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Part:BBa_K729005:Experience

Designed by: Bouran Sohrabi   Group: iGEM12_UCL_London   (2012-07-10)


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Applications of BBa_K729005

UCL iGEM 2017 has further characterized improved its functionality and tested the part IrrE that has never been tested by UCL iGEM 2012 and 2016. We proved that IrrE transformed E. coli cells could withstand high salinity, oxidative stress and viscosity.

Specifically what we have improved for this part is that we repurposed IrrE as a peroxide resistance marker.

Therefore, peroxide will be a cheap chemical you could use as chemical switch or disinfectant in a future bio-streetlamp

Purpose

If IrrE harbouring cells can successfully withstand certain environmental stresses, we can apply this part onto our project e.g. producing 3D bacterial PHB structures under extreme conditions (Martian brine) and resistance to cleaning reagents when it comes to cleaning our light bulb (Oxidative stress and viscosity).

IrrE Introduction

The protein IrrE originates from Deinococcus radiodurans, where it confers resistance to radiation. When transformed into E. Coli however, it protected against salt, oxidative and thermal shock. IrrE appears to function as a global regulator of stress factor genes. So far it has been demonstrated to upregulate transcription of recA and pprA – genes which encode Recombinase A and Radiation Inducible Protein. With respect to salt tolerance, IrrE upregulates the production of several stress responsive proteins, protein kinases, metabolic proteins, and detoxification proteins. It also downregulates glycerol degradation. With this global regulatory effect, E. Coli becomes more salt tolerant.

Experimental Design

We used strain NEB 10ß and simulated three different environments by using 1M NaCl, 1.5% H2O2 and 40%v/v lubricant. All cultures started with OD600 of 0.01.

1. Control Both IrrE transformed cells and plasmid-free cells were cultured in LB media

2. 1M NaCl/LB Cells were grown in LB with 1 Molar NaCl.

3. 1.5% H2O2/LB Cells were pelleted down firt and re-suspended with 2ml of 1.5% H2O2 containing LB and incubated at 37˚C for 20 mins. Then the cells were pelleted and re-suspended to adjusted the optical density to inoculate into LB media.

4. 40%v/v lubricant/LB Cells were cultured in 40%v/v Lubricant/LB media for 30 hours. The purpose of this is to obtain more growth data than previous years and prove that it can also be applied to our light bulb.

All cultures were incubated in triplicates at 37˚C at 250 r.p.m. Optical density at 600 nm was taken over 30 hours.

Results and Summary

IrrE Full Dataset Line.png

Optical Density (OD600) vs. Time (hours) for E.coli IrrE Positive and Negative Control, averaged from triplicate experiments. (Standard Deviation shown).

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Optical Density (OD600) vs. various growing media for E.coli IrrE Positive and Negative Control cultured after 30hrs , averaged from triplicate experiments. (Standard Deviation shown).

The graphs above have shown optimistic results as we expected.


Plasmid-free cells have outgrown IrrE-transformed cells over 30 hours in control culture(pure LB)as the former one did not use the energy to express plasmid. In 1M NaCl containing LB media, normal cells showed nearly no growth whereas transformed cells were growing faster than normal ones although it has not reached an optical density as high as the ones did in pure LB.

In 1.5% H2O2 containing LB, normal cells had similar optical density but IrrE harbouring cells outgrew greatly which may demonstrate that 1M NaCl and 1.5% H2O2 inhibited the growth of both cells while IrrE plasmid can promote the resistance to these extreme conditions so that IrrE transformed cells grew better than normal ones.

In 40%v/v Lubricant/LB media, we saw both cells grew greatly but IrrE transformed cells grew better, which leads to different situation from previous year. We think it might be the other components in the lubricants killing cells because this year we used pure lubricant. This experiment further characterized the previous team’s data.


UCL iGEM 2016 proved that IrrE could be repurposed for assisting bacterial growth within lubricant.

IrrE Concept


IrrE is a part (5) from a former UCL iGEM team, where, when E. coli was transformed, protected against salt, oxidative and thermal shock. In our case we would see if E. coli transformed with IrrE is better adapted to living in lubricant than wild type E. coli. If so the IrrE transformed E. coli cells could be used as a chassi to introduce pathogen detecting and reporting systems into. Originally IrrE protects against radiation as it is a protein originating from Deinococcus radiodurans.


Experimental Design


The experiment to be conducted is to see whether IrrE transformed E. coli can more readily survive in lubricant than wild type E. coli. To test this firstly wilt type E. coli will be introduced into different concentrations of Superdrug, head quartered in Croydon, Surrey, (Code: 380900) lubricant and LB and be grown over several hours. Then the same will be done with the transformed cells and we will check whether there is a significant difference between the growth curves of the different cells in different concentrations.

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Diagram to show different growth of wild type E.coli vs. IrrE E.coli in 40% Lubricant 60% LB using Absorbance at 600nm


From the data shown in the graph above it clearly shown that the E.Coli transformed with IrrE grows better in the 40%Lubricant/60%LB solution in direct comparison with the Wild type E.Coli, which hardly grew at all. This data thus suggests that with the IrrE the E.Coli is better adapted to living in, which would allow further studies being conducted on the maximum concentration of Lubricant it can be grown in. Furthermore once that has been determined the pathogen detecting aspect of the concept can be realised. Thus overall this experiment has allowed us to show that IrrE increases the growth of the E. Coli in the Superdrug Lubricant containing: purified water, glycerine, Carbopol 940, Triethanolamine and Sodium Butyl Paraben. This supports the previous experiments that concluded that IrrE allows for better growth in saline conditions[iv], except this time in Superdrug Lubricant.

For further conceptualisation and experimentation it should be taken into consideration that within a commercialisable product the bacteria will not be under ideal growth conditions and the nutrients will be a limiting factor, especially when considering that the lubricant concentration will have to be a lot higher than in this experiment.

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