Difference between revisions of "Team:Nottingham/Human Practices"

Safety and Ethics

Lab photos

Project description

We are planning to use dCas9 and asRNA to silence the production of toxins A and B in C. difficile which is known for causing hospital-acquired diarrhoea in the western world. We want to prove a concept of asRNA and/or dCas9 as an alternative to antibiotic treatment against C. difficile. As a result, we will neutralise the negative impacts of bacteria on human health rather than completely killing it. We will create and test a promoter library in both E. coli and C. difficile, express anti-sense RNAs in C. difficile, express dCas9/sgRNA in E. coli and C. difficile. dCas9 is planned to be used to repress the reporter gene gusA, by dCas9 binding to the toxin promoters placed upstream of gusA, in E. coli and C. difficile. If this repression is successful the system will be used to repress the C. difficile toxin genes.

Whole organisms

Species used as a chassis

Parts

See our parts page

Experiments undertaken with organisms and parts

All parts will be used to create and test a promoter library in both E. coli and C. difficile, express asRNAs in C. difficile express dCas9/sgRNA in E. coli and C. difficile. The asRNAs will be used to repress the expression of toxin A and B in C. difficile. dCas9 will be used to repress the reporter gene gusA (Cas9 binding to the toxin promoters placed upstream of gusA) in E. coli and C. difficile.If this repression is successful the system will be used to repress the C. difficile toxin genes.

If toxin suppression in E. coli is successful, we plan to use E.coli DH5 alpha, E.coli Sexpress (A derivative of NEB Express containing the R factor R702) to conjugate E. coli containing asRNA or dCas9 vector into C. difficile SBRC 078 to test toxin suppression in the host organism. Given enough time, we plan to insert asRNA/dCas9 system into C. difficile bacteriophage phiSBRC as an ultimate test of our project efficacy.

How real people would use our product

Our product would be used in the human body, or in food (Examples: anti-cancer bacteria, bread made with engineered yeast, engineered rice plants)

How experts have overseen our project to manage identified risk

Michelle Kelly, one of the safety officers at the lab our team has been working in, carried out a full laboratory safety induction and introduced the risk assessments and standard operating procedures that are adhered to when working in a Category 2 laboratory. She has also set up training records for each team member which requires signing off by advisors when each lab skill has been efficiently performed. She has been working on C. difficile for more than 5 years and possesses deep knowledge into proper project design that ensures safety of the researcher.

Nigel Minton (PI) has worked on C. difficile and Clostridia for over 20 years and has overseen the project development. Four other advisors who will also oversee the work that is completed, all of whom have worked on Clostridia for at least 3 years. These researchers will be responsible for ensuring SOPs and RAs are followed on a day to day basis. All parts of our project fall under university biosafety regulations as was checked by PI and safety officer.

Rules and guidance cover that helped manage identified risks

Safety training

Our team recieved security and safety training

Topics learnt about

Areas where we handled biological materials

Biosafety level of lab

How rules, training, containment and other procedures helped manage risks identified:

Each team member has completed a building safety induction and wet lab team members have completed a laboratory work induction. Each requires the successful passing of a quiz. Each lab team member has read and signed risk assessments for all of the work they will be completing and has a training record which has to be completed by a supervisor when training has been completed.

UK Regulations and their associated guidance are implemented, the University of Nottingham has defined appropriate standards for the growth, storage, disposal and transport of biological agents and genetically modified organisms within its premises. All these requirements are written down in the safety regulation forms. Our project was reviewed by the university safety committee to ensure that it follows the biosafety regulations of the laboratory we use.

All wet lab members were trained to work in group risk 2 laboratory and using the facilities. All regulations regarding waste disposal, equipment use and handling of dangerous chemicals were introduced by the safety officer. At least one supervisor was always present in the lab to ensure that the safety protocols are followed thoroughly. In addition to standard procedures for working within a category 2 laboratory due to the nature of C. difficile no member of the team can enter the lab when they are taking antibiotics.

Compliance

Releasing organisms from our project

We will not be realsing any organisms from our project.

Gene drives

Our project does not use gene drives.

Resistance factors

We have used resistance factors, and have thus submitted an iGEM check-in form.

Use of animals

We do not use any animals in our project.

Parts not on the whitelist

We have used parts not on the whitelist, and have therefore submitted an iGEM check-in form.

Parts obtained outside of lab

We don't plan on using any parts outside of the lab.