UnivLyon1-INSALyon

Logo BIO Snare

Experiments



BIO Snare’s goal was to produce functionalized cellulose to make it adaptable for specifically trapping insects through the use of different colors and bioglue. We were inspired by an article and decided to use a coculture of the bacterium K. rhaeticus, which overproduces cellulose, along with a genetically modified yeast, S. cerevisiae.

Our main plasmid, plasmid D, has been constructed as following:

This biobrick contains:

Click on each of the components to learn more about what this biobrick contains.

  • Constitutive GAP promoter
    • Drives the continuous expression of both YFP and the bioglue.
  • fwYellow gene
    • Encodes Yellow Fluorescent Protein (YFP), the chromoprotein we selected. The sequence has been optimized for expression in S. cerevisiae. The fwYellow gene is a BioBrick created by the Uppsala iGEM team in 2013.
  • Bioglue genetic sequence
    • Encodes a bioglue made from a fusion of two natural proteins to significantly enhance adhesion properties: Masp1 from spider silk and Cp19k from barnacles. These proteins have individual adhesion strengths of 22.2 mJ/m² and 2.2 mJ/m², respectively, but their fusion increases this to 39.9 mJ/m² (Ye et al. (2023). The sequences were optimized for expression in S. cerevisiae.
  • Cellulose Binding Domain (CBD)
    • Each protein (both the colored and the adhesive) contains a CBD to allow binding to cellulose. The CBD sequence was adapted from Gilbert et al. (2021).
  • Alpha factor signal peptide
    • Derived from the MAT alpha mating system, this sequence acts as a signal peptide to facilitate protein secretion. Once secreted, the signal peptide is cleaved, leaving only the mature protein.
  • P2A system
    • Enables co-expression of both YFP and the bioglue protein from a single mRNA transcript. This system is optimized for eukaryotic cells and allows simultaneous synthesis of YFP and the adhesive protein in our context.
  • URA3 gene
    • Selected as a marker for yeast transformation. This gene allows the URA- yeast strain to synthesize uracil, enabling cells that have successfully integrated the plasmid containing URA3 to grow in uracil-deficient medium.
  • Homologous sequences (URA5’ and URA3’)
    • Facilitate plasmid integration into the yeast chromosome via homologous recombination.

This plasmid was designed to make the cellulose sticky and yellow-colored, attracting insects. However, the ultimate goal of our project is to make the trap adaptable and specific to targeted insects, avoiding any negative impact on biodiversity. To achieve this, we aim to modify features like the color or other attractants in order to capture specific insects while minimizing effects on non-target species.

The steps we followed are outlined below. Diagrams for each construction and key stages are provided. Additionally, you will find a toolbox containing detailed protocols used for each step.



Main steps of our project



Click on the buttons to discover a diagram for each step of our project.

Here is a diagram to sum up the different steps to produce the plasmid D, the original BIO Snare plasmid coloring the bacterial cellulose in yellow and making it sticky.

Here is a diagram to sum up the different steps to transform S. cerevisiae with the plasmid D encoding for the yellow protein and the bioadhesive protein.
The insertion of the fragment in the yeast chromosome is possible thanks to the homologous sequences URA3’ and URA5’.

Here is a diagram to sum up the different steps to do the coculture of our two organisms to produce the trap. K. rhaeticus naturally overproduces cellulose and S. cerevisiae that has been genetically modified to produce both a yellow and a bio-adhesive proteins.


Tool box


Here are detailed all protocols used in labs. They are separated in 3 main categories: microbiological methods with all media used for each microorganisms we used, genetic methods and molecular biology with all the protocols related to DNA and biochemical methods with protocols related to proteins.

Click on the buttons to discover the protocols associated.


We have used different media for each organism alone, and others for their coculture.

Click on the organism you want to study to discover the media to use.

K. rhaeticus medium

For 1L of medium:

Glucose 20 g
Tryptone 5 g
Yeast extract 5 g
Agar (for plates) (25 g)
HCl 37% ~ 200 µL

S. cerevisiae media

Minimum medium:

Components Quantities for 1L Quantities for 0.25L
YNE (without (NH4)2SO4) 1.7 g 0.425 g
Glucose 20 g 5 g
(NH4)2SO4 5 g 1.25 g
Agar 24 g 6 g
Demineralised water qsf 1L qsf 250mL

Selective yeast medium -URA:

Add sterilely each component to a minimum medium kept cooled down at 60°C.

Solutions Quantities for 1L Quantities for 0.25L
Minimum medium 10mL 2.5mL
1% ADE 1mL 0.25mL
1% TYR 5mL 1.25mL

YPD medium

Composition:

Component Concentration (g/L)
Yeast extract 10
Bacto Peptone 10
Glucose 20
To make agar plates 20

Protocol:

Dissolve all components in the required amount of demineralized water.
Sterilize for 30 min at 110°C.
Store at 4°C.

Coculture media

YPD + 1% Cellulase

Before the coculture, K. rhaeticus has to grow in a YPD+1%cellulase medium because the cellulase improves its growth. So, to an YPD medium, 0.5 g of cellulase have to be added. Then, the medium is filtrated.

YPS medium

To avoid the dominance of K. rhaeticus on S. cerevisiae, a YPS medium was used in the coculture. Indeed, K. rhaeticus can't metabolize sucrose and need S. cerevisiae to survive in YPS medium.


Composition:

Component Concentration (g/L)
Yeast extract 10
Bacto Peptone 10
Sucrose 20
To make agar plates 20

Protocol:

Dissolve all components in the required amount of demineralized water.
Sterilize for 30 min at 110°C.
Store at 4°C.

In this section, you can find all protocols related to DNA: extraction, digestion, amplification, analysis on agarose gel, DNA purification, DNA cloning and finally transformation in E. coli firstly and then in S. cerevisiae

Click on the buttons to discover the protocols used in the lab.





DNA Extraction

Quick and Dirty

Aim: extract DNA from bacterial colonies for a PCR reaction to check the efficiency of the recombination.
For each clone:

Put 25 µL of NaOH 20 mM in a microtube.
With a sterile cone, take one colony and resuspend it in the tube.
Incubate 15 min at 95°C.
Vortex.
Spin 1 min.
For a PCR, take 1 µL of supernatant.

Plasmid Extraction


Aim: extract plasmid from bacteria. Kit “Nucleospin Plasmids” (Macherey-Nagel).
Prepare 2 heat blocks: 50°C and 70°C, 1 tube of 2 mL, 1 column, 1 tube of 1.5 mL per culture. Put aliquots of AW (500 µL per culture) at 50°C and of AE (60µL per culture) at 70°C.

Pick colonies.
Inoculate 5 mL of LB medium with a colony and incubate bacterial culture O/N at 37°C.
Transfer 2 mL in a micro tube, centrifuge 1 min at 13,000 rpm, discard supernatant.
Resuspend the pellet with 250 µL of A1, vortex.
Lyse cells by adding 250 µL of A2, incubate 3 min at RT.
Neutralize lysate by adding 300 µL of A3, spin 10 min at 13,000.
Load supernatant on the column.
Spin 1 min at 13,000 rpm.
Discard supernatant.
Add 500µL of Aw at 50°C.
Spin 1 min at 13,000 rpm.
Discard supernatant.
Add 600 µL of A4 to wash DNA.
Spin 1 min at 13,000 rpm.
Discard supernatant.
Dry the column by spinning 1 min at 13,000 rpm.
Put the column in a new micro-tube , add 30 µL of AE at 70°C and incubate 2 min at 70°C.
Pick colonies
Inoculate 5 mL of LB medium with a colony and incubate bacterial culture O/N at 37°C.
Transfer 2 mL in a micro tube, centrifuge 1 min at 13,000 rpm, discard supernatant.
Resuspend the pellet with 250 µL of A1, vortex.
Lyse cells by adding 250 µL of A2, incubate 3 min at RT.
Neutralize lysate by adding 300 µL of A3, spin 10 min at 13,000 rpm.
Load supernatant on the column.
Spin 1 min at 13,000 rpm.
Discard the supernatant.
Add 500 µL of Aw at 50°C.
Spin 1 min at 13,000 rpm.
Discard the supernatant.
Add 600 µL of A4 to wash DNA.
Elute DNA by spinning 1 min at 13,000 rpm.
Quantify DNA with Nanodrop.

Extraction of chromosomal DNA yeast


Aim: extract DNA from yeast clones for a PCR reaction to check the efficiency of the transformation.

Add 25 mL of NaOH 20 mM in a 1.5 mL tube.
With a sterile cone, take one colony and resuspend it.
Incubate 15 min at 95°C.
Vortex and spin 1 min.
Use 1-2 µL/PCR.

Digestion by restriction enzymes

Aim: Cut a plasmid on targeted sites thanks to specific restriction enzymes. It enables either to check the integrity or to linearize a plasmid.
The specific restriction enzyme has to be chosen using the Snapgene software. Be careful to take the buffer corresponding to the specific enzyme.
The conditions of the reaction are as follow:

10X CutSmart Buffer Restriction enzyme (HF) DNA Water
5 µL 1 µL 200 ng complete to 50 µL
Incubate at 37°C for 4h.

DNA Amplification by PCR


PCR on bacterial and yeast colonies


  • Take a colony and put it in 20 µL of UP water and then streak it on the required medium to have a pure culture.
  • Put the colonies at 95°C in 20 µL of UP water and centrifuge 1 min at 13,000 rpm.

PCR PrimeSTAR max


Mix:

DNA matrix 1 ng
Primer 1 (10 µM) 1.2 µL
Primer 2 (10 µM) 1.2 µL
PrimeSTAR max master mix (2X) 20 µL
Pure water qsf 40 µL

30 cycles:

98°C 10 s
55°C 10 s
72°C 5s/kb*

*In the case of gDNA, as the DNA is less available, a longer time is required.


PCR Dream Taq


For colonies PCR, suspend each colony in 50 µL of pure water. Depending on the strain, place 5 to 10 min at 98°C.
Mix:

Bacterial suspension 6.3 µL
Primer 1 (10 µM) 0.6 µL
Primer 2 (10 µM) 1.2 µL
Primer 2 (10 µM) 0.6 µL
DreamTaq Mastermix (2X) 7.5 µL

Cycles:

98°C 30 s
98°C 10 s
Depending of the primers (NEB sites to know) 30 s
72°C 25s/kb
72°C 5 min

Agarose gel

Aim: Separate DNA fragments by molecular sizes.
If you only want to control sizes of your fragments, perform an EtBr gel coloration. If you want to purify a specific DNA fragment, perform the gel coloration with Gel Green.
Firstly, choose the required agarose gel percentage according to the following table:

Gel percent DNA Size Range (bp)
0.5% 1000 - 30,000
0.8% 800 - 12,000
1.0% 500 - 10,000
1.5% 200 - 3,000
2.0% 50 - 2,000

Protocol:

  • In an Erlenmeyer, add the required quantity of agarose in 0.5X TAE buffer.
  • Heat in the microwave to melt agarose.
  • When the liquid is transparent, take it out of the microwave and pour in a casting stand.

Coloration:

EtBr gel Gel green
Bath the gel into an EtBr solution after migration. Add 2 µL of the Gel Green solution

Purification

Aim: Extracted DNA band from an agarose gel.

Excise band of interest on blue light, weight it.
Add Gel dissolving buffer NT1 (200 µL / 100 mg agarose).
Incubate at 50°C until the gel is dissolved.
Load the dissolved gel on a column.
Spin 1 min at 13,000 rpm, discard supernatant.
Wash the column with 700 µL of NT3, spin 1 min at 13,000 rpm, discard supernatant x2.
Dry the column by spinning 1 min at 13000.
Put the column on an micro tube.
Incubate 2 min at 70°C.
Add 30 µL of NE Buffer in the column, incubate 2 min at 70°C x2.
Spin 1 min at 13000 rpm.
Quantify DNA.

Cloning

Hifi Cloning


Aim: Assembling fragments by recombination thanks to their identical ends.
The conditions of the reaction are as followed:

Volume
NEBuilder HiFi DNA Assembly Master Mix 10µL
2X
DNA fragments Use NEBuilder Calculator (required length and concentration of each fragments)
H2O Up to 20 µL
Incubate 15 min (up to 60 min) at 50°C

TEDA Cloning


Principle: This technique enables building DNA assembly with overlapping ends thanks to T5 exonuclease.

Mix:

Vector : Insert Ratio 1:3 or 1:4
TEDA reagents 4 µL
Pure water qsf 20 µL

Control tube:

Vector
TEDA reagents 4 µL
Pure water qsf 20 µL

Reaction: incubation 40 min at 30°C.
Transformation: use 10 µL, see protocol.

Reagents:

TrisHCl 1 M pH 7.5 500 µL
MgCl2 1M 50 µL
DTT 50 µL
PEG8000 250 mg
T5 exonuclease 1 µL
Water qsf 1 mL 1 mL

Transformation


Transformation in E. coli


Aim: Introducing a plasmid into a competent E. coli.

Preparation before starting:

  • Put 1 heat block at 42°C + 1 Thermomixer micro tube eppendorf at 37°C.
  • Preheat 900 µL of SOC per transformation (37°C).
  • Preheat petri dishes at 37°C, beads on the lid. Let competent cells thaw on ice.

Protocol:

Add a maximum of 1 ng/µL of DNA Add a maximum of 1ng of DNA per µL of competent cells.
Mix by tapping on tube.
Incubate 45 min on ice.
Heat shock : incubate at 42°C for 45 sec.
Incubate 5 min on ice.
Add 900 µL of SOC preincubated at 37°C.
Centrifuge 1h at 37°C at 900 rpm.
Spread 100 µL on a Petri dish.

Transformation integrative in S. cerevisiae


Aim: Introduction of a DNA in S. cerevisia genome.

Preparation of cells:
The day before the transformation:
Preparing a preculture by inoculating a colony in 2 mL of YPD overnight at 30°C 120 rpm.
The day of the transformation:
Inoculate the preculture (2 mL) in 50 mL of YPD. Measure the OD600. When OD600 is between 1 and 2, it means the cells are in the exponential phase. Transformation can start.

Protocol:
Solutions:

  • Lithium acetate 10X: 1M Lithium acetate pH 7.5 (adjust pH with diluted acetic acid).
  • TE 10X: 100 mM Tris-HCl pH 7.5; 10 mM EDTA pH 8.0.
  • 50% PEG: 50% polyethylene glycol 4000 (weight/volume).
  • LiAcTE: 1vol 10X TE, 1vol 10X lithium acetate, 8 vol H2O.
  • LiAcTE/PEG: 1vol 10X TE, 1vol 10X lithium acetate, 8 vol PEG.
Make the cells competent:

Transfer 50 mL of yeast preculture in a 50 mL tube.
Centrifuge 3 min at 3000 rpm.
Discard supernatant and add 10 mL of LiAcTE buffer.
Incubate 15 min at RT (room temperature).
Centrifuge 3 min at 3000 rpm.
Discard Supernatant.
Resuspend in 1 mL of LiAcTE buffer.

Transformation:

In a 1.5 mL tube, add between 0.5-1 ng of DNA fragment.
Add 10 µL of ssDNA (salmon sperm DNA) at 10 mg/mL predenaturated 5 min at 95°C. Keep on the ice.
Add 100 µL of competent cells for each transformation. Mix by tapping..
Incubate 5 min at RT.
Add 280 µL of solution LiAcTE/PEG. Mix by inverting..
Incubate 1h at 30°C.
Add 43 µL of DMSO (10% final) and mix by inverting.
Heat shock : 42°C for 10 min.
Centrifuge at 5000 rpm for 2 min at RT.
Discard the supernatant.
Resuspend in 1 mL of YPD, incubate at 30°C O/N.
Centrifuge.
Resuspend in 200 µL of H2O
Spread 100 µL / petri dish (make 2 petri dish/transformation)

In this section, you can find all protocols related to proteins: microplate reader to analyze the fluorescence in the media, checking if YFP is produced, PAGE and western blot.

Click on the buttons to discover the protocols used in the lab.

Microplate reader

Aim: measuring the production of fluorochrome.
• From a culture, keep a sample. If you have several cultures to test, be careful to have the same concentration of cells, or you can take the absorbance at 600 nm.
• From the same culture, take 108 cells (knowing that for a DO600 = 1, there are 3x107 cell/mL).
• Spin 5 min at 5000 rpm.
• Keep 200 µL of supernatant, this is the second sample.
• Read with Tecan.


Protein Excitation 𝛌 Emission 𝛌
GFP 488 nm 510 nm
YFP 522 nm 536 nm
mRuby 558 nm 605 nm
Venus 515 nm 528 nm

SDS PAGE

Aim: separating proteins according to their molecular mass.

Preparation Buffers

  • APS 10% (stored: -20°C ∞, 4°C 1 month):
    • APS 1 g
    Water 10 mL
    Aliquot per 1 mL


  • Stacking gel preparation:
    • Water 23.7 mL
    Tris 0.5M pH 6.8 2 mL
    Acrylamide 30% 4 mL
    SDS 10% 300 µL
    APS 10% 40 µL
    Temed 5 µL


  • Running buffer SDS-PAGE 10X:
Tris 30.3 g
SDS 10 g
Glycine 144.1 g
Water qsf 1L

Start by assembling the plates and putting in water to check that the system is not leaking. Remove the water and wipe with whatmann paper.

Resolving Gel preparation:

Components Volume for 2 gels 10% Volumes for 2 gels 12.5% Volumes for 2 gels 15%
Water 3.89 mL 3.06 mL 2.22 mL
Tris 1.5M pH 8.8 2.64 mL 2.64 mL 2.64 mL
Acrylamide 30% 3.33 mL 4.16 mL 5 mL
SDS 10% 100 µL 100 µL 100 µL
APS 10% 40 µL/ 40 µL 40 µL
Temed 5 µL 5 µL 5 µL


Pour in the casting stand Put a layer of water on top to smooth out and avoid air bubbles.

Sample preparation and migration
Buffers



  • 2X SDS-PAGE sample buffer, RT.
    • Tris 0.5M pH 6.8 16 mL
    SDS 10% 16 mL
    Glycerol 10% 10 mL
    Bromophenol blue 1% 800 µL
    Water 7.2 mL


  • Cracking buffer (-20°C)
    • Water 4.5 mL
    2X SDS-PAGE Sample buffer 5 mL
    ß mercaptoéthanol 500 µL
Protocol
Sample preparation:
Crude extract:
  1. Pick colonies
  2. Inoculate
  3. Incubate bacterial culture, O/N, 30°C for S. cerevisiae
  4. Inoculate in 5 mL of YPD the required volume to have OD = 0.2. Incubate until OD = 1, to be in the exponential phase.
  5. Take 108 cells, spin 5 min 5,000 rpm.
  6. Keep the supernatant if you want to precipitate secreted proteins. Wash the pellet with 1 mL of cold water and transfer in a tube, spin.
  7. Discard supernatant with pipetman, resuspend pellet with 100 µL NaOH 0.2M.
  8. Incubate 10 min at RT.
  9. Spin 3 min at 7830 rpm, discard supernatant.
  10. Add 50 µL of cracking buffer 1X and mix by pipetting.
  11. Incubate 5 min at 95°C.
  12. Spin 2 min at 13,000 rpm.
  13. Store at -20°C or put 18 µL in SDS Page Gel
Supernatant:
  1. Mix 300 µL of supernatant with 900 µL of acetone.
  2. Incubate 15 min at 4°C.
  3. Spin 10 min at 12000 rpm at 4°C.
  4. Discard supernatant.
  5. Short spin 5/10 seconds to dry the pellet.
  6. Let the pellet dry O/N, cover it with alumnium.
  7. The next day, add 50 µL of SDS 0.5%.
  8. Take 30 µL of this sample and add 10 µL of cracking buffer 4X.
  9. Incubate 5 min at 95°C.
  10. Spin 2 min at 13,000 rpm.
  11. Store at -20°C or put 18 µL in SDS Page.

Western Blot

Transfer
Buffers:

  • Transfer buffer 10X
    • Tris 3.1 g
    Glycine 144 g
    Water qsf 1L


  • Transfer buffer 1X (4°C)

Transfer buffer 10X 100 mL
Ethanol 100% 200 mL
SDS 10% 10 mL
Water 690 mL

Protocol
20 min before the end of the SDS-PAGE migration, soak the whatmannspaper in 1X transfer buffer.
Activate membrane:

  • PVDF membrane 3 min in ethanol, then soak it in transfer buffer.
  • Soak the gel in transfer buffer.
Transfer
Order of the sandwich: (-) whatmann, gel, membrane, whatmann (+) Rake between each layer to remove bubbles, remove excess pad with paper towel. For one gel: 20 V, 100 mA, for two gels: 20V, 200 mA, 20 min.

Antibodies binding
Buffers:

  • TBS (4°C)

    • Tris-HCl 1M, pH8 20 mL
    NaCl 5M 27 mL
    Water qsf 1L


  • Blocking solution (to be prepared the same day)

    • TBS 50 mL
    Skim milk 2.5 g


  • TBST (4°C)

TBS 50 mL
Skim milk 2.5 g
Tween 20 1 mL
Water qsf 1L

Protocol

Aspecific sites Blocking: put PVDF membrane in blocking solution at least 1h at RT.
Incubate with ab I O/N at 4°C.
Wash 3 times in TBST 15 min at RT.
Incubate with ab II 2h at RT.
Wash 4 times in TBST 15 min at RT.
Put in TBS.
Reveal: PVDL membrane in substrate HRP 1-2 min at RT, Streatchable film plastic, chemidoc..