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Protocols

Safety

General

We are working with two new (to iGEM) bacterial strains to pick our chassis from: Pseudomonas putida & Pseudomonas fluorescens, as well as a new organism: microalgae (Chlamydomonas reinhardtii and Dunaliella tertiolecta). All are non-pathogenic lab strains, not able to produce any substance that might be harmful to humans.

The final goal of our system is to create a bacterium that will be placed in a sealed container and when a tailings sample is introduced it will express an enzyme in response to the naphthenic acids present in the sample. This enzyme will cleave a substrate that will be introduced to the same and when a voltage is applied the cleaved product will be ionized. This ionization will be detectable with an electrical apparatus we have designed. The bacteria will not escape the sealed system and if it did, the bacteria is native to the tailings ponds and will have no biological advantage due to any modifications we have made.

Modifications made involved the introduction a lacZ reporter gene into the bacterium. The only side effect of this modification is that the bacteria will be able to break down and digest lactose when the naphthenic acid sensitive promoter is activated. This is not harmful to bacteria. The bacteria is usually found in oil sands tailings ponds, an environment where the ability to digest lactose is not of advantage due to the lack of lactose.

The other modification being made is that in the plasmid we are introducing to the bacterium contains oriT and ori1600 parts. These allow the plasmid to conjugate between Pseudomonas and Escherichia species. This will not be problematic, as in the testing apparatus the only bacterium present will already have the plasmid. If the bacteria did escape, then the plasmid would only be able to travel to those two genuses. If the plasmid did manage to transfer to a new host then no advantage would be given in the environment, as the only modification would be the ability to degrade lactose. This degradation could only happen in the presence of naphthenic acids that could activate the promoter for the lacZ gene.

Any mutation to the biobricks we are using would just render them inoperative. It is highly unlikely that the bacteria would start producing harmful substances as a result of random mutation. Neither the bacteria we are using, nor the parts that we are introducing to them, associated with pathogenicity, infectivity, or toxicity.

Overall it can be said that the bacteria don’t pose a risk to humans. The only risk they pose to the environment is the remote chance that they might outgrow un-modified bacteria. However, this can only happen in a lactose rich environment, which the oil sands tailings ponds are not. The media which the bacteria are in, tailings pond water (TPW), is more harmful than the bacteria due to the toxic substances like heavy metals and NAs.

Safety of the People in the Lab

All of the work is being done in level 2 Bio-safety labs. All researchers have received appropriate training and are aware of all applicable safety procedure and guidelines. All harmful chemicals, including tailings pond samples and bacteria, were disposed of appropriately due to the University guidelines. Samples were stored in the correct places according to their MSDS.

Public Safety

Our goal is to design a kit which is extremely user friendly. One way to design it would be that the kit includes a test tube with the bacteria to which the TPW sample is added. If the user follows the instructions, the bacteria will be contained and won’t pose a risk to the public. If the bacteria should be released on accident, it still doesn’t pose a risk because it is neither toxic nor infectious as it is a lab strain that will not survive in the environment.

Environmental Safety

We will determine the most suitable way to treat the samples in order to destroy the bacteria. The three options we are considering are autoclaving, addition of bleach (or other similar chemical) to the sample, and suicide genes in the bacteria. The sample will be less hazardous than regular tailings pond water, as any bacteria will be destroyed. Should the bacteria be released it still doesn’t pose a danger to the environment, since it can’t outgrow the naturally occurring strains in their native environment unless there are large quantities of lactose present.

Security Risks

Pseudomonas putida and fluorescens are benign and do not pose a risk to the people working in the lab, the general public, or the environment the potential for misuse is virtually non-existent. The parts we are using also have no potential for misuse. The lacZ gene allows bacteria to breakdown and digest lactose, which is harmless. The plasmid backbone we are using has oriT and ori1600 parts, which means there is the potential for conjugation of the plasmid from one bacterium to another and between Pseudomonas and Escherichia species. It will only work between these species as the origin of replication is only compatible with them. This does not give any bacterium an advantage, as the only possible benefit would be the ability to metabolise lactose if there was some present in the case that the bacteria escaped from the testing apparatus.

Safety of Parts Submitted

The only part with any potential safety issues is the oriT / ori1600 plasmid, as it can travel between species of Pseudomonas and Escherichia. This does not pose any major safety risks when used with lab strains of both organisms. Although the ability to transfer DNA between species seems dangerous, it can only be transferred to species in which the origin of replication is functional, reducing the risk of transferring harmful parts to other organisms.

A Statement from the Biosafety Officer

We have talked to multiple people who are on the Biosafety Committee before and during our research. We didn’t need to make any drastic changes based on our discussions with them. The Biosafety officer for our area has prepared a statement that is linked below.

Click here to read the official statement of endorsement.