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 a commercial gene gun costs 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 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 protect itself from bacterial biofilms.

Key Components

gene gun body, solenoid, and timer relay

Image: gene gun without protective features

Image: macrocarrier made of washers and parafilm

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 firing 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, releases 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 box: a laser cut birch box to contain electrical components. Firing time can be adjusted from outside the box by pressing buttons on the timer.
protective shield: a 5.4mm thick acrylic shield to prevent any damage to the surroundings.

Image: box for electrical components and protective shield

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.

Gene Gun setup Gene Gun Set up with Lettuce
Images of our gene gun.

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 United States. A table of corresponding parts in the previous and current design can be downloaded through the following link: Part by Part Comparison between WPI 2018 and Cambridge 2016 Gene Guns (Microsoft Excel File)

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 it could penetrate cell walls. They tested by shooting their biobrick part into algae, but didn’t confirm successful expression due to limited time. Here, our team provided 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).

plasmid diagram

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: Protocol for Preparing Microcarriers for Plasmid Delivery (PDF)

The team tested with two different types of lettuce: butterhead and romaine. Butterhead was chosen because of the uniformity and strength of its leaves and involvement with the biofilm formation test. Romaine lettuce was chosen because they are a more common type of lettuce on the market and part of the inspiration for our project. The team picked fresh leaves from the lettuce of roughly 100 square centimeters, then washed and labeled the leaves.

eleven lettuce leaves laid out on a lab bench on top of a paper towel

The team tested various heights and firing pressure to determine the best firing conditions for the gene gun. Here is a table detailing the firing conditions:

Leaf Number	Leaf Type	Bombardment Type	Height	Pressure
1	butterhead	Bio-Rad	6cm	500psi
2	butterhead	Bio-Rad	6cm	1100psi
3	butterhead	Bio-Rad	9cm	1100psi
4	butterhead	WPI iGEM	2cm	300psi
5	butterhead	WPI iGEM	4cm	200psi
6	butterhead	Bio-Rad	6cm	1100psi
7	romaine	Bio-Rad	6cm	1100psi
8	romaine	Bio-Rad	9cm	1100psi
9	romaine	WPI iGEM	3cm	200psi
10	romaine	WPI iGEM	4cm	200psi
11	romaine	WPI iGEM	4 cm	175 psi

The bombarded leaves were then covered with wet paper towel to stop them from drying out. Lettuce leaves were stored at room temperature for the first two days and then in the fridge for one day.

Fluoresence was checked under a fluorescence microscope (SteREO Discovery.V12 microscope). Since injured parts of the leaves could also fluoresce under the microscope, the team checked for specific spots on the lettuce leaves that fluoresces under the filter EGFP 480 but not DSRED to make sure the fluorescence is generated by expressed GFP.

Fluorescence of the leaves were checked 24, 48, and 72 hours after the bombardments.

Lettuce under the U V light of the microscope Beck looking at the lettuce with the microscope

Results

Lettuce under G F P filter Lettuce under R F P filter
Figure. cell fluorescing under microscope (left or above: EGFP 480, right or below: DSRED)

Evidence of expressed GFP on lettuce leaves was found 72 hours after the bombardments. The leaf found to be expressing GFP was leaf number 4, which was transformed using the low-cost gene gun at pressure of 300 psi and height of 2 cm. Several spots on the leaf was found to be fluorescing under EGFP 480 but not DSRED, which confirms expression of GFP.

The previous pictures shows one of the cells that was transformed by the gene gun. In the middle of the image a cell is fluorescing under the GFP channel (EGFP 480, picture to the left or above) but not RFP channel (DSRED, picture to the right or below). Since parts of the lettuce leaf that are autofluorescent will be fluorescent under both channels, but the fluorescence generated by GFP will only be fluorescent under the GFP channel, behavior of this cell confirms expression of GFP.

Future Work

Though proven to be functional, various features of the gene gun could still be improved. Currently when the low-cost gene gun is fired at a lettuce leaf, it tends to rupture a hole at where the particle is fired due to high flow speed of air. Further improvements on securing the target could be developed to prevent this from happening.

Ideal firing conditions also need to be further specified, though this may depend on the specific types of target. More tests also need to be carried out to replicate the current results. A more efficient way of confirming expression could also be developed.

The reason for the creation of the gene gun was to be able to transform leaves with AFPs to determine whether the plant could produce AFPs to protect itself from human pathogens that form biofilms. This is still a line of research open to testing.

Gene Gun Construction Manual

Materials

A list of materials needed for building the gene gun can be downloaded through this link: WPI iGEM 2018 Gene Gun Parts List (Microsoft Excel File)

Downloads

Laser Cut Plan for the Box and Shield (Zipped Folder)

Installation Steps

Referring to the image, put together all parts for the gun body except for the CO2 cartridge and the solenoid valve (note that in the image, the schrader valve on top of the gun is replaced by a reducer, and the pipe fitting at the bottom is of a different size from the actual one).
Tighten all connections above the solenoid valve by applying PTFE tape to the connections, and then screwing with wrench until the connections cannot be tightened anymore. Connections below the solenoid valve could be tightened by hands, since this part is not involved with storage of the high pressure and need to be removed to place the macrocarrier.
Connect the timer relay with the solenoid valve, cord and switch button according to the instruction that comes with the timer relay. Make sure to connect the solenoid valve to the ground port in the cord.
Plug in the cord. Test if pushing the switch button successfully opens the solenoid valve for a certain amount of time.
Connect the solenoid valve to other parts of the body of the gun. Tighten connection between the solenoid valve and the upper part of the body using the same method as step 2.
Fasten the gene gun body to a stand (a ring stand would be preferred).
Put electronic parts of the gene gun in the box and put shield in front of gene gun.
Prepare the macrocarrier by putting a piece of parafilm in between 2 washers. The parafilm should be evenly stretched to obtain even rupture when firing. Place the macrocarrier into the hose fitting.
Screw the CO2 cartridge and pump onto the gun body.
Test fire with just the parafilm. Increase pressure up to 100 psi and press the switch button. A loud pop should be heard to indicate rupturing of the parafilm. Check rupturing condition of the parafilm.

Key Points for Firing

Turn the CO2 pump slowly so that the pressure increases slowly in the gun body.
Test for best conditions (height and pressure) for the specific kind of target you are using. Recommended firing pressure is roughly between 200-300 psi.
Check if the parafilm has ruptured after firing the gene gun. If not, increase pressure and fire again.
Always release excess pressure through the needle valve and not by pressing the switch button

Storage

Remove CO2 cartridge and pump to avoid accidental pressure increase in the body of the gun, or open needle valve. The shield and box can be taken apart for storage if needed.

References

GFPGUSPlus, a new binary vector for gene expression studies and optimising transformation systems in plants. Vickers CE, Schenk PM, Li D, Mullineaux PM, Gresshoff PM. Biotechnol Lett. 2007 Nov;29(11):1793-6. Epub 2007 Aug 9. 10.1007/s10529-007-9467-6 PubMed 17687623

iGEM 2016 Cambridge JIC Team

Wernick, Jay Hanson, Kyle Taylor, Arnie. (2016). How to build and use a gene gun. Retrieved from https://www.oreilly.com/ideas/how-to-build-and-use-a-gene-gun

Gene gun. (2018). Retrieved from https://en.wikipedia.org/w/index.php?title=Gene_gun&oldid=859011687

Ruhlman, T. A. (2014). Plastid transformation in lettuce (lactuca sativa L.) by biolistic DNA delivery. Chloroplast biotechnology (pp. 331-343) Humana Press, Totowa, NJ. Retrieved from https://link.springer.com/protocol/10.1007/978-1-62703-995-6_21

Team:WPI Worcester/Hardware