed clump formation in the SOC medium. As a result, we decided to utilize the transformants grown in LB medium for further experiments.

Take 3:

The transformation was repeated to further improve transformation efficiency. To visibly observe the transformed cells in the selection medium containing chloramphenicol antibiotic, an incubation period of 48 hours was required. The transformants were selected and the colonies were subcultured in a selection medium containing chloramphenicol antibiotic to maintain a pure culture of the transformed bacteria.

Effect of pH on the biobrick BBa\_K608011 activity

Optimization of Chloramphenicol Concentration

As the first step of our characterization experiment, we decided to optimize the chloramphenicol concentration for our DH5alpha cells, transformed with biobrick Bba\_K608011- consisting of a medium constitutive promoter, a medium RBS and Green Fluorescence Protein (GFP). We decided to vary the chloramphenicol concentrations in the LB broth as - 10 µg/mL, 20 µg/mL and 30 µg/mL. This was done to identify the most suitable concentration for the transformed cells to grow, without creating a stressful condition for the bacteria, all the while allowing optimal production of GFP, measured in spectrofluorometer. From our experiment, we concluded that 20 µg/mL was the optimal concentration for the growth of our transformed cells and decided to keep this as our standard concentrations for all further experiments.
![](https://static.igem.org/mediawiki/2019/0/0d/T--SASTRA_Thanjavur--R5.jpg)

Identifying the Optimal pH conditions for the growth of our transformed cells

Once the optimal chloramphenicol concentration was identified, we went on to characterize the biobrick Bba\_K608011 by identifying the ideal pH conditions under which our transformants can grow, with optimal expression of GFP. We varied the pH conditions of LB broth as- pH 5, 6, 7.2 (standard pH of LB Broth), 8 and 9 and allowed the transformants to grow under these conditions for 48 hours. After measuring the cell Optical Density (OD) at 595 nm and normalizing the cell OD to 0.35, we measured the fluorescence intensity of the cell solutions in a spectrofluorometer.
Our results indicate that the fluorescence intensity was maximum at pH 7.2 (The standard pH of LB broth). Surprisingly, the intensity at pH 8 did not vary significantly from the intensity at pH 7.2 while the intensity at pH 6 deviated much more drastically. At pH values of 5 and 9, we found that the cell growth was extremely low. As we expected, the absorbance at 595 nm of pH 5 and 9 broth with transformant inoculum were similar to that of control LB broth.
We were thus able to conclude that pH 7.2 and 8 were optimal conditions for the expression of our biobrick. On the other hand, pH 6 was found to be suboptimal for GFP production. We suspect that this variation in GFP intensity could be due to the effect of pH on the plasmid copy number and the binding affinity of proteins involved in the transcription and translation machinery, such as RNA polymerases and ribosomes.
![](https://static.igem.org/mediawiki/2019/3/38/T--SASTRA_Thanjavur--FinalpH.jpeg)

PCR - Standardization and Amplification of Toehold

![](https://static.igem.org/mediawiki/2019/c/cb/T--SASTRA_Thanjavur--R6PCR.png)

##### Run 1

PCR Run 1: Toehold for miR20a

Gradient PCR was set up to optimize the annealing temperature. The PCR products were then run on a 1.5% agarose gel and TBE buffer. The results were observed as follows:
Clear 1 kb bands were observed at all 6 temperatures (61-66°C). The size of our toehold construct is around 1 kb and hence, this PCR run proved to be a successful attempt. The annealing temperature was optimised to 63°C.

|  |  |
| --- | --- |
| 98°C | 30s |
| 98°C | 10s |
| 61°C-63°C/ 64°C-66°C | 20s |
| 72°C | 30s |
| 72°C | 3 mins |
| 4°C | ∞ |

PCR Run 2 - Toehold for miR21 and miR29

The PCR reaction was set up for three temperatures (62-64°C). A gradient PCR was set up as the toeholds for miR21 and miR29 were amplified as opposed to toehold for miR20a in the first run. No definite bands were observed in 1.5% agarose gel. This could be due to low concentration (1 in 100 dilution of the stock) of the template DNA used.

PCR Run 3 - Toehold for miR20a and miR21

The PCR reaction was set up with an annealing temperature of 63°C with more concentration of DNA (1 in 3 dilution of stock). Clear 1 kb bands were observed on increasing the sample concentration.

PCR Run 4 - Toehold for miR20a and miR21

The PCR reaction was set up with an annealing temperature of 63°C using 1 µL of the stock sample directly (no dilution was performed). Five reactions were pooled and results were observed in 1.5% agarose gel. Clear bands were observed.

PCR Run 5 - Toehold for miR20a and miR21

The PCR reaction was set up with an annealing temperature of 63°C using 1 µL of the stock sample directly (no dilution was performed). Five reactions were pooled and results were observed in 1.5% agarose gel. Clear bands were observed.Purification and Quantification of PCR product
The PCR product from PCR Run 4 and Run 5 were purified and quantified.
The PCR product from the two runs were pooled into three different tubes and purified.
Concentration of Toehold miR21 -
1 - 93.8 ng/µL
2 - 128.5 ng/µL
3 - 118.9 ng/µL
Concentration of Toehold miR20a -
1 - 26.5 ng/µL
2 - 30.7 ng/µL
3 - 33.7 ng/µL

Second Gen Toehold switch characterization

Sensor characterization was designed with toehold switch for hsa-miR-21-5p as the sensing element, miR21:antimiR21 complex as the analyte and the fluorescence of GFP reported as the sensor output. The working of the sensor follows the basic principle of second-generation toehold switches wherein upon binding of miR:antimiR complex, the toehold switch unfolds to its linear conformation exposing the ribosome binding site. This triggers the translation of reporter gene (GFP) following which its fluorescence could be measured. Thus, the concentration of analyte (miR21:antimiR21 complex) is expected to follow a linear relation with the GFP fluorescence intensity.

The fluorescent plate reader (Biotek Synergy H1 Multimode Reader) was calibrated using sodium fluorescein with varying concentration and the linearity plot was constructed with R2 = 0.99. The fluorescence was measured with excitation at 395 nm and emission at 520 nm with the instrument gain set at 100. We were quite intrigued initially on the obtained fluorescence values for excitation of GFP at 395 nm, because this excitation wavelength is a characteristic of the wt GFP while we had used a mut3b version of GFP which has its characteristic excitation wavelength at 485 nm. So, what we observe here is a blue shift of the absorption spectra, which could possibly be due to anything in the PURExpress Protein Synthesis kit that is interfering in the fluorescence phenomenon. We carried out a control study to validate this and we obtained fluorescence value of ~400 units for the kit solution alone. In addition, the wt and mut3b variants of the GFP vary from one another by 3 mutations in the sequence leading to a protonated & deprotonated variations of the chromophore. The ratio of the protonated and deprotonated chromophore species in the sample decides the excitation wavelength of the GFP, plausibly from our understanding. Hence, in our case, we obtained the maximum fluorescence intensity at 385 nm excitation with 520 nm being the emission wavelength, possibly due to protonation effects in the reaction system. This was fixed as the instrument parameters for the specificity and concentration study.

These instrumental parameters were set for all the following studies. The NEB (#E6800) kit was tested using the DHFR control provided by the manufacturer. The protein expression was confirmed by SDS PAGE using a standard protein ladder for 20 kDa.

Specificity of toehold for miR-21-5p:

In order to comment on the specificity of the proposed toehold switch, non-specific analytes such as miR-200a-3p and miR-29a-3p with a concentration of 100 nM each, were used as sources of interfering analytes (Figure 1). The readings at 4 h were taken into consideration as 2 h reading had less significant intensity and hence the trend could not be inferred. The values were normalized with the control wherein miR-200-3p showed a four-fold decrease in intensity while the miR-29a-3p had effectively ‘zero’ intensity in comparison to that of miR-21-5p. The minimal leakiness towards miR-200a-3p might be attributed to its sequence similarity with miR-21-5p in the region which binds to the trigger domain of the toehold switch. Thus, the toehold for miR-21-5p was found to be highly unobtrusive for miR-200-3p and miR-29a-3p implicating its specificity.

![](https://static.igem.org/mediawiki/2019/e/ef/T--SASTRA_Thanjavur--StudySpec.jpeg)

**Figure 1 – Specificity study of toehold for miR-21-5p**

Concentration study of toehold for miR-21-5p:

The concentration study was performed for 3-fold range of concentration (100 pM, 1 nM, 10 nM and 100 nM). The readings taken at 4 h were considered as the 2 h readings had less significant intensity for lower concentrations and hence the trend was not discernable. The graph illustrates an increasing trend from 100 pM to 10 nM and the intensity was found to saturate at 100 nM (Figure 2). This implicates the linear range to be between 100 pM and 10 nM. The limit of detection (LOD) was found to be 100 pM implying its responsiveness even at very low concentration. A calibration curve was plotted by taking log of concentration of miR21:antimiR21 complex normalized with 10 pM (Figure 3). The sigmoidal curve was seen to saturate at about 10 nM validating the linear range (R2 = 0.99). A heat map was also constructed to better substantiate the saturation and the expected linear range was corroborated.

![](https://static.igem.org/mediawiki/2019/2/2d/T--SASTRA_Thanjavur--ConcStudy.jpeg)
**Figure 2 – Concentration study of the toehold switch for miR-21-5p after after 4 h**
![](https://static.igem.org/mediawiki/2019/a/ae/T--SASTRA_Thanjavur--MapHeat.jpeg)
**Figure 3 – Heat map of fluorescence for concentration study of the toehold switch for miR-21-5p after 4 h**
![](https://static.igem.org/mediawiki/2019/1/16/T--SASTRA_Thanjavur--CalibCurve.jpeg)
**Figure 4 – Calibration curve for the biosensor with toehold switch for miR-21-5p as the sensing element**

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