iGEM 2017 — Team TecCEM

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:TecCEM/Notebook

IGEM\_TECCEM

[![](https://static.igem.org/mediawiki/2017/a/a6/TEC-CEM-logos.png)](https://2017.igem.org/Team%3ATecCEM)

* [Home(current)](https://2017.igem.org/Team%3ATecCEM)
* [Project](https://2017.igem.org/Team%3ATecCEM/Description)

  [Description](https://2017.igem.org/Team%3ATecCEM/Description)
  [Design](https://2017.igem.org/Team%3ATecCEM/Design)
  [Experiments](https://2017.igem.org/Team%3ATecCEM/Experiments)
  [Notebook](https://2017.igem.org/Team%3ATecCEM/Notebook)
  [Interlab](https://2017.igem.org/Team%3ATecCEM/InterLab)
  [Model](https://2017.igem.org/Team%3ATecCEM/Model)
  [Results](https://2017.igem.org/Team%3ATecCEM/Results)
  [Demonstrate](https://2017.igem.org/Team%3ATecCEM/Demonstrate)
  [Improve](https://2017.igem.org/Team%3ATecCEM/Improve)
  [Attributions](https://2017.igem.org/Team%3ATecCEM/Attributions)
* [Parts](https://2017.igem.org/Team%3ATecCEM/Parts)

  [Basic Parts](https://2017.igem.org/Team%3ATecCEM/Basic_Part)
* [Safety(current)](https://2017.igem.org/Team%3ATecCEM/Safety)
* [Human Practices](https://2017.igem.org/Team%3ATecCEM/PublicEngagement)

  [Silver HP](https://2017.igem.org/Team%3ATecCEM/HP/Silver)
  [Integrated and Gold](https://2017.igem.org/Team%3ATecCEM/HP/Gold_Integrated)
  [Public Engagement](https://2017.igem.org/Team%3ATecCEM/Engagement)
* [Awards](https://2017.igem.org/Team%3ATecCEM/software)

  [Applied Design](https://2017.igem.org/Team%3ATecCEM/Applied_Design)
  [Hardware](https://2017.igem.org/Team%3ATecCEM/Hardware)
  [Model](https://2017.igem.org/Team%3ATecCEM/Model)
  [Software](https://2017.igem.org/Team%3ATecCEM/Software)
* [Team](https://2017.igem.org/Team%3ATecCEM/team)

  [Team](https://2017.igem.org/Team%3ATecCEM/Team)
  [Collaborations](https://2017.igem.org/Team%3ATecCEM/Collaborations)

![](https://static.igem.org/mediawiki/2017/0/07/TEC-CEM-home_hojas_header.png)

Notebooks

# NOTEBOOK

This document contains all the laboratory work developed throughout this year.

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2016/e/ec/T--Imperial_College--LabBook.pdf)

IGEM\_TECCEM

![](https://static.igem.org/mediawiki/2017/0/0f/TEC-CEM-idt.png)

![](https://static.igem.org/mediawiki/2017/8/84/TEC-CEM-biolabs.png)

![](https://static.igem.org/mediawiki/2017/f/fc/TEC-CEM-mathworks.png)

![](https://static.igem.org/mediawiki/2017/3/3b/TEC-CEM-snapgeneLogo.png)

![](https://static.igem.org/mediawiki/2017/3/39/TEC-CEM-agilent.png)

![](https://static.igem.org/mediawiki/2017/6/63/TEC-CEM-donadora.png)

![](https://static.igem.org/mediawiki/2017/e/e7/TEC-CEM-logoItesm.png)

[![](https://static.igem.org/mediawiki/2017/1/1e/TEC-CEM-facebook.png)](https://www.facebook.com/igemteccem/)

[![](https://static.igem.org/mediawiki/2017/7/7d/TEC-CEM-instagram.png)](https://www.instagram.com/igemteccem/)

[![](https://static.igem.org/mediawiki/2017/7/74/TEC-CEM-twitter.png)](https://twitter.com/igemteccem)

[![](https://static.igem.org/mediawiki/2017/8/89/TEC-CEM-youtube.png)](https://www.youtube.com/channel/UClG-DbIbK6KBlFsepIX40jA/videos)

Mail:
igem.tec.cem@gmail.com

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:TecCEM/Experiments

IGEM\_TECCEM

[![](https://static.igem.org/mediawiki/2017/a/a6/TEC-CEM-logos.png)](https://2017.igem.org/Team%3ATecCEM)

* [Home(current)](https://2017.igem.org/Team%3ATecCEM)
* [Project](https://2017.igem.org/Team%3ATecCEM/Description)

  [Description](https://2017.igem.org/Team%3ATecCEM/Description)
  [Design](https://2017.igem.org/Team%3ATecCEM/Design)
  [Experiments](https://2017.igem.org/Team%3ATecCEM/Experiments)
  [Notebook](https://2017.igem.org/Team%3ATecCEM/Notebook)
  [Interlab](https://2017.igem.org/Team%3ATecCEM/InterLab)
  [Model](https://2017.igem.org/Team%3ATecCEM/Model)
  [Results](https://2017.igem.org/Team%3ATecCEM/Results)
  [Demonstrate](https://2017.igem.org/Team%3ATecCEM/Demonstrate)
  [Improve](https://2017.igem.org/Team%3ATecCEM/Improve)
  [Attributions](https://2017.igem.org/Team%3ATecCEM/Attributions)
* [Parts](https://2017.igem.org/Team%3ATecCEM/Parts)

  [Basic Parts](https://2017.igem.org/Team%3ATecCEM/Basic_Part)
* [Safety(current)](https://2017.igem.org/Team%3ATecCEM/Safety)
* [Human Practices](https://2017.igem.org/Team%3ATecCEM/PublicEngagement)

  [Silver HP](https://2017.igem.org/Team%3ATecCEM/HP/Silver)
  [Integrated and Gold](https://2017.igem.org/Team%3ATecCEM/HP/Gold_Integrated)
  [Public Engagement](https://2017.igem.org/Team%3ATecCEM/Engagement)
* [Awards](https://2017.igem.org/Team%3ATecCEM/software)

  [Applied Design](https://2017.igem.org/Team%3ATecCEM/Applied_Design)
  [Hardware](https://2017.igem.org/Team%3ATecCEM/Hardware)
  [Model](https://2017.igem.org/Team%3ATecCEM/Model)
  [Software](https://2017.igem.org/Team%3ATecCEM/Software)
* [Team](https://2017.igem.org/Team%3ATecCEM/team)

  [Team](https://2017.igem.org/Team%3ATecCEM/Team)
  [Collaborations](https://2017.igem.org/Team%3ATecCEM/Collaborations)

![](https://static.igem.org/mediawiki/2017/0/07/TEC-CEM-home_hojas_header.png)

Experiments

# Protocols

Chemocompetent cell preparation

### Preparation of chemocompetent cells

#### Materials

* LB Medium
* CaCl2 0.1 M
* CaCl2 0.1M + 15% glycerol
* Desired bacterial strain

#### Steps

1. Inoculate an isolated colony of the desired bacterial strain in a sterile tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
2. Inoculate 100 mL of LB broth with 1 mL of the previous culture (1:100 proportion).
3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
4. Place on ice for 10 minutes.
5. Divide the culture in sterile Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and incubate on ice for 10 minutes.
7. Centrifuge at 4000 rpm for 10 minutes.
8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and incubate on ice for 10 minutes.
9. Prepare 200 μl aliquots and store at -80 °C.

Chemocompetent cell transformation

### Chemocompetent cell transformation

#### Materials

* Chemocompetent cells
* Plasmid DNA
* LB or SOC medium

#### Previous Steps

* Prepare a water bath at 42°C.

#### Steps

1. Thaw a tube of competent cells on ice.
2. Add 5-10 μL of DNA (concentration between 1 pg/mL- 100 ng) to 125μL the competent cells.
3. Incubate the mixture in ice for 30 minutes.
4. Place in the water bath at 42°C for 30 seconds and immediately incubate on ice for 5 minutes.
5. Add 950 μL with SOC or LB medium under sterile conditions.
6. Incubate at 37°C for 60 minutes at 250 rpm.
7. Centrifuge at 5000 rpm for 2 minutes to obtain a pellet.
8. Discard 800 μL from the supernatant and resuspend the pellet in the remaining 200 μL.
9. Spatulate the volume on LB plates with the selection antibiotic and incubate for 16 hours at 37°C.

Annealing of oligonucleotides

### Annealing of oligonucleotides

#### Materials

* Oligonucleotides
* Injectable water
* NEBuffer 2.1

#### Previous Steps

* Prepare a water bath at 95°C.

#### Steps

1. Perform the corresponding calculations for a desired concentration (μg/μL) and the volume needed to use 5 μg of each oligonucleotide.
2. For each oligonucleotide, make a mix with 1X NEBuffer 2.1 for a total volume of 20 μl with the following materials:

* Injectable water
* NEBuffer 2.1
* Sense oligonucleotide
* Antisense oligonucleotide

3. Incubate the mixture at 95°C for 5 minutes on the water bath.
4. Leave in the water bath to cool down until room temperature is reached.
5. Place on ice until further use.

Plasmid digestion

### Plasmid digestion

#### Materials

* NEBuffer 2.1
* Restriction enzymes
* Injectable water

#### Steps

1. Make a Master Mix with the following:

* 18 μL of injectable water
* 5 μL of NEBuffer 2.1
* 1 μL of Enzyme 1
* 1 μL of Enzyme 2

2. Add 6 μL of the Master Mix to 6 μL of linearized plasmid in a sterile tube.
3. Incubate at 37° for 2 hours.
4. Inactivate in water bath at 80°C for 20 minutes.
5. Incubate on ice for immediate use or store at -20 oC.

Ligation

### Ligation

#### Materials

* Injectable water
* T4 DNA ligase buffer (10X)
* T4 DNA ligase
* Aligned oligonucleotides
* Digested linearized plasmid (25ng/μL)

#### Steps

1. Prepare a master mix (if necessary) with the following components:

* Digested plasmid
* Water °MB
* T4 DNA ligase
* T4 DNA ligase buffer

2. Distribute the master mix among the reaction tubes and place the oligonucleotides aligned to have a final volume of 10 μL per reaction.
3. Leave incubating at room temperature overnight and transform the next day.

Lowry Method por protein quantification

### Lowry Method for protein quantification

#### Standard curve protocol

1. To identify the protein concentration, quantify a BSA standard curve.
2. Preparation of samples with known proteins for a BSA stock (1mg/ml):

**Table 1.** Known protein concentrations with the volumes of each reactant for its preparation.

| Sample | BSA (μg) | BSA vol (1ug/ul) | H2O (ul) | C Reactant (ul) | Folin Reactant (ul) |
| --- | --- | --- | --- | --- | --- |
| 1 | 0 | 0 | 100 | 500 | 50 |
| 2 | 10 | 5 | 95 | 500 | 50 |
| 3 | 20 | 10 | 90 | 500 | 50 |
| 4 | 30 | 15 | 85 | 500 | 50 |
| 5 | 40 | 20 | 80 | 500 | 50 |
| 6 | 50 | 25 | 75 | 500 | 50 |
| 7 | 60 | 30 | 70 | 500 | 50 |
| 8 | 70 | 35 | 65 | 500 | 50 |
| 9 | 80 | 40 | 60 | 500 | 50 |
| 10 | 110 | 55 | 45 | 500 | 50 |
| 11 | 120 | 60 | 40 | 500 | 50 |

3. After the samples of each concentration are prepared, add 500 μl of solution “C” (Biuret reactant) to each one and incubate for 10 minutes at room temperature.
4. Add 50 μl of Folin reactant to each sample and then incubate 45 minutes at room temperature.
5. Add 150μl per sample in a 96-well plate and read absorbance.

#### Protein quantification for the curve sample protocol

1. Resuspend samples in lysis buffer RIPA (200μl).
2. Dilute 1:20 using 10μl of sample in 190μl of milliQ water.
3. Add 1ml of “solution C” to the diluted samples and incubate for 10 minutes at room temperature.
4. Add 10μl of Folin reactant per sample and resuspend. Incubate 45 min at room temperature.
5. In a 96 well plate add 150μl per sample and read absorbance at 660.

Plasmid extraction

### Plasmid extraction

#### Materials

* Transformed E. coli DH5a cells.
* LB + CAM
* STET Buffer
* Lysozyme
* RNAse A
* 3 M Sodium acetate
* Isopropanol
* 70% ethanol
* Injectable water

#### Steps

1. Seed an isolated colony of each transformed Escherichia coli strain in 10 mL of LB+CAM in a test tube and incubate for 16 hours at 37oC at 260 rpm.
2. Centrifuge all the culture in 2 mL sterile microtubes at 13,000 rpm for 1 minute in order to retrieve the pellet.
3. Remove the supernatant and wash the pellets without resuspending with 350μL of STET Buffer (0.1M NaCl, 10 mM TRIS-HCl pH 8, 1 mM EDTA pH 8 and Triton X-100 5%)
4. Resuspend with 350 μL of STET Buffer and add 25μL of fresh lysozyme (2 mg/mL).
5. Incubate for 4 minutes at room temperature, and then place in a water bath at 100oC for 1 minute.
6. Centrifuge at 12,000 rpm for 10 minutes and remove the pellet with a stick.
7. Add 5 μL of RNAse A and incubate for 20 minutes at 37°C .
8. Add 75 μL of sodium acetate 3M pH 5.2 and 250 μL of isopropanol and incubate for 10 minutes at room temperature.
9. Centrifuge at 12,400 rpm for 10 minutes and discard the supernatant.
10. Wash the pellet twice with 750 μL of 70% ethanol to eliminate salts.
11. Resuspend the pellet in 150 μL of water for injections.

SDS-PAGE

### SDS-PAGE

#### Steps

1. Resuspend each sample in Laemmli buffer (B-Mercaptoethanol, 0.1%, Bromophenol blue 0.0005% ,Glycerol, 10%, SDS (electrophoresis-grade) 2% and Tris-HCl, 63 mM (pH 6.8). Make the necessary calculations so all samples have the same amount of protein.
2. Homogenize samples with the polytron.
3. Place the samples on a water bath at 100oC for 5 minutes.

#### Pouring the resolving gel

1. Clean glass plates with soap and water, then with ethanol. Assemble the glass plates and spacers.
2. Once the gel holder has been assembled, it is verified that there is no leak, the boundary of the separating phase is then marked 0.5 cm below the teeth of the comb.
3. Prepare 10 mL of separating phase. Due to the size of RFP (26 kDa), 10% or 12% gel would do.

Table 1: Reagents required to prepare the separating phase in different % gels.

| Acylamide percentage | 6% | 8% | 10% | 12% | 15% |
| --- | --- | --- | --- | --- | --- |
| H2O | 5.2 ml | 4.6 ml | 3.8 ml | 3.2 ml | 2.2 ml |
| Acrylamide/Bis-acrylamide (30%/0.8% w/v) | 2 ml | 2.6 ml | 3.4 ml | 4 ml | 5 ml |
| 1.5M Tris (pH 8.8) | 2.6 ml | 2.6 ml | 2.6 ml | 2.6 ml | 2.6 ml |
| 10% w/v SDS | 0.1 ml | 0.1 ml | 0.1 ml | 0.1 ml | 0.1 ml |
| 10% w/v ammonium persulfate | 100 μl | 100 μl | 100 μl | 100 μl | 100 μl |
| TEMED | 10 μl | 10 μl | 10 μl | 10 μl | 10 μl |

4. Add 5 mL the separating phase mixture taking care not to exceed the previously marked limit.
5. So that the phase is even and without bubbles, add a layer of ethanol, which is not miscible with the above mixture.
6. When the separating phase has gelled, the ethanol is removed with the help of a filter paper.
7. Prepare 5 mL of the concentrating phase.

Table 2: Reagents required to prepare the concentrating phase.

| H2O | 2.97 ml |
| --- | --- |
| 0.5 M Tris-HCl (pH 6.8) | 1.25 ml |
| 10% w/v SDS | 0.05 ml |
| Acrylamide/Bis-acrylamide (30%/0.8% w/v) | 0.67 ml |
| 10% w/v ammonium persulfate | 0.05 ml |
| TEMED | 0.005 ml |

8. The concentrating phase is added and the comb is placed so that there are no empty spaces. After waiting until it gels, the comb is removed.
9. Place the gel in the electrophoresis, cover with the running buffer.
10. Load the samples and the molecular weight in the wells.
11. Run the gel at given conditions.
12. Stain with Coomassie Blue dye overnight.
13. Destain with Destaining Solution (30% Methanol, 7% acetic acid) for 30 minutes. After the time is up, replace the solution with fresh one. Repeat until the gel is visible.

Plasmid extraction with Monarch Miniprep Kit

### Plasmid extraction with Monarch ® Plasmid DNA Miniprep Kit from New England Biolabs TM

#### Previous step

* Prepare Plasmid Wash Buffer 2, add 4 volumes of ethanol (> 95%) to one volume of Buffer. Add 24 mL of ethanol for a final volume of (30 mL) of Plasmid Wash Buffer 2.

\*All centrifugations steps should be carried out at 13,000 rpm.

#### Steps

1. Pellet 10 ml bacterial culture by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in 400 μL plasmid Resuspension Buffer (B1). Vortex to ensure cells are completely resuspended. There should be no visible clumps.
3. Add 400μl Plasmid Lysis Buffer (B2), gently invert tube 5-6 times, and incubate at room temperature for 1 minute. Color should change to dark pink, and solution will become transparent and viscous. Do NOT vortex.
4. Add 800μl of Plasmid Neutralization Buffer (B3), gently invert tube until neutralized, and incubate at room temperature for 2 minutes. Sample is neutralized when color is uniformly yellow and precipitate forms. Do NOT vortex.
5. Centrifuge lysate for 10 minutes.
6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
7. Re-insert column in the collection tube and add 200 μL of Plasmid Wash Buffer 1. Centrifuge for 1 minute. Discarding the flow-through is optional.
8. Add 400 μL of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
9. Transfer column to a clean 1.5ml microfuge tube. Use care to ensure that the tip of the column does NOT come into contact with the flow-through. If there is any doubt, re-spin the column for 1 min.
10. Add 100 μl of injectable water to the center of the matrix. Wait for 1 minute, then spin for 1 min to elute DNA. A second eluted DNA was realized with 50 μl of water.

DNA band purification

### DNA band purification with GenElute TM Gel Extraction Kit from Sigma-Aldrich.

#### Materials

* Scalpel
* Ethanol 100%
* Isopropanol 100%
* 3M Sodium Acetate Buffer, pH 5.2
* Water bath at 60°C
* Molecular Biology water
* All the centrifuge are performed at 16,000 xg or 13,155 rpm

#### Previous steps

* Prepare the Wash Solution Concentrate G with 48 mL of EtOH 100%

#### Steps

1. Excise the DNA fragment of interest from the agarose gel with the scalpel.
2. Weigh the gel slice in a microtube.
3. For every 100 mg of agarose gel, add 300 mL of Gel Solubilization Solution.
4. Incubate the gel mixture at 50-60 °C for 10 minutes, or until the agarose has dissolved. Vortex briefly every 2-3 minutes during incubation to help dissolve the gel.
5. Prepare the binding column in a microtube of 2 mL, add 500 μL of the Column Preparation Solution, centrifuge for 1 min and discard flow-through liquid.
6. If the mixture is red, add 10 μL of Sodium Acetate Buffer, until the mixture turns yellow.
7. Add 1 gel volume isopropanol and mix.
8. Load the gel solution to the binding column. If the volume of the gel mixture is >700 mL, load the sample onto the column in 700 mL portions. Centrifuge for 1 minute after loading the column each time. Discard the flow-through liquid.
9. Add 700 μL of Wash Solution to the binding column, centrifuge 1 minute and discard the flow- through. Centrifuge again 1 minute.
10. Elute DNA, transfer the column to a new microtube, add 50 μL of Elute Solution, incubate for 1 minute and centrifuge 1 minute.

Polymerase Chain Reaction

### Polymerase Chain Reaction

#### Steps

1. Prepare the Master Mix for the PCR reaction adding

* 0.75μL 50 mM MgCl2
* 22. 5 μL PCR Buffer 10X
* 0.5 μL 10 mM dNTP Mix
* 1 μL 10 mM FWD Primer
* 1 μL 10 mM REV Primer
* 0.2 μL Taq DNA Polymerase
* 18.05 μL MBo Water

2. Add the Master Mix and 1 μL of DNA template for each reaction. Final volume: 45 μL per reaction.
3. Reactions are placed in the thermal cycler with the following program:

* 94°C for 5 minutes
* 31 cycles:

+ 94°C for 30 seconds
+ 55°C for 30 seconds
+ 72°C for 30 seconds

* 72°C for 5 minutes
* 12°C indefinitely

4. For PCR product analysis, an agarose gel electrophoresis must be performed.

\*Note: Number of cycles and alignment temperatures may vary in each case.

Schneider's Insect Medium preparation

### Schneider’s Insect Medium Preparation Protocol

#### Steps

1. Measure 80% of final volume of water at 15-20°C.
2. While gently stirring the water, add the powdered medium. Stir until dispersed.
3. Rinse original package with a small amount of water to remove all traces of powder. Add to solution in step 2.
4. To the solution in step 3, add 0.4 g of sodium bicarbonate for each liter of solution.
5. While stirring, adjust the pH to at least 9.2 ± 0.2 with 1N NaOH. Stir for a minimum of 10 minutes. Solution may become turbid.
6. While stirring, adjust the pH to 6.7 ± 0.2 with 1N HCl, solution will clear.
7. Prepare a calcium chloride solution by dissolving 0.6 g of anhydrous calcium chloride in 50 mL of tissue culture grade water for each liter of final volume being prepared. Slowly add the calcium chloride solution dropwise to the medium with rapid mixing to avoid precipitate formation.
8. While stirring, adjust the pH of the medium to 0.1-0.3 pH units below the desired pH with 1N HCl or NaOH.
9. Add additional water to bring the solution to final volume.
10. Sterilize immediately by filtration using a membrane with a porosity of 0.22 microns or less.
11. Aseptically dispense medium into sterile container.

Sequencing Biobricks

### Sequencing Biobricks

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2017/9/9b/TEC-CEM-Protocol_for_biobricks.pdf)

BSLA SiRNA confirmation protocol

### BSLA SiRNA confirmation protocol

#### Steps

1. Transform BSLA devices in HT115
2. Culture a blue colony of HT115 with BSLA devices for 2 days in liquid medium
3. Extract RNA (Without using RNAse)
4. Quantify the RNA samples
5. Prepare a hybridization buffer (30 mM Na2HPO4 pH 8, 0.3 M NaCl, 10 mM EDTA)
6. Place 16 μL fo the hybridization buffer in a 0.6 ml microtube and then add 4 μL of oligos (100 μM)
7. Add 2 μg of RNA sample
8. Mix the samples with the micropipette
9. Incubate the samples at 94°C for 5 minutes
10. Incubate the samples at 42°C for 60 minutes
11. Add 20 units of exonuclease 1
12. Incubate at 37°C for 30 minutes
13. Place the samples in ice or store they at -80°C until its use
14. Run a non-denaturing gel

# Experiments

RFP characterization curve for DH5a strain

### RFP characterization curve for DH5a strain

#### IPTG induction

1. Prepare the seed culture by resuspending an isolated colony of transformed Escherichia coli DH5a+BBa\_J04450 in 10 mL of liquid LB broth with chloramphenicol (25μg/mL) and incubate it at 37°C overnight.
2. Inoculate 100 mL of LB + CAM (25μg/mL) with 1 ml of the seed culture and incubate at 37°C and 260 RPM until the OD reaches 0.6.
3. Divide the culture in two different flasks (50 mL each one).
4. Induce one of the flasks with 1 mL of a 1 M stock of IPTG, resulting in a final concentration of 20 mM.
5. Divide the content of each flask in 8 glass tubes (1 per hour of the kinetics) with the objective of not interrupting the bacterial growth and incubated for 3 and a half hours at 37°C and 260 RPM.
6. Each hour, take a single tube from the incubator.
7. Use 100 μl to measure absorbance at 600 nm using the multiwell plate and 100 μl to measure fluorescence using the Synergy H1 Hybrid Multi-Mode Reader of BioTek. Program the excitation and emission wavelength at 550 nm and 584 nm respectively.
8. Divide the remaining volume in two different microtubes and centrifuge them at 5000 rpm for 2 minutes, and discard the supernatant.
9. Conserve the sample at -20°C for further analysis by Lowry Method and SDS-PAGE.
10. Repeat steps 7, 8 and 9 for each hour of the experiment.

Chitosan encapsulation

### Chitosan encapsulation

#### Materials

* Chitosan 0.15 g
* Tripoliphosphate 0.05 g
* Pluronic F-68 0.5 g
* H2O BM 100ml
* Acetic Acid 1 ml

#### Steps

1. Prepare a 1% acetic acid solution and adjust to pH 4 using NaOH.
2. Divide the solution in two 50 mL containers.
3. In one container, add and hydrate the chitosan and mix until dissolution.
4. Filter the chitosan using filter paper.
5. Add Pluronic F-68 to the filtered solution, hydrate and mix avoiding the formation of foam.
6. To the other 50 mL acetic acid 1% solution, add the tripolyphosphate and mix.

#### RacI siRNA encapsulation

1. Add 1.25 mL chitosan solution to a sterile 15 mL Falcon tube.
2. Add 250 uL of RacI siRNA 100 ng/uL.
3. Add 1.25 mL of tripolyphosphate solution.
4. Mix by vortex until the color changed from colorless to translucent white.
5. Store at 5°C.

#### Awd+Alexa siRNA encapsulation

1. Add 225 μL chitosan solution was added to a 15ml falcon tube.
2. Add 50 μL of Awd+Alexa siRNA 100 ng/μL.
3. Add 225 μL of tripolyphosphate solution.
4. Mix by vortex until the color changed from colorless to translucent white.
5. Store at 5°C.

Extraction of DNA from filter paper

### Extraction of DNA from filter paper

#### Steps

1. Cut out the area of the filter with spotted cDNA (circle marked on the paper).
2. Put the piece of paper in a sterile microtube.
3. Apply 75 µl of Water Molecular Biology Grade.
4. Vortex 30 seconds.
5. Centrifuge 1 min to elute the DNA and pellet the solution and paper.
6. Store the solution toghether with the filter paper at 4°C.
7. Transform E. coli with 1 µl of the plasmid DNA.

*Diaphorina citri* Total RNA Extraction

### Diaphorina citri Total RNA Extraction

#### Materials

* 20 *Diaphorina citri* live specimens
* Liquid nitrogen
* Trizol
* Chloroform
* RNaseOUT
* Isopropanol
* 75% ethanol
* Injectable water

#### Steps

1. Place 20 individuals at 4°C for 10 minutes.
2. Place on a mortar with liquid nitrogen, and crush with pestle until homogeneous.
3. Add 500 μL of Trizol and mix.
4. Transfer the mixture to a 1.5 mL Eppendorf tube. To ensure the transfer of all the material, any remnants in the mortar may be washed with an additional 300 μL of Trizol.
5. Leave the mixture for 5 minutes at room temperature to allow complete dissociation.
6. Add 160 μL of chloroform to the mixture and leave for 2 to 3 minutes.
7. Centrifuge at 12,000 g and 4°C for 15 minutes.
8. Transfer the aqueous phase to a new microtube and add 5 μL of RNAseOUT.
9. Add 400 μL of isopropanol and incubate for 10 minutes at room temperature.
10. Centrifuge at 10,000 g and 4°C for 10 minutes.
11. Discard the supernatant with a micropipette and resuspend in 800 μL of 75% ethanol.
12. Vortex the sample briefly.
13. Centrifuge at 7,500 g and 4°C for 5 minutes.
14. Discard the supernatant with a micropipette and let the pellet dry by placing the tube downwards on a clean sheet of paper.
15. Resuspend the pellet in 30 μL of injectable water.

Two-Step Reverse Transcriptase PCR

### Two-Step Reverse Transcriptase Polymerase Chain Reaction

#### Steps

1. Prepare the RNA using RevertAid H Minus First Strand cDNA Synthesis Kit from ThermoFisher:

* 3 μL (250 ng/ μL) of Diaphorina citri RNA
* 1 μL of Reverse primer (The mix was made using 10 μL of each primer)
* 8 μL of injectable water.

2. As only 38.5 % of D. citri genome is composed by GC’s the step of incubating the sample at 65°C for 5 minutes can be omitted.
3. Prepare the Mix for cDNA synthesis as follows:

Table 1: Preparation of the Master Mix using RevertAid H Minus First Strand cDNA Synthesis Kit.

| Reagent | 1X |
| --- | --- |
| Buffer 5X | 4 μL |
| Ribolock RNAse inhibitor (20 U/μL) | 1 μL |
| dNTPs Mix (10 μM) | 2 μL |
| Reverse Transcriptase (200 U/μL) | 1 μL |

4. Give a spin in the centrifuge.
5. Incubate at 52°C for 1 hour in the thermal cycler.
6. Prepare the Mix for PCR as follows:

Table 2: Preparation of the Master Mix for PCR.

| Reagent | 1X |
| --- | --- |
| Buffer 10X | 2.5 μL |
| MgCl2 10 mM | 0.75 μL |
| dNTP Mix 10 μM | 1 μL |
| Forward Primer (10 μM) | 1 μL |
| Reverse Primer (10 μM) | 1 μL |
| Taq DNA polymerase | 0.25 μL |
| D. citri cDNA | 2 μL |
| Injectible water | 14.5 μL |

7. Add the Master Mix to the different microtubes, and complete the volume to 25 μL with 1 μL of each corresponding primer.
8. Place the reactions in the thermal cycler in the following conditions:

* 94°C for 3 minutes
* 40 cycles:

+ 94°C for 30 seconds
+ 54°C for 30 seconds
+ 72°C for 20 seconds

* 72°C for 5 minutes
* 12°C indefinitely

9. A 2.5%-3% agarose gel electrophoresis is required to verify PCR products.

Diaphorina citri siRNA soaking

### Diaphorina citri siRNA soaking

#### Steps

1. Collect 10 Diaphorina citri per essay.
2. Starve them for 2 hours.
3. Cool the Diaphorina citri at 4°C, and keep them on ice while working with them.
4. Take 8 μL of concentration of 100 ng/μL of siRNA.
5. Use a cool clean petri dish, place the Diaphorina citri and quickly put the droplet of SiRNA in the middle abdomen.
6. Wait 5 minutes, then remove the remaining water with a filter paper.
7. Place the psyllids on an apical meristem or a fresh shoot.

1. If it is for a real time PCR test, harvest them after 4 days, perform the RNA protocol as done before.
2. If it is for mortality analysis watch and report the number of dead individualsevery 6 hours.

Diaphorina citri RNA extraction

### Transfected Diaphorina citri RNA extraction

#### Steps

1. Collect 10 Diaphorina citri from each soaking essay.
2. Prepare the utensil to smash the psyllid. It is recommended to use a micropipette tip, this could be done by burning the tip and smashing it in order to close the hole of the tip.
3. Put the Diaphorina citri in the microtube and submerge it liquid nitrogen.
4. Take the tip and grind over the Diaphorina citri until an homogenous mix is obtained.
5. Take 250 μL of TrizolTM and add it to the microtube, wash the tip.
6. Mix and grind.
7. Add 250 μL of TRIzolTM and wash the tip again.
8. Centrifuge the lysate for 5 minutes at 12,000 × g at 4–10°C.
9. Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex.
10. Add 0.2 mL of chloroform per 1 mL of TRIzolTM Reagent used for lysis, then securely cap the tube.
11. Incubate for 2–3 minutes.
12. Centrifuge the sample for 15 minutes at 12,000 × g at 4°C. The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase.
13. Transfer the aqueous phase containing the RNA to a new tube.
14. Transfer the aqueous phase containing the RNA to a new tube by angling the tube at 45° and pipetting the solution out.
15. Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of TRIzolTM Reagent used for lysis.
16. Incubate for 10 minutes.
17. Centrifuge for 10 minutes at 12,500 × g at 4°C. Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.
18. Discard the supernatant pouring the solution out.
19. Resuspend the pellet in 1 mL of 70% ethanol per 1 mL of TRIzolTM Reagent used for lysis.
20. Centrifuge for 5 minutes at 12500× g at 4°C. c.
21. Discard the supernatant, make sure the pellet is still in your microtube.
22. Air dry the RNA pellet for 5–10 minutes resuspend in 20-40 μL of water depending on the color of the aqueous phase in step 13, if it is almost transparent add 20 μL.

Two-Step qRT-PCR

### Two-Step Quantitative Reverse Transcriptase Polymerase Chain Reaction

#### Steps

1. Prepare the RNA using RevertAid H Minus First Strand cDNA Synthesis Kit from ThermoFisher:

* 250 ng/ μL of Diaphorina citri RNA-siRNA.
* 2 μL of Reverse primer (The mix was made using 10 μL of each primer)
* Injectable water up to 24 μL.

2. As only 38.5 % of D. citri genome is composed by GC’s the step of incubating the sample at 65oC for 5 minutes can be omitted.
3. Prepare the Mix for cDNA synthesis as follows:

Table 1: Preparation of the Master Mix using RevertAid H Minus First Strand cDNA Synthesis Kit.

| Reagent | 1X |
| --- | --- |
| Buffer 5X | 4 μL |
| Ribolock RNAse inhibitor (20 U/μL) | 1 μL |
| dNTPs Mix (10 μM) | 2 μL |
| Reverse Transcriptase (200 U/μL) | 1 μL |

4. Give a spin in the centrifuge.
5. Incubate at 52°C for 1 hour in the thermal cycler.
6. Prepare the Mix for qPCR as follows:

Table 2: Preparation of the Master Mix for PCR.

| Reagent | 1X |
| --- | --- |
| Maxima SYBR Green/ROX qPCR Master Mix (2X) | 12.5 μL |
| Forward Primer (10 μM) | 1 μL |
| Reverse Primer (10 μM) | 1 μL |
| D. citri cDNA | 2.5 μL |
| Injectible water | Adjust to 25 μL |

7. Add the Master Mix to the different microtubes.
8. Place the reactions in the thermal cycler in the following conditions:

* 94°C for 3 minutes
* 40 cycles:

+ 94°C for 30 seconds
+ 54°C for 30 seconds
+ 72°C for 20 seconds

* 72°C for 5 minutes
* 12°C indefinitely

9. A 2.5%-3% agarose gel electrophoresis is required to verify PCR products in addition to the generated plots.

Primary cell culture

### Establishment of Diaphorina citri primary cell culture

#### Steps

1. Collect 50 Diaphorina citri and keep in ice.
2. Pick each with sterile tweezers.
3. Wash in .11% sodium hypochlorite for 10 seconds.
4. Wash in 75% of ethanol for 30 seconds.
5. Wash in sterile H20MiliQ water for 20 seconds.
6. Place on an sterile petri dish under a stereoscopic microscope.
7. Grab the head of the insect with tweezers then slowly remove the lower abdomen. If removed slowly, green strings will appear indicating the digestive tract. In order to avoid contamination, remove completely.
8. Collect the removed digestive tracts and place in a separate container.
9. Collect the head and rest of the body and place in a microtube.

For the digestive tract:

10. Place a Corning 10 μm cell strain filter over a sterile 50 mL Falcon Tube and place the material in it.
11. Using a 5 ml syringe plunger, grind the tissue until it is completely homogenous.
12. Add 500 ul of Hank's Salt Solution.
13. Keep grinding the remains.
14. Add another 500ul of Hank's Salt Solution.
15. In a different 50mL Falcon Tube, place a 40 μm Corning cell strain filter and filter the previously obtained solution.
16. Add 300 uL of Schneider's Insect medium and place on a cell culture plate.

For the rest of the body:

17. Centrifuge at 250 g for 5 minutes.
18. Remove the supernatant with a micropipette and place on a 10 μm cell strain filter.
19. Grind with a syringe plunger until homogeneous.
20. Wash the filter with 400 uL of Hank’s Salt Solution.
21. Transfer the filtered liquid to a 40 μm cell strain filter and add 200 uL of Hank’s Salt Solution.
22. Centrifuge the filtered liquid at 250 g for 10 minutes.
23. Add the supernatant to a cell culture plate well, then resuspend the pellet in 300 uL of Schneider’s Insect medium and add to a different well.

IGEM\_TECCEM

![](https://static.igem.org/mediawiki/2017/0/0f/TEC-CEM-idt.png)

![](https://static.igem.org/mediawiki/2017/8/84/TEC-CEM-biolabs.png)

![](https://static.igem.org/mediawiki/2017/f/fc/TEC-CEM-mathworks.png)

![](https://static.igem.org/mediawiki/2017/3/3b/TEC-CEM-snapgeneLogo.png)

![](https://static.igem.org/mediawiki/2017/3/39/TEC-CEM-agilent.png)

![](https://static.igem.org/mediawiki/2017/6/63/TEC-CEM-donadora.png)

![](https://static.igem.org/mediawiki/2017/e/e7/TEC-CEM-logoItesm.png)

[![](https://static.igem.org/mediawiki/2017/1/1e/TEC-CEM-facebook.png)](https://www.facebook.com/igemteccem/)

[![](https://static.igem.org/mediawiki/2017/7/7d/TEC-CEM-instagram.png)](https://www.instagram.com/igemteccem/)

[![](https://static.igem.org/mediawiki/2017/7/74/TEC-CEM-twitter.png)](https://twitter.com/igemteccem)

[![](https://static.igem.org/mediawiki/2017/8/89/TEC-CEM-youtube.png)](https://www.youtube.com/channel/UClG-DbIbK6KBlFsepIX40jA/videos)

Mail:
igem.tec.cem@gmail.com

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:TecCEM/Results

IGEM\_TECCEM

[![](https://static.igem.org/mediawiki/2017/a/a6/TEC-CEM-logos.png)](https://2017.igem.org/Team%3ATecCEM)

* [Home(current)](https://2017.igem.org/Team%3ATecCEM)
* [Project](https://2017.igem.org/Team%3ATecCEM/Description)

  [Description](https://2017.igem.org/Team%3ATecCEM/Description)
  [Design](https://2017.igem.org/Team%3ATecCEM/Design)
  [Experiments](https://2017.igem.org/Team%3ATecCEM/Experiments)
  [Notebook](https://2017.igem.org/Team%3ATecCEM/Notebook)
  [Interlab](https://2017.igem.org/Team%3ATecCEM/InterLab)
  [Model](https://2017.igem.org/Team%3ATecCEM/Model)
  [Results](https://2017.igem.org/Team%3ATecCEM/Results)
  [Demonstrate](https://2017.igem.org/Team%3ATecCEM/Demonstrate)
  [Improve](https://2017.igem.org/Team%3ATecCEM/Improve)
  [Attributions](https://2017.igem.org/Team%3ATecCEM/Attributions)
* [Parts](https://2017.igem.org/Team%3ATecCEM/Parts)

  [Basic Parts](https://2017.igem.org/Team%3ATecCEM/Basic_Part)
* [Safety(current)](https://2017.igem.org/Team%3ATecCEM/Safety)
* [Human Practices](https://2017.igem.org/Team%3ATecCEM/PublicEngagement)

  [Silver HP](https://2017.igem.org/Team%3ATecCEM/HP/Silver)
  [Integrated and Gold](https://2017.igem.org/Team%3ATecCEM/HP/Gold_Integrated)
  [Public Engagement](https://2017.igem.org/Team%3ATecCEM/Engagement)
* [Awards](https://2017.igem.org/Team%3ATecCEM/software)

  [Applied Design](https://2017.igem.org/Team%3ATecCEM/Applied_Design)
  [Hardware](https://2017.igem.org/Team%3ATecCEM/Hardware)
  [Model](https://2017.igem.org/Team%3ATecCEM/Model)
  [Software](https://2017.igem.org/Team%3ATecCEM/Software)
* [Team](https://2017.igem.org/Team%3ATecCEM/team)

  [Team](https://2017.igem.org/Team%3ATecCEM/Team)
  [Collaborations](https://2017.igem.org/Team%3ATecCEM/Collaborations)

![](https://static.igem.org/mediawiki/2017/0/07/TEC-CEM-home_hojas_header.png)

Results

# Results

# siRNA confirmation

The siRNA confirmation protocol was made to test, with the real time PCR, the presence of the siRNAs designed by the iGEM Tec Cem team. The four devices capable of producing the siRNAs were tested: BSLA-SOD, BSLA-WNT, BSLA-RACI and BSLA -AWD. The protocol used for this purpose is based on the liquid northern hybridization proposed by Wang X., Tong Y. and Wang S. (2010). The protocol is described as a novel form to detect miRNAs. We expect to detect the siRNAs that our different constructs can produce taking advantage of their hybridization with the forward and reverse templates of their DNA sequences and the ability of the exonuclease to digest the non-hybridized RNA sequences.

![](https://static.igem.org/mediawiki/2017/5/59/TEC-CEM_diaresults.jpeg)

It appears you don't have a PDF plugin for this browser. No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2017/b/b5/TEC-CEM_Methodology1.pdf)

![](https://static.igem.org/mediawiki/2017/e/e0/TEC-CEM_gel1a.png)

Figure 1. RNA from the HTT115 strain witouth the siRNA construct and with the siRNA construct (BSLA-AWD) with different hybridization oligos and treatments in a 12% no-denaturing gel at 100 Volts..

![](https://static.igem.org/mediawiki/2017/1/1c/TEC-CEM_gel1b.png)

Figure 2. RNA from the HTT115 strain with the siRNA construct (BSLA-SOD, BSLA-WNT and BSLA-RACI) with different hybridization oligos and treatments in a 12% no-denaturing gel at 100 Volts.

Second BSLA siRNA confirmation

It appears you don't have a PDF plugin for this browser. No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2017/5/5a/TEC-CEM_meth2.pdf)

![](https://static.igem.org/mediawiki/2017/a/a2/TEC-CEM_gel2a.png)

Figure 3. RNA from the HTT115 strain witouth the siRNA construct and with the siRNA construct (BSLA-AWD) with different hybridization oligos and treatments in a 12% no-denaturing gel at 100 Volts.

![](https://static.igem.org/mediawiki/2017/2/28/TEC-CEM_met2b.png)

Figure 4. RNA from the HTT115 strain with the siRNA construct (BSLA-SOD, BSLA-WNT and BSLA-RACI) with different hybridization oligos and treatments in a 12% no-denaturing gel at 100 Volts.

The first test of the control (only RNA of HTT115) revealed the presence of two bands in the no-denaturing gel when no exonuclease is added to the reaction of approximately 150 pb and 100 pb respectively. When exonuclease is added to the same sample a little band is observed below the 50 bp. When 4 μL of a 100μM dna oligo was added to the same sample, the samples seem to migrate better than the last two and a great amount of material stayed in the low part of the gel, probably because of the high amount of non-hybridized oligo.

For the BSLA samples, interestingly, the behavior between the samples treated with the forward and reverse DNA template was different. The RNA samples treated with the forward template sequence migrated less than the ones treated with the reverse, probably because they really hybridized with the siRNA. The RNA samples treated with the reverse sequence migrated really similar to the RNA of HT115 treated with the oligo and exonuclease, a behavior that might mean that the oligos didn´t hybridize.

We expected to observe hybridization with both templates (forward and reverse), since the construction for each siRNA has their own sense and anti-sense sequence. The results in both confirmations might mean that the siRNA might hybridize with the forward sequence but no with the reverse. Together with the real time PCR we can conclude that we have a certain amount of siRNA but to characterize their hybridization, we should changes the amount of material for the experiment.

The second confirmation of siRNA was repeated with less RNA material (2 μg instead of 5 μg) and more units of exonuclease (20 instead of 10) expecting to distinguish more defined bands. Nevertheless, since it was used a lower dilution of GelRed seems brighter than the first confirmation and the observed material is similar. Next studies might use less RNA and oligos as in the protocol proposed by Wang X., Tong Y. and Wang S. (2010) where they used 1 μg of RNA.

# Blue BSLA colonies

The following figures show blue fluorescent colonies of E. coli HT115. The color of the bacteria represents the presence of the BSLA construct and production of siRNA.

![](https://static.igem.org/mediawiki/2017/0/02/TEC-CEM_bslaA.jpg)

Figure 11. First transformation in DH5a of the BSLA-AWD device from Genscript. The characteristic blue color from the blue chromoprotein is observed in the colonies.

![](https://static.igem.org/mediawiki/2017/b/b5/TEC-CEM_bslaaaaa.jpg)

Figure 12. First transformation in DH5a of the BSLA-AWD device in PSB1C3. The characteristic blue color from the blue chromoprotein is observed in the colonies.

![](https://static.igem.org/mediawiki/2017/1/11/TEC-CEM_bslac.jpg)

Figure 13. First transformation in HT115 of the BSLA-AWD device in PSB1C3. The characteristic blue color from the blue chromoprotein is observed in the colonies.

# Sequencing

It is fundamental for us to make sure that all our BioBricks have the correct sequence before sending them to Boston for the parts registry. Therefore, we sequenced every single piece, to confirm that the ligated products into the pSB1C3 backbone correspond to the original sequence.
The BioBricks cloned by the iGEM TEC CEM 2017 team in PSB1C3 were sequenced in order to test if the inserts for the siRNAs and T7 terminator were really ligated. For the following samples of BSLA devices (Blue chromoprotein, forward siRNA, loop and reverse siRNA): 1, 2, 3, 4 and 5 the sequencing was made using the VF2 primer. Although it was possible to confirm the presence of the blue chromoprotein, the reaction couldn´t show us the presence of the siRNAs constructs because of the length of the protein gene before them. In these cases, a reaction using the VR primer is recommended since there are approximately in each case a total of 220-230 pb since the reverse primer to the siRNAs constructs.
The single presence of the siRNAs sequence (5’-3’) in PSCB1C3 without all the machinery (blue chromoprotein, loop and siRNA anti sense) was confirmed for the RacI and SOD constructs. The Wnt construct wasn’t sequenced and the results for the Awd construct weren´t conclusive. Also, the presence of the sequenced used as the control of the siRNAs (CNT) was confirmed to be cloned in PSB1C3 (sample 7). The sample 8 correspond to the single sequence of the T7-Terminator used during the construction of the BSLA devices, it was confirmed to be cloned in the PSB1C3. For the samples of BSLA device, we have to repeat the sequencing using the VR primers for the BSLA-SOD and BSLA-RACI, so for this samples we have the sequence forward and reverse with the objective to seek the siRNA construction before the blue chromoprotein, however the results weren't conclusive since the alignment showed the correct base pairs just after the reverse primer but not the same sequence before the terminator. And finally the sequencing for BSLA-AWD in PSB1C3 wasn’t repeated, since it was given to the team by the Gene script sponsorship, and once cloned in PSB1C3 the colonies became blue.\*All the samples were sequenced in a ABI PRISM 310 equipment

It appears you don't have a PDF plugin for this browser. No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2017/e/e0/TEC-CEM_Secuenciacion.pdf)

# In vivo transfection

The In vivo transfection was done by submerging the psyllids for 4 minutes in a 20 ul siRNA drop. The results were used to extract RNA to perform the real time PCR.

![](https://static.igem.org/mediawiki/2017/b/b4/TEC-CEM_soakinggggg.png)

Figure 21. A specimen was placed in a microtube and then the siRNA drop was added.

# RNA Extraction

For the RNA extraction, psyllids were collected and stored at -80°C. The extraction had a high concentration using this method and the RNA samples were used to perform a RT-PCR. The process for this protocol is shown below.

![](https://static.igem.org/mediawiki/2017/9/92/TEC-CEM_ran111111.jpeg)

![](https://static.igem.org/mediawiki/2017/3/37/TEC-CEM_rna2.jpeg)

Figure 19. 1% agarose gel with TBE Buffer 1X for the Diaphorina citri RNA extraction. The gel was loaded with Fast-DNA Ladder in lanes 3 and 7, and lanes 4 and 8 with RNA. Lanes 3 and 4 were loaded first and ran for 30 minutes at 100 V. Due to the high concentration, lanes 7 and 8 were loaded with less RNA sample and ran for 30 minutes at 100 V.

![](https://static.igem.org/mediawiki/2017/9/92/TEC-CEM_rna3.png)

Figure 20. Left, the psyllids were captured and placed on liquid nitrogen for instant death. Right, the yellow supernatant is indicative that the protocol was done succesfully.

# cDNA Synthesis

Table 1. Expected products for the amplifications in the Two-step RT-PCR.

| Product | Expected size |
| --- | --- |
| Tubulin | 195 bp |
| Rac I | 193 bp |
| WNT | 200 bp |
| AWD | 180 bp |
| SOD | 199 bp |

![](https://static.igem.org/mediawiki/2017/b/b3/TEC-CEM_resultsgel.png)

Figure 10. 2.5 % agarose gel with TBE Buffer 1X for the RT-PCR. The gel was loaded with Fast Ladder in lane 2 and 8 and with the samples listed in table N. The gel was stained with GelRed and run for 60 minutes at 100 volts.

As we can see in the figure, all the products for the Tubulin, AWD, SOD and WNT rely within the 0.150 and 0.300 kb marker bands, corresponding to the expected base pair sizes. The only product that remains outside the expected size is the RacI amplification product, which was observed in last week’s gel. The primers when tested in silico using PrimerBlast show the 193 bp product, but this does not happen in the gels. Since the reported sequence states “like-sequence” it is possible that there are mismatches between the reported sequence and the real one. It is still unclear why do the primers not generate the expected product. There are other predicted sequences such as XM\_008482020.1 and XM\_017443825.1, as this sequences are not specific the primers do not amplify this sequences either.

# Real Time PCR Melt Curves

SYBR® Green dye will detect any double-stranded DNA, including: primer dimers, contaminating DNA, PCR product due to mis-annealed primers and the PCR product of interest. By viewing a dissociation/melt curve, you ensure that the desired amplicon was detected. The presence of a single peak indicates that you have a specific product that can be corroborated by electrophoresis. For all amplicons we got a single peak that is in accordance with the expected size of the amplicons except for RacI. We checked primer specificity with blast tools and after a revisión of NCBI databases of transcriptomics we found that RacI have other isoform. It suggests that the fragment of 50 bp could belong to a different isoform for which the primers were designed. Furthermore both isoforms share the region where siRNA was designed.

![](https://static.igem.org/mediawiki/2017/b/bd/TEC-CEM_melt2.png)

Figure 5. Melting curves for Tubulin, Awd, RacI, SOD and WNT.

# Real Time PCR

Now knowing that our siRNA molecules generate all the expected products (except RacI), we proceeded to the qRT-PCR, which allowed us to measure the amount of mRNA for the corresponding genes at any given time in the cells. We used SYBR Green as an intercalating agent and got the following results. The experiment results were divided on three groups:

Experiment A: We performed the soaking method with all the siRNA, the synthetic ones; AWD and RAC1. The ones we created using our raw BSLA Method; SOD and WNT. The purified samples of SOD and WNT, the sp and wp stand for pure. And finally a mix of RAC1, WNT, AWD and SOD to learn about the expression when these siRNA are mixed.

Experiment B: By applying chitosan to the leaves and feeding the diaphorina we tested two genes RAC1 and AWD.

Experiment C: We tested different concentrations to observe which one was the best option for our essay, we also performed this experiment in order to prove our mathematical modeling.

The following figures represent our Real Time PCR results.

![](https://static.igem.org/mediawiki/2017/5/5f/TEC-CEM_rtaaa.png)

Figure 6: Effect of the different siRNA molecules over the mRNA production in Diaphorina citri after in vivo soaking application.

![](https://static.igem.org/mediawiki/2017/d/dd/TEC-CEM_rtB.png)

Figure 7: Effect of the siRNA molecules mix over the mRNA production in Diaphorina citri after in vivo soaking application.

![](https://static.igem.org/mediawiki/2017/0/0e/TEC-CEM_graficac.png)

Figure 8: Effect of the chitosan-encapsulated siRNA molecules over mRNA production in Diaphorina citri after direct application over the plant leaves.

![](https://static.igem.org/mediawiki/2017/c/c4/TEC-CEM_graph.png)

Figure 9: Effects of different concentrations (ng/uL) of siRNA-AWD tested in Diaphorina citri after in vivo soaking application to prove the proposed mathematical modeling.

The results from Figure 6 show that both synthetic and E. coli HT115 produced siRNA molecules were able to hybridize with its target sequence, inducing effective gene silencing by reducing the mRNA production. As it can be appreciated, the WNT siRNA reduced its target mRNA up to three times, being the highest-impact molecule, followed by AWD.

Figure 7 shows the effect of treating the psyllids with a mixture of the different siRNA molecules rather than with each siRNA separately. Once again, the WNT gene showed the strongest level of mRNA reduction, but in a much higher scale than with the solo treatment. The AWD siRNA is second, with almost the same impact as in the previous figure. The importance of this graph relies within the siRNA molecules for WNT and SOD produced with the novel BSLA BioBrick in Escherichia coli HT115 are functional on their own, which means that the BSLA has a great potential for future siRNA production in bacterial systems. It is important to notice that the siRNA have a higher activity in the Diaphorina citri mRNA production when applied together in a mix rather than individually.

The results observed in Figure 8 give us crucial information about the possible upscaling of our project via chitosan-encapsulated siRNA molecules and their direct application over the plant leaves. The main question was if the psyllids were attracted to the molecules and could feed on them. Since we observe reduction in the levels of mRNA, we can say that Diaphorina citri in fact fed on the chitosan. When comparing the treated psyllid with the negative control, it can be concluded that the AWD siRNA has the greatest reduction of the mRNA, while RacI has a lower direct impact in the mRNA expression.

In Figure 9, we tested different concentrations of the AWD siRNA to prove the mathematical modeling. All the concentrations yielded mRNA change expressions in the psyllids, but the most effective one was 60 ng/uL, followed by 20 ng/uL. The math model was developed to graph amount of siRNA to mRNA expression this helps us validate the results by analyzing the reduction of this expression, it also helps us determine which is the best concentration to be used to control the HLB plague.
qRT calculations

It appears you don't have a PDF plugin for this browser. No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2017/f/fc/TEC-CEM_resultsrtpcr.pdf)

# Diaphorina citri primary cell culture

The main objetive was to develop a primary culture of Diaphorina citri cells, to be transfected with our siRNA and analyzed through flow cytometry. The transfected siRNA sequences were tagged with Alexafluor so they would be visible. This protocol was repeated several times under different conditions, and different compositions of the culture medium were used, which can be seen below. The cells that were cultured in Medium B were able to survive after twelve hours, and replicated at a slow rate. Because of this, the flow cytometry was not performed.

Table 2. Composition of different mediums for cell culture.

| Reagent | Medium A | Medium B |
| --- | --- | --- |
| 200 mM L-Glutamine | 1 mL | 1 mL |
| Penicillin and Streptomycin 1000X | 1.5 mL | 1.5 mL |
| Fungizone 500X | 0.2 mL | 0.2 mL |
| Schneider Medium | 72 mL | 80 mL |
| 199 Medium 10X with Hanks’ salts | 6 mL | ----- |
| Fetal Bovine Serum (heat-inactivated) | 20 mL | 20 mL |
| 0.06 M Histidine solution | 1 mL | ----- |

![](https://static.igem.org/mediawiki/2017/7/79/TEC-CEM_cellculture12hr.png)

Figure 14. Cell culture 12 hours after extraction.

![](https://static.igem.org/mediawiki/2017/e/e8/TEC-CEM_cellculture2day.png)

Figure 15. Cell culture 2 days after extraction, observed under an inverted microscope at 10x. No significant cell replication can be observed compared to the first day,

![](https://static.igem.org/mediawiki/2017/d/d4/TEC-CEM_cellculture2week.png)

Figure 16. Cell culture 2 weeks after extraction, observed under an inverted microscope at 10x. Cell replication can be observed.

![](https://static.igem.org/mediawiki/2017/a/a2/TEC-CEM_intestinos.jpeg)

Figure 17. The body was separated in three parts: 1. head and wings, 2. intestines, 3. abdomen. Cell cultures were made with parts 1 and 3, while number 2 was discarded to avoid contamination.

# Chitosan test

The test consists in encapsulating siRNA, in this case of Rac1 and AWD. The concentration is of 100ng/ul of the siRNA used.

![](https://static.igem.org/mediawiki/2017/2/23/TEC-CEM_chit1.jpeg)

The results of the silencing can be viewed in the protocol involving the real time PCR, but it is important to note that the insect does it the leave and it does show mRNA reduction.

![](https://static.igem.org/mediawiki/2017/9/9b/TEC-CEM_chit2.jpeg)

Figure 18. Damage observed in the leaves following chitosan application.

The only problem of the chitosan protocol is suspected to be the chitosan concentration as it produces a thin film over the leaves, which caused them to be blocked and the leaves died.

# Diaphorina citri laboratory development

Special cages were built for rearing and reproduction of Diaphorina citri specimens across three months. Each cage was double lined with fine white mesh to prevent the insects from escaping while allowing us to inspect them. On top, a set of bulbs was placed and connected to a timer, to control daily light hours, which were set at 16 hours of light and 8 of darkness. Additionally, two fan heaters were placed at a distance of 1 meter, which increased the temperature inside the cages to up to 30ºC. While this is not the ideal temperature for reproduction, the presence of eggs and nymphs could be observed after two weeks.

The initial population across all cages was of 80 insects, and after approximately one month an increase of almost double was observed. However, due to the psyllid's lifespan, a rapid decline was observed shortly after. In addition to this, the increase in population made it harder for each psyllid to feed on one plant, which increased mortality.

![](https://static.igem.org/mediawiki/2017/f/fe/TEC-CEM_diaphoresults1.png)

Figure 22. Detail of mesh lining the interior of the cages. The size of the mesh prevents the insects from escaping while providing light, air flow and visibility.

![](https://static.igem.org/mediawiki/2017/7/75/TEC-CEM_diaphoresults3.png)

Figure 23. Presence of dew in the plant's leaves, resulting from nymphs feeding on the plant, which indicates reproduction.

![](https://static.igem.org/mediawiki/2017/7/7b/TEC-CEM_diaphoresults2.png)

Figure 24. Detail of plant leaves after two weeks of rearing. The presence of eggs in the stems of the plant can be observed.

![](https://static.igem.org/mediawiki/2017/9/98/TEC-CEM_diaphores.png)

Figure 25. Complete set-up for rearing of Diaphorina citri, consisting in two mesh-lined cages and complete with light and temperature conditioning.

![](https://static.igem.org/mediawiki/2017/f/f4/TEC-CEM_diaphoresults0.png)

Figure 26. Presence of adult Diaphorina citri specimens after one month of rearing.

# Morphology

Different specimens were observed under a stereoscopic microscope and identified by gender.

![](https://static.igem.org/mediawiki/2017/7/76/TEC-CEM_diaphoa.jpeg)

Figure 27. Male specimen.

![](https://static.igem.org/mediawiki/2017/8/89/TEC-CEM_nena.jpeg)

Figure 28. Female specimen.