Team:Nottingham/Lab

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
Home
Lab

Ideas

Immediately after meeting for the first time, we were tasked with finding a project that we would be working on over the summer. At first, the idea seemed daunting but before we knew it, amazing (sometimes outrageous) ideas were being suggested.

Almost a month later, after numerous presentations, we narrowed down our potential projects to five:

Exploiting the PET-degrading qualities of Ideonella sakaiensisto tackle the plastic crisis
Creating a biosensor to detect harmful substances in water bottles
using Bdellovibrio bacteriovorus (and its enzymes) to attack and kill harmful/unwanted bacteria in food processes
using mRNA interference to silence Streptococcus mutanstoxins
using mRNA interference to silence Clostridium difficiletoxins

Ideonella sakaiensis

Ideonella sakaiensisis a bacterium that can degrade plastic (PET) using two enzymes. It was discovered outside a plastic recycling facility in Japan and subsequently isolated by research teams at Kyoto Institute of Technology and Keio University(Yoshida et al., 2016). The idea of being able to tackle the plastic crisis affecting the oceans and marine life was a particularly appealing one. However, we quickly realised that we didn’t know how to transform the organism and the time-scale for how long it would take. Also, our project was very similar to other research being conducted and so it would be difficult to come up with a novel way to address the crisis.

Water bottle biosensor

According to the House of Commons Environmental Audit Committee, in their first report of the season (2017-19), in the UK alone we use ‘13 billion plastic bottles every year’ and ‘only 7.5 billion are recycled’. This prompted the team to suggest a biosensor that detects harmful substance in plastic bottles. As a result, the average consumer would buy fewer plastic bottles and reuse their bottle. However, many solutions immediately were proposed that counteracted the need for the biosensor-this included the fact that many people buy permanent water bottles that they wash and reuse; it would be more cost effective to campaign recycling efforts than to market a biosensor and it is cheaper to just recycle a plastic bottle and buy a new one than to buy a biosensor. Indeed in the report by the Environmental Audit Committee (potential stakeholders), they suggest increasing the number of water fountains in open spaces and improving recycling through a number of methods including Deposit Return Schemes (which incentivise consumers).

Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a predatory bacterium that preys on other (Gram-negative) bacterial species (Rendulic et al., 2004). Its enzymes and their mechanisms are being studied in order to better understand the bacterium for future use as a possible therapeutic agent or as a method to penetrate biofilms (Kadouri and O'Toole, 2005; Sockett and Lambert, 2004). Our team was interested in engineering the bacterium to use it (and its enzymes) as a ‘pathogen eating machine’in food processes-for example targeting Clostridium botulinum, which releases the botulinum toxin. On top of it being an interesting species to work on, we had a leading expert on the Bdellovibrio species at our university. However, the main bacterium we were targeting (Clostridium botulinum) is a Gram-positive bacterium so is not recognised by Bdellovibrio. Also, it would take many weeks to engineer Bdellovibrio for single gene mutations, which is difficult to transform using random transposon mutagenesis. This was a project that required many years which our team did not have!

mRNA interference of Streptococcus mutans

Streptococcus mutansis one of a number of bacteria involved in tooth decay and is the most prevalent.According to Public Health England, in the UK, ‘almost a quarter (24.7%) of 5 year olds have tooth decay’of which 3 or 4 teeth are affected. Dental health problems also have a heavy financial burden on the NHS-it spends around £3.4 billion per year on dental care.What makes S. mutans particularly hard to deal wit his its ability to form biofilms regulated by glucosyltransferases which catalyse sucrose to adhesive glucan. In particular, GtfB and GtfB seem to be the most important in biofilm production-mutations in the gtfB and gtfC genes disrupted microcolony formation on saliva coated surfaces(Koo et al., 2010). Our idea was to use a bacteriophage (a virus that only infects bacteria) to deliver micro RNAsor small interfering RNAs to silence those genes. One of our supervisors works with phage so she would be able to guide the wet lab team. We opted to silence the toxins rather than kill the bacteria because we wanted a way of preventing glucan formation without disturbing the balance of the oral microbiome

mRNA interference of Clostridium difficile

Clostridium difficile is an anaerobic bacterium capable of forming spores (meaning it persists in the environment).Clostridium difficil einfection(CDI) is a major infection which causes hospital and community acquired-diarrhoea.It particularly affects those who have long-term hospital stays (especially the elderly), are immunocompromised/immunodeficient (for example due to chemotherapy) and/or are on broad-spectrum antibiotics. CDI has a heavy financial burden-according to Zhang et al. (2017), the annual costs due to C. difficile infections in the US alone are an estimated $6.3 billion with almost 2.4 million days spent in hospitals.According to the Centers for Disease Control and Prevention, between 1999 and 2007 there was an increase in the estimated number of deaths due to CDI from 3,000 to 14,000 which was seen across Europe and Canada as well (McDonaldet al., 2012; Lessa et al., 2012). This has been linked to a hypervirulent, resistance strain of C. difficile.

Within the SBRC, our supervisors work with a wide range of Clostridial species in the Clostridia Research Group. Unlike with S. mutans, there was more experience within the team and so the supervisors would be able to work with us more closely and advise us on this project.

It was hard to decide between working with Ideonella sakaiensis, Streptococcus mutansor Clostridium difficile. But in the end,the team voted and chose mRNA interference of Clostridium difficile which became our project-ClostridiumdTox.

Bibliography

Design

C. difficile and Phage Characterisation

dCas 9

AS RNA

Promoters

Results

C. difficile and Phage Characterisation

dCas 9

AS RNA

Promoters

Conclusion

Future Work

InterLab

Labfolder

Lab book antisense RNA experiments

Lab book promoter experiments

Parts

BBa_K2715001

The Ribosomal Binding Site was taken from Clostridium acetobutylicum as it is a well characterized strong RBS driving transcription of the Thiolase gene. The promoter is from a previous team (iGEM2006_Berkeley), part BBa_J23114 which is from a family of constitutive promoters known as “Anderson promoter after the founder Prof. Chris Anderson, J23114 was a relatively weak member compared to the rest of the family. The GFP gene is a commonly used reporter gene as it allows expression to be measured in fluorescence units.
BBa_K2715002

Promoter taken from Clostridium sporogenes ferrodoxin gene, the RBS is also from the ferrodoxin gene.

BBa_K2715003

Clostridium difficile toxin B gene promoter. This was an important promoter to test as it was the target for the asRNA team to reduce toxin production.

BBa_K2715004

BBa_K2715114

The Ribosomal Binding Site was taken from Clostridium acetobutylicum as it is a well characterized strong RBS driving transcription of the Thiolase gene. The promoter is from a previous team (iGEM2006_Berkeley), part BBa_J23114 which is from a family of constitutive promoters known as “Anderson promoter after the founder Prof. Chris Anderson, J23114 was a relatively weak member compared to the rest of the family. The GFP gene is a commonly used reporter gene as it allows expression to be measured in fluorescence units.

BBa_K2715106

The promoter is from a previous team (iGEM2006_Berkeley), part BBa_J23106 which is from a family of constitutive promoters known as “Anderson promoter after the founder Prof. Chris Anderson, J23106 was a relatively weak member compared to the rest of the family. The Ribosomal Binding Site was taken from Clostridium acetobutylicum as it is a well characterized strong RBS driving transcription of the Thiolase gene. The GFP gene is a commonly used reporter gene as it allows expression to be measured in fluorescence units.

BBa_K2715119

The promoter is from a previous team (iGEM2006_Berkeley), part BBa_J23119 which is from a family of constitutive promoters known as “Anderson promoter after the founder Prof. Chris Anderson, J23119 was a relatively weak member compared to the rest of the family. The Ribosomal Binding Site was taken from Clostridium acetobutylicum as it is a well characterized strong RBS driving transcription of the Thiolase gene. The GFP gene is a commonly used reporter gene as it allows expression to be measured in fluorescence units.

BBa_K2715007

BBa_K2715008

BBa_K2715010

BBa_K2715011

BBa_K2715012

BBa_K2715013

BBa_K2715009

BBa_K2715020

BBa_K2715022

BBa_K2715024

BBa_K2715019

BBa_K2715021

BBa_K2715023

BBa_K2715014

BBa_K2715015

BBa_K2715016

BBa_K2715017

BBa_K2715018

Ideas Design Results Conclusion Future Work Interlab Labfolder
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