Team:WPI Worcester/Hardware

Hardware: Low-Cost Gene Gun

Introduction

A gene gun, or a biolistic particle delivery system, is a device to transform cells with exogenous DNA using DNA-coated microparticles. Biolistics is a versatile and important method for genetic modification, yet the price of a commercial gene gun is anywhere between $10,000 and $30,000, making it difficult for some labs to afford.

Inspired by the 2016 iGEM University of Cambridge team and online sources (Wernick, Hanson, Taylor & Arnie, 2016), our team built a low-cost gene gun costing only $416.35, with parts easily purchased online. Our gene gun has improved safety features and simplified electronic components of previous designs.

This low-cost gene gun has been proved to be functional through test fires on lettuce leaves, which had cells successfully transformed with GFP. We look forward to testing the gene gun with AFPs to see if it could transform lettuce to produce AFP to protect itself from bacteria forming biofilms.

Key Components

  • CO2 cartridge: provides air pressure in the gun in order to fire microparticles into cells.
  • solenoid valve: briefly opens upon closing the switch to release compressed CO2 in the gun.
  • time delay relay: controls firing time of the gene gun. Normal fireing time is around 80 ms.
  • body: holds compressed CO2 until it is release by the solenoid valve. Mainly composed of standard pipe parts.
  • pressure gauge: monitors air pressure in the gun. Normal firing pressure is 200-300 psi. The gun is built to withstand air pressure at 600 psi
  • needle valve: in case the body of the gun gets over-pressurized, release the pressure in the body.
  • macrocarrier: part of the gene gun that holds the microparticles before they get fired into cells. For this gene gun, the macrocarrier is a piece of parafilm sandwiched in between two M8 washers.
  • hose nozzle: holds the macrocarrier and aims at the target plant.
  • protective features
    • box: a laser cut birch box to contain electric components. Firing time could be adjusted from out of box through pressing buttons on the timer.
    • shield: a 5.4mm thick acrylic shield to prevent any damage to the surroundings.

Comparison with iGEM 2016 University of Cambridge Gene Gun

Our gene gun is largely based on the design of the iGEM 2016 University of Cambridge team, with some improvements and adjustments.

Part University of Cambridge 2016 Worcester Polytechnic Institute 2018
Electronic Consists of switched-mode power supply, IEC filter, time delay relay, switch and kettle cord. Electric part is simplified to only time delay relay, switch and cord.
Box Box is sealed and closed. Time delay relay could only be adjusted after opening the box. Opening on the top for adjusting relay from out of box. Front board could be removed if needed.
Shield 3mm thick acrylic boards glued together. 5.4mm thick acrylic boards connected with nuts and screws. Reinforced corners to increase strength of shield. Can be taken apart for easy storage.
Macrocarrier M3 washers wrapped with parafilm. Mesh added to stop parafilm from going into target. M8 washers wrapped with parafilm. Mesh removed since it could stop the parafilm from rupturing.

All parts in the previous design were also converted into standard American parts that could be easily purchased in the U.S.. A table of corresponding parts in the previous and current design can be downloaded through the following link: https://static.igem.org/mediawiki/2018/a/ab/T--WPI_Worcester--part_comparison.xlsx

We have also implemented a different method for testing functionality of the gene gun, which will be further explained in the following section.

Testing

Functionality of the gun was tested through transforming lettuce leaves with green fluorescent protein (GFP). The iGEM 2016 University of Cambridge team tested their gene gun with onion epidermal tissue to see if the gene gun could penetrate cell walls, and then tested shooting their biobrick part into algae, but didn’t confirm successful expression due to limited time. Here our team provide a simpler test for functionality of the gene gun, which could yield results in a few days. Our test also proves that the gene gun could successfully transform plants other than onions.

For firing the gene gun, the team used 0.6 micrometer gold particles coated with a plasmid containing binary plant vector expressing GFP and GUS, both driven by a 35S promoter (link to the vector used: https://www.addgene.org/64401/).

For comparison of functionality between the low cost gene gun and a commercial gene gun, the team fired using both the low-cost gene gun and a common commercial gene gun available at the school biology department, the Biolistic PDS-1000/He Particle Delivery System. Directions on how to operate the PDS-1000/He system and how to prepare the microcarriers can be found through the following link: https://static.igem.org/mediawiki/2018/d/dd/T--WPI_Worcester--gene_gun_operation.pdf

The team tested with two different types of lettuce: butterhead and romaine. Butterhead was chosen because of their strength in leaves and involvement with the biofilm formation test. Romaine lettuce was chosen because of they are a more common type of lettuce on the market. The team picked fresh leaves from the lettuce of roughly 100 cm^2, then washed and labeled the leaves.

Leaf #

Leaf Type

Bombardment type

Height

Pressure

1

butterhead

Biorad

6cm

500psi

2

butterhead

Biorad

6cm

1100psi

3

butterhead

Biorad

9cm

1100psi

4

butterhead

DIY

2cm

300psi

5

butterhead

DIY

4cm

200psi

6

butterhead

Biorad

6cm

1100psi

7

romaine

Biorad

6cm

1100psi

8

romaine

Biorad

9cm

1100psi

9

romaine

DIY

3cm

200psi

10

romaine

DIY

4cm

200psi

11

romaine

DIY

 4cm 175psi