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

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We are planning to use dCas9 and asRNA to silence the production of toxins A and B in C. difficile. C.difficile 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.
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We are planning to use dCas9 and asRNA to silence the production of toxins A and B in <i>C. difficile</i>. <i>C.difficile</i> 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 <i>C.difficile</i>. As a result, we will neutralise the negative impacts of bacteria on human health rather than completely killing it.
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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.  
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We will create and test a promoter library in both <i>E. coli</i> and <i>C. difficile</i>, express anti-sense RNAs in <i>C. difficile</i>, express dcas9/sgRNA in <i>E. coli</i> and <i>C. difficile</i>. 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 <i>C. difficile</i>. If this repression is successful the system will be used to repress the <i>C. difficile</i> toxin genes.  
 
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Revision as of 08:53, 17 October 2018

Clostridium dTox Project
Project description Abstract
Human Practices
Overview Silver Gold Safety and Ethics
Public Engagement
Discovery day School workshops Media
Lab
Ideas Design Results Conclusion Future Work Interlab Labfolder
Modelling
Introduction Summary Lytic Phage Models Temporate Phage Models Comparsion of all three models Methodology Parameters References
Collaborations
The University of Warwick Team BioMarvel- Korea The University of Warwick and Imperial College London UK Meetups
Achievements
Medal Criteria Parts Improved parts Demonstrate General conclusion
Team
Students Supervisors Principal Investigators
Attributions

Overview

Welcome to the Nottingham iGEM 2018 Human Practices pages. This overview will introduce you to the projects that we investigation for our Human Practices efforts, as well as why we investigated them and what our results showed.

For our silver medal criteria, we complied 3 reports on the impact of our project, how people feel about phage therapy and what current legislation there is on phage therapy. The objective of compiling these reports was to investigate whether our work was responsible and good for the world. We engaged with a variety of communities including field experts, community discussion groups and scientific articles and research papers to gain a wide scope of information to demonstrate the positive impact of our work.

For our gold medal criteria, we analysed how we could integrate the findings of our discussion group and field expert interviews into our project. We chose to document this via a clear flowchart so that we can explain how we have taken on board feedback and modified our project as a result. We hope enjoy learning about our Human Practices work on this year’s project. Click the dropdown menu and pages to find our more details about our work!

Silver

How will our project positively impact the world
Clostridium difficile infections are the dominant cause of healthcare associated diarrhoea in the western world. With increasing outbreaks of hyper virulent strains, due to the alarming rates at which bacterial pathogens are developing antibiotic resistance...
Read more
Exploring the emotions that people feel towards phage therapy through a representative sample video
June 18 2018
This report intends to explore what emotions people feel towards phage therapy.
Read more
Legistlation report
Legistlation description here.
Read more
Do the elderly have difficulties taking prescription medication?
This report will analyse a range of literature with the objective of identifying whether the elderly have any difficulties in taking prescription medications and if so, why this is...
Read more

Gold

For our gold medal criteria, we analysed how we could integrate the findings of our discussion group and field expert interviews into our project. We chose to document this via a clear flowchart so that we can explain how we have taken on board feedback and modified our project as a result. We hope enjoy learning about our Human Practices work on this year’s project. Click the dropdown menu and pages to find our more details about our work!

HP Gold guide
July 2018
Read more
Report on Discussion Groups and interviews
July 2018
Read more
Integration flowchart
July 2018
We created a detailed flowchart, explaining how we met and achieved the Gold Medal criteria through our Human Practices.
Read more
Information leaflet
July 2018
Following our discussion group, we decided there was a need to create a leaflet to educate people about Phage Therapy.
Read more

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. C.difficile 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

E.coli DH5 alpha, E. coli sexpress (NEB express + R702)
C.difficile 1801 (clinical isolate), C.difficile bacteriophage

Risks the organisms pose

E.coli is the model lab organism and has low, but not non-existent, virulence. It is very unlikely to cause disease in healthy adults. The risk group mostly includes young, elderly, immunocompromised people and is spread through injection or contamination of broken skin with bacteria which can cause inflammation. The release of bacteria in the environment poses the risk of spreading of antibiotics resistance. By following appropriate laboratory safety precautions and waste disposal regulations, we can minimise unsafe exposure to the bacteria and prevent release into the environment.

Species used as a chasis

E.coli DH5 alpha, E. coli sexpress (NEB express + R702)
Clostridium difficile 1801 - ribotype 078

Risks the chasis species pose

The SBRC have risk assessments and waste disposal management that will ensure that chassis are not released into the environment as they possess antibiotic resistance that could be spread to other bacteria. Except that, E.coli strains used do not possess pathogenic properties and therefore pose minimal to no risk to human health.

C. difficile could pose a risk to human health in immunocompromised patients or patients receiving antibiotic therapy. However, they would need to ingest spores, which can be removed with hand washing. Colleagues or team members are not authorised to enter the lab whilst on antibiotic therapy. Providing correct PPE is worn and hand washing procedures are followed, C. difficile will not pose a risk to the lab-based team members. Release of C.difficile into environment could cause a hazard due to production of spores that can survive in the environment for a long time. The lab follows specific disposal rules for C.difficile specifically.

E.coli sexpress (NEB express +R702) is a new strain created at SBRC Nottingham and the research paper on it has not been published yet. Clostridium difficile 1801 is a clinical isolate of C. difficile belonging to PCR ribotype 078.

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 supression in E.coli is successful, we plan to use E.coli sexpress (NEB express + R702) to conjugate E.coli containing asRNA or dCas9 vector into C.difficile 1801 to test toxin supression in the host organism. Given enough time, we plan to insert asRNA/dCas9 system into bacteriophage specific to C.difficile 1801 (ribotype 078) as an ultimate test of our project efficacy.

Risks that could arise from experiments

In our project we use all the basic microbiology techniques and equipment which may possess risks of causing disease after long exposure or physical injuries. To ensure safety of all lab-based team members, specific training for each technique was given with all safety precautions explained. All team members could use bunsen burner without burning themselves or others; bench centrifuges were handled properly due to aerosols production; SYBR Safe was handled in the designated area to minimise carcinogenic-related risks; sharp equipment was handled safely to prevent injuries, etc. Throughout the work in the lab, all team members wore PPE at all times, with extra precautions taken when working in the lab.

The introduction of antibiotic-resistant genes into bacteria can pose a threat to risk groups if released. To minimise the risk and prevent the unwanted release, the safety protocols for handling and disposal of genetically-modified bacteria were followed at all times (e.g. putting waste in appropriate bins, autoclaving, washing hands after leaving the laboratory, etc). Woking in biosafety level 2 anaerobic cabinet can be challenging for someone with not much experience due to relatively hot environment and continuous limitation of movement. To minimise the risk of sudden loss of consciousness or other extreme situations, at least one other person was present during work in the cabinet. This was applied to all our work in the lab - team members worked at least in pairs to ensure maximum safety during experimental procedures. At least one supervisor was present in the lab at all times during lab work. When working with phage, it is important to follow strict safety regulations as inappropriate handling may cause risk to both the person who carries out experiment as well as other people around him/her. People working with bacteriophage received proper safety training and were introduced to all specific rules.

How real people would use our product

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

Risks associated with real use

Our project is basically focused on proving the concept that one can use bacteriophage therapy against C.difficile. We wanted to explore alternative treatment options due to the increasing problem of antibiotic resistance. Of course, we do not claim in any way that our final product will be straight away ready for therapeutic use. In the best scenario, it would need to be tested in vivo in preclinical studies and in clinical trials to assess their safety and test for any side effects. This would be done with ethical approval. Pharmacokinetic studies and biochemical experiments would be carried out to determine optimum conditions for use.

Our project is focused on using phage. Even though it is specific to C.difficile, its specificity may vary depending on different conditions. More research would be needed to investigate possible non-specific infection.

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

University of Nottingham safety management checklist.
The Control of Substances Hazardous to Health Regulations. This document provides regulations on laboratory biosecurity and biosafety.

Safety training

Our team recieved security and safety training

Topics learnt about

Lab access and rules (including appropriate clothing, eating and drinking, etc.)
Responsible individuals (such as lab or departmental specialist or institutional biosafety officer)
Differences between biosafety levels
Biosafety equipment (such as biosafety cabinets)
Good microbial technique (such as lab practices)
Disinfection and sterilization
Emergency procedures
Transport rules
Physical biosecurity
Personnel biosecurity
Dual-use and experiments of concern
Data biosecurity
Chemicals, fire and electrical safety

Areas where we handled biological materials

Open bench
Biosafety cabinet

Biosafety level of lab

Level 2 (moderate risk)

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 Nottinghamhas 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.

Safety form
Overview Silver Gold Safety and Ethics
About our sponsors