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Biosafety

Biosafety

Safety is incredibly important not just to the team members in the lab but also the external implications of our project. Therefore, we comprehensively covered all of the dangers of our project as part of our safety form. Our review had to pass through a panel of academics which made up the health and safety commission. It was then accepted. Standard laboratory safety protocols were carried out whenever we were working in the lab. These included the following precautions: The team made sure that all the written and verbal instructions were followed closely. Team members made sure not to come in contact with any hazardous equipment or chemicals until they obtained permission. Team members were also clarified about the operating procedures of all safety equipment and their locations such as fire alarm, fire extinguisher and first aid kit. Gloves were worn at all times whilst present in the lab or before coming in contact with any hazardous equipment or toxic agent. Overall, all the safety guidelines were followed carefully in order to keep the team members safe and meet the safety requirement needed. We also complied with all of the safety requirements set out by iGEM.

UNIVERSITY OF WESTMINSTER RISK ASSESSMENT OF GENETICALLY MODIFIED MICROORGANISMS (GMM)

Part 1

(a) Title of project

Metabolic engineering of Acyl-Homoserine Lactone (AHL) genes ppuR,ppuI and RsaL in the quorum-sensing pathway of Pseudomonas putida.

(b) Scientific goals of project

AHL is a quorum-sensing molecule secreted by bacteria and is essential in the formation of biofilm. Biofilm is a collection of extracellular proteins and DNA which decreases sensitivity of bacteria to antibiotics. This is extremely problematic as antibiotic resistance is emerging as a world crisis with the NHS spending roughly £1 billion just on treating nosocomial infections. Multidrug resistant (MDR) gram negative bacteria such as Pseudomonas aeruginosa in particular are emerging as increasingly problematic bacterial species, especially in secondary infections. Biofilms can also be considered a virulence factor as in infectious endocarditis, where septic emboli break off from the biofilm and cause symptoms.

Therefore, we are aiming to develop strategies to inhibit biofilm formation from the molecular level, with the specific genes ppuR,ppuA, ppuI and RsaL being found to form the quorum sensing molecule AHL. We aim to downregulate ppuR, ppuA and ppuI while up-regulating the suppressor RsaL. This should result in the decreased production of AHL resulting in decreased biofilm formation and an increased sensitivity of the bacteria to antibiotics.

We will be using P. Putida as it is more compatible with the iGEM registry, less virulent and most importantly has only one dominant quorum-sensing pathway.

The data we gather can be used to extrapolate our knowledge onto the more dangerous Pseudomonas strain: P. aeruginosa. Both have a very similar genetic pathway with similar products.

Once proof of concept is achieved, we can then move on towards applications, such as a potential genetic kill-switch via biosensor P. putida cells which can be incorporated into a spray, allowing for biofilm destructing cells. Our findings can also help potentially create a zone of therapeutic antimicrobial drugs.

(c) An overview of the different types of GMM that will be constructed

(i) List of recipient strain(s) and what hazards are associated with this, consider if the pathogenic traits of the recipient strain been altered?

Pseudomonas putida IsoF

PCL1445 is a wild-type

Laboratory strain KT2440

Certain strains of P. putida are not pathogenic due to the lack of certain genes including those for enzymes that digest the cell membranes and walls of humans and plants.

Although P. putida is saprophytic and deemed a safe bacteria, other species in the genus are opportunistic pathogens such as P. aeruginosa and P. syringae. P. aeruginosa has been isolated from the human body through many different locations, it also infects the urinary and respiratory tracts. This bacteria is associated with: pneumonia, enteritis, vaginitis, and mastitis. Both opportunistic pathogens have the ability and enzymes to release toxins and degrade cell membranes, something that P. putida lacks.

(ii) List of vector(s)

pSB1A - Bacterial expression vector using LacI inducible promoter. Containing Ampicillin resistance for bacteria and have class 1 restriction enzyme cut sites allowing them to be RFC-10 Biobrick standard compatible.

pSB1C - Bacterial expression vector using LacI inducible promoter. Containing Chloramphenicol resistance for bacteria and have class 1 restriction enzyme cut sites allowing them to be RFC-10 Biobrick standard compatible.

pSB1T - Bacterial expression vector using LacI inducible promoter. Containing Tetracycline resistance for bacteria and have class 1 restriction enzyme cut sites allowing them to be RFC-10 Biobrick standard compatible.

pSB1K - Bacterial expression vector using LacI inducible promoter. Containing Kanamycin resistance for bacteria and have class 1 restriction enzyme cut sites allowing them to be RFC-10 Biobrick standard compatible.

pSB3A - Bacterial expression vector using LacI inducible promoter. Containing Ampicillin resistance for bacteria and have class 1 restriction enzyme cut sites allowing them to be RFC-10 Biobrick standard compatible.

pSB3C - Bacterial expression vector using LacI inducible promoter. Containing Chloramphenicol resistance for bacteria and have class 1 restriction enzyme cut sites allowing them to be RFC-10 Biobrick standard compatible.

pSB3T - Bacterial expression vector using LacI inducible promoter. Containing Tetracycline resistance for bacteria and have class 1 restriction enzyme cut sites allowing them to be RFC-10 Biobrick standard compatible.

pSB3K - Bacterial expression vector using LacI inducible promoter. Containing Kanamycin resistance for bacteria and have class 1 restriction enzyme cut sites allowing them to be RFC-10 Biobrick standard compatible.

Pseudomonas vectors provided from the Standard Vector Architecture.

(iii) List functions of inserted gene(s)

RsaL: Genetic studies show that the RsaL protein keeps the AHL system at low expression levels, it does this by competition for ppul promoter binding with ppuR. This role is very similar to the function of las/l in Pseudomonas aeruginosa. This control is however much together in RsaL and Pseudomonas Putida. The biological role of this strong repression is unknown! It could possibly be responding via de-repression of ppul to an environmental and/or metabolic signal, very similar to what occurs in P. aeruginosa. Therefore, it is negatively regulated by RsaL repressor and positively regulated by ppuR-AHL. RsaL is also negatively auto regulated.

ppuR: The putisolvins were found to inhibit biofilm formation of P. putida. Mutant ppuR show significant decrease in putisolvin production, allowing biofilm formation. transcription of ppuI is positively regulated by the PpuR/AHL complex. RsaL reported to play repression of ppul P.putida. However, the molecular mechanism can not be explained.

Both ppul and ppuR responsible for AHL production and regulation of putisolvin expression. Mutation of RsaL leads to increased AHL production, this indicates that rsaL is involved in repressing ppul and ppuR. Leading to a positive effect on putisolvin production.

Upstream of ppuR is ppuA gene, which is involved in biofilm formation. ppuA also adopts a AHL dependant expression manner. However, the ppuA gene expression (AHL dependant) alters fatty acid composition of cell membrane. Affecting cell surface properties, in turn having an effect on biofilms.

ppuI: The ppuI and LasI genes code for the enzymes that produce the signal molecule N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL), the autoinducer of QS.

The ppu QS locus identified in P. putida WCS358 is 99% identical to those described for the P. putida strains IsoF and PCL1445.

(d) An indication of the most hazardous GMM

None if the GMM is more hazardous than another. They all fall within class 1 regulations.

(e) Are you confident that for all of the GMMs covered by this assessment there are no harmful properties associated with the recipient strain, the vector, or the inserted material?

Yes, all P. putida strains being used are well known laboratory strains. There is very little risk of the strains becoming pathogenic with the genes inserted as the genes themselves are non-disease causing genes and will not change bacterial classification. The vector is also a common vector used for protein expression and is recommended under iGEM guidelines as they ship all “BioBricks” in the pSB1C(3) vector therefore it is very safe and unlikely to survive In the environment, as are the strains. Even though they contain antibiotic resistance they still require specific conditions supplemented in media to survive and the genes pose no threat. Furthermore, the expression of these genes is induced using lactose to activate the LacI pathway required to begin polymerisation and transcription. The study will follow protocols that are standard, well established and safe procedures at the University of Westminster.

(f) Are you confident that none of the final GMMs could be hazardous to humans or the environment?

Yes the final GMMs are not hazardous to any human or the environment. All laboratory strains of P. putida used in this study are without recombination or endonuclease activity, which will limit its transferability of the inserted gene sequence. There is zero risk that gene transfer would occur outside the laboratory conditions.

Signature of Proposer: R.sarwary

Date: 04/07/17

Risk Assessment approved by Genetic Modification Safety Committee

Signature of Safety Officer or Biological Safety Officer: S.Thompson

Date: 11/07/17