Team:TU Delft/Notebook

Notebook

Subtitle

Overview

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Work Space

Our lab and office. Our new home for the summer.

About our lab

The iGEM lab, inside the Applied Sciences building of the TU Delft, has been the place where our science took place. It has the certification for ML-1 experiments, which was enough for our experiments. Here we learned and improved our skills in cloning and manipulating biological materials. We also tried to apply our ideas from paper to the real world. All in all, we discovered the world of synthetic biology from the inside.

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About our office

Our office has been our meeting point and our dry-lab zone during the project. Here, amazing talks about biology, engineering and philosophy took place. And here we debated about science and lab topics. To sum up, the office that you can see in the picture in the left has been our home this summer: the place where we built the project, but also where we built our team spirit.

Safety

Lab and Project Safety

Safety in our Lab

Our lab is classified as Level 1 of biosafety. This is the lowest safety level, meaning that our experiments involve low or no risk. The biological materials used for our experiments are handled in an open bench All the members of the team have received safety training, including:

Introduction to sterile working

RNA handling

Laser safety

Microscopy training

ML-1 safety test completion

Chemical safety training

General safety information, regarding contact persons and locations

Computer infrastructure

The safety of our experiments was supervised by Susanne Hage (Safety Manager of the TNW faculty) and Marinka Almering (Safety officer of the TU Delft). The research has been conducted with respect to the regulations of biosafety for The Netherlands, that can be found here

Safety in our Project

Our harmless bacteria produces curli subunits in order to make an inducible biofilm. The curli is natively produced by several safe strains, like the TOP10 used for transformations. Our strain contains a CsgA deletion and can only produce the curli when transformed with the designed plasmid and induced. This part of the project is based on sufficient scientific publications and previous iGEM information and parts. Also, one assay is designed to test the efficiency of the biofilm created carrying a specific affinity tag. For the assay teeth pieces were used, taking care of all the existing regulations and safety recommendations.

The chassis organism used for our project is a modified strain of the lab organism Escherichia coli K-12. It is called E. coli K-12 MG1655 PRO ΔcsgA ompR234.

In addition to our chassis organism, Escherichia coli Top 10 cells were used for cloning procedures

A cow tooth is used for testing our affinity application to hydroxyapatite. This will be arranged following the lab regulations, and the tooth will be provided following all the existing regulations. We will handle the part in the ML1 lab, and using gloves while working with the part. Furthermore, the teeth are cleaned properly before the use. The use of that samples is regulated by the Ministry of Economic affairs, Agriculture and Innovation of the Government of The Netherlands. The waste of all animal byproduct is designated as Specific Animal Waste (following Euralcode 180102). Later on, it is transported to the ZAVIN in Dordrecht for its destruction. For further information, you could contact the Residual Management Department of TU Delft (ReststoffenBeheer@tudelft.nl)

One of the problems to be faced with this project is the production of csgA as biofilm-maker unit. It has been reported previously as a virulence factor from harmful strains of E.coli, because the amyloid make the attachment to surfaces easier. However, we implemented two safety points for minimizing the risk of plasmid transmission to pathogen strains. First of all, the gene which codifies for csgA is preceded by a Rhamnose promoter. That prevents curli (csgA+csgB polymer) to be produces when no inductor is applied to the system. Furthermore, as rhamnose is a metabolizable inductor the gene expression could be avoided just by stopping the rhamnose supply. On the other hand, we aim our project for product testing or industrial production. So, the good laboratory practices and good manufacturing practices in these two fields should avoid any safety issue.

If our project could be fully developed into a real product, it would aim the following fields:

Factories

Lab applications

Consumer products

Test products to be used in the human body

Test products on food

We would like to encourage every interested iGEM team or researcher in general to contact us for further details about the safety in our lab and project!

Day Notes

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General Procedures

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Interlab Study

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Curli Formation

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3D Printer Design

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Printing Studies

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Protocols

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  1. Plate Top10 cells and incubate at 37ºC overnight

  2. Pick one colony, inoculate in LB media and incubate overnight while shaking at 37ºC

  3. Dilute the culture in fresh medium, and continue the incubation until the OD600= 0.4-0.6

  4. Centrifugation 5 min. at 4000 rpm.

  5. Pellet cells and resuspend in 100mM of CaCl2 solution

  6. Incubate on ice for 20 min

  7. Centrifugation 5 min at 3000 rpm

  8. Pellet cells and suspend again in 100mM CaCl2 solution

  9. Incubate on ice for 60 min

  10. Centrifugation 5 min at 3000 rpm

  11. Add a solution of 100mM CaCl2 + 40% glycerol

  12. Store immediately at -80ºC

  1. Take the competent cells from the storage at -80ºC and leave them on ice for 10-15 min

  2. Add 1-2 µL of plasmid solution to the 50 µL cell tube

  3. Incubate on ice for 30 min

  4. Heat-shock the cells at 42ºC for 45s

  5. Incubate on ice for 2 min

  6. Add 500 µL of LB media and incubate 60 min at 37ºC

  7. Plate the cultures on agar plate

  1. Pick a single colony from a plate or cryostock

  2. Put the colony in a 50 mL sterile tube and add 5-10 mL of LB fresh medium

  3. Put the tube to incubate for at least 16 h, at 37ºC and 200 rpm

Gel making

  1. Prepare 200 mL of TAE buffer

  2. Mix the TAE solution with 2g of agarose (for 1%)

  3. Heat the solution to boiling, and then cool it to 50ºC aprox.

  4. Add 5 µL of Ethidium bromide to the solution

  5. Pour the solution in the electrophoresis vessel. Apply the combs.

  6. Let it polymerize, and then cover it with TAE

Gel running

  1. Add 1/6 of total volume of Loading buffer to every DNA sample.

  2. Remove the combs from the gel, and pipette DNA samples and DNA ladder

  3. Run at 100-130V for 30-60 min (depends on the fragments)

  1. Add 20-100 ng of vector DNA (can be calculated from the DNA concentration in the sample)

  2. Add X ng of insert DNA. X is calculated using the length of both vector and insert and the molar ratio desired.

  3. Add 2µL of ligation buffer

  4. Add MQ water to set the final volume to 15-20

  5. Add 1 µL of T4 ligase (always at the end to keep the enzyme in optimal conditions)

  6. Incubate for at least 3 hours at 16ºC

  1. Take 1.5 mL from a freshly grown culture and put it in a 1.5 mL tube

  2. Spin the tube for 10 min at 2000 rpm

  3. Decant the supernatant without disturbing the pellet

  4. Add 0.5 mL of LB media and 0.5 mL of glycerol 80% solution

  5. Mix by vortexing

  6. Save in the -80ºC freezer

  1. Add 1 µg of DNA (can be calculated from concentration in the sample)

  2. Add 5 µL of NEB buffer

  3. Add 1 µL of restriction enzyme 1

  4. Add 1 µL of restriction enzyme 2

  5. Add MQ water to set the final volume at 50 µL

  6. Mix the solution by flicking the tube

  7. Spin-down in a microcentrifuge for 15 s

  8. Incubate at 37ºC for 1-2 hours

  1. Add 1.5 mL of bacterial culture in LB medium to a 1.5 mL micro-centrifuge tube. Centrifuge that tube at max speed for 3 min

  2. Remove the supernatant, and add 600 µL of MQ water to the pellet

  3. Add 100 µL of Cell Lysis Buffer, and mix by inverting 6 times. The color change to blue indicates complete lysis

  4. Add 350 µL of cold (4-8ºC) Neutralization Buffer, and mix by inverting the tube. The color change to yellow indicates total neutralization

  5. Centrifugate at maximum speed for 3 minutes, and transfer the supernatant to a PureYield Minicolumn

  6. Place the minicolumn into a PureYield Collection Tube and centrifuge at maximum speed for 15 seconds

  7. Discard the flowthrough and place the minicolumn again into the same PureYield Collection tube

  8. Add 200 µL of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed for 15 seconds. Do not empty the Collection Tube now

  9. Add 400 µL of Column Wash Solution to the minicolumn, and centrifuge at maximum speed for 30 seconds

  10. Transfer the minicolumn to a clean 1.5 mL tube, and 30 µL of hot (50ºC, pre-warmed) MQ water directly to the minicolumn matrix. Let stand for 5 minutes at room temperature

  11. Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute plasmidic DNA. Cap the tube, and store the DNA solution at -20 ºC (or use it directly for cloning experiments)

  1. Weigh a 1.5 mL microcentrifuge tube for each DNA fragment to be isolated, and record the weight

  2. Visualize the DNA in the agarose gel using a long-wavelength UV lamp and an intercalating dye (Ethidium bromide). Irradiate the gel the minimum possible time to reduce nicking

  3. Excise the DNA fragment of interest in a minimal volume of agarose using a clean scalpel or razor blade. Transfer the gel slice to a weighted 1.5 mL tube and record the weight, again. Subtract the previously measured tube weight to obtain the weight of the gel slice containing the DNA fragment

  4. Add Membrane Binding Solution at a ratio of 10 µL of solution per 10 mg of agarose gel slice

  5. Vortex the mixture and incubate at 50-65ºC for 10 minutes, or until the gel slice is completely dissolve in the liquid. You can vortex the tube every few minutes to increase the rate of agarose melting

  6. Centrifuge the tube briefly at room temperature to ensure the contents are at the bottom of the tube. Once the agarose gel is melted, the gel will not re-solidify at room temperature

  7. Place one SV Minicolumn in a Collection Tube for each dissolved gel slice

  8. Transfer the dissolved gel mixture to the SV minicolumn assembly and incubate for 1 minute at room temperature

  9. Centrifuge the SV Minicolumn assembly in a microcentrifuge at max speed for 1 minute. Remove the SV Minicolumn from the Spin Column assembly and discard the liquid in the Collection Tube. Return the SV Minicolumn to the Collection Tube afterwards

  10. Wash the column by adding 700 µL of Membrane Wash Solution, previously diluted with 95% ethanol to the SV Minicolumn. Centrifuge the SV Minicolumn assembly for 1 minute at maximum speed

  11. Empty the Collection Tube as before, and place the SV Minicolumn back in the Collection Tube. Repeat the wash with 500 µL of Membrane Wash Solution, and centrifuge the SV Minicolumn assembly for 5 minutes at maximum speed

  12. Remove the SV Minicolumn assembly from the centrifuge (not wetting the bottom of the column with the supernatant). Empty the Collection Tube and centrifuge the assembly for 1 minute with the microcentrifuge lid open (or off) to allow ethanol evaporation

  13. Carefully transfer the SV Minicolumn to a clean 1.5 mL tube. Apply 50 µL of Nuclease-Free Water (at 50ºC) directly to the center of the column, without touching the membrane with the pipette. Incubate at room temperature for 5 minutes

  14. Centrifuge for 1 minute at 14000 rpm. Discard the SV Minicolumn, and store the tube containing the eluted DNA at 4ºC or -20ºC

  1. Add an equal volume of Membrane Binding Solution to the restriction product tube

  2. Place one SV Minicolumn in a Collection Tube for each restriction product solution

  3. Transfer the mixture to the SV Minicolumn assembly and incubate for 1 minute at room temperature

  4. Centrifuge the SV Minicolumn assembly in a microcentrifuge at max speed for 1 minute. Remove the SV Minicolumn from the Spin Column assembly and discard the liquid in the Collection Tube. Return the SV Minicolumn to the Collection Tube afterwards

  5. Wash the column by adding 700 µL of Membrane Wash Solution, previously diluted with 95% ethanol to the SV Minicolumn. Centrifuge the SV Minicolumn assembly for 1 minute at maximum speed

  6. Empty the Collection Tube as before, and place the SV Minicolumn back in the Collection Tube. Repeat the wash with 500 µL of Membrane Wash Solution, and centrifuge the SV Minicolumn assembly for 5 minutes at maximum speed

  7. Remove the SV Minicolumn assembly from the centrifuge (not wetting the bottom of the column with the supernatant). Empty the Collection Tube and centrifuge the assembly for 1 minute with the microcentrifuge lid open (or off) to allow ethanol evaporation

  8. Carefully transfer the SV Minicolumn to a clean 1.5 mL tube. Apply 50 µL of Nuclease-Free Water (at 50ºC) directly to the center of the column, without touching the membrane with the pipette. Incubate at room temperature for 5 minutes

  9. Centrifuge for 1 minute at 14000 rpm. Discard the SV Minicolumn, and store the tube containing the eluted DNA at 4ºC or -20ºC

Resources

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Contact Details

Address:

Faculty of Applied Sciences

Room F109

TU Delft

Lorentzweg 1

2628 CJ Delft

The Netherlands

Phone:

+31 15 278 18 62

Email:

tudelft.igem@gmail.com