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<p> Our first step was to try a proof of concept knockdown technique; Antisense RNA gene silencing. This is where the reverse complement of the coding strand is transcribed and interferes with the ispB mRNA hence affecting translation. | <p> Our first step was to try a proof of concept knockdown technique; Antisense RNA gene silencing. This is where the reverse complement of the coding strand is transcribed and interferes with the ispB mRNA hence affecting translation. | ||
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Revision as of 17:48, 16 October 2014
The ultimate safety tool
“Any technological advance can be dangerous. Fire was dangerous from the start, and so (even more so) was speech - and both are still dangerous to this day - but human beings would not be human without them.” - Isaac Asimov
The wide use of genetically modified organisms causes concerns on how they will interact in the natural environment. In particular could the genetically modified microbes escape our constrains, and outcompete the organisms found in the natural ecosystem? Could the DNA we inserted into a specific bacteria be transmitted, with unknown spread of information?
Xenobiology is the part of synthetic biology that mostly implements the term "synthetic" by creating organisms that are unable to survive in the natural environment and necessitate an artificial intervention from man to exist. It aims to create a synthetic "man-made" version of Biology that respects the definition of life, but is based on entirely different mechanisms to function. The biochemistry of a xeno-organism uses different materials from the ones explored by Biology and is therefore incompatible with other forms of life. This allows a much higher level of control: a xeno-organism will not be able to find the xenocompounds in the natural environment, and will not be able to use bacterial communication systems.
We explored this possibility with the longer term vision of creating an X. coli which lives is metabolically dependent on azo dyes. An alien form of life, different from the one we know, will merge synthetic chemistry with synthetic biology - allowing the remediate the damage that the first one caused and making the remediating agent dependent on the toxic compounds. This system would be completely incompatible and invisible to regular biology, now we can ask: is alien life safe enough?
Designing Xeno-Coli
1. Choosing a metabolic pathway
In order to create a xenobiological organism with a metabolic firewall we decided to try to design cofactor that would be essential to E-Coli metabolism that could be derived from our Azo Dye waste products. This cofactor would need to be functionally similar to an existing molecule in the E-Coli metabolism.
Image Credits: EcoCyc Metabolic Database To see the full and interactive tree visit: http://goo.gl/bEK46y
Given the vast number of cofactors in the e-coli we needed a method to select the molecular structures closest to azo dye waste products. To solve this problem we developed a computer program that would do just that
2. Designing the Xeno-quinones
The pathway chosen from XenoRank was that of the cofactors ubiquinone and menaquinone. They are the electron buffer substituent of coenzyme Q in complex III of the electron transport chain that creates ATP on the membrane of E-Coli.
Ubiquinone is the electron buffer used in aerobic conditions and menaquinone in anaerobic conditions. Both quinols produce protons and electrons to create the electrochemical gradient used later in Complex IV and V to yield ATP.
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<img src=”https://static.igem.org/mediawiki/2014/8/8a/Sir-Xenoquinone-Pathway-from-Mordant-Brown-33.png”>
<img src=”https://static.igem.org/mediawiki/2014/d/de/Sir-Bondiquinone-Pathway-from-Acid-Orange-7.png”>
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<h4> 3. Replacing the Natural Quinones </h4>
<p>In order for our organism to become an autotroph to our xenobiological quinones it was necessary for us to remove the original quinones. The best approach it seemed was to do a gene knock out for a biosynthesis step of ubiquinone and menaquinone. The metabolic pathway provided a number of options
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<!--Insert picture of tube map for biosynthesis-->
<p>A search of the literature showed that a knockout of ispB successfully killed e-coli colonies. ispB codes for the protein that attaches the hydrophobic side chain on the quinones, allowing them to dock in the membrane. As ispB is used to synthesise all electron buffer quinones, it was the perfect knock out as it meant that only one was required.</p>
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See paper here: http://goo.gl/MlMY3c
Antisense RNA gene Silencing
Our first step was to try a proof of concept knockdown technique; Antisense RNA gene silencing. This is where the reverse complement of the coding strand is transcribed and interferes with the ispB mRNA hence affecting translation.
The design for the primers that would amplify the gene contained the bbk prefix and suffix. The forward primer contains suffix, reverse primer contains prefix hence sequence inserted as reverse complement into the vector
The gene was then cloned into pSB1C3 under the control of lac inducible promoter to observe effect on growth.
The knockdown showed to be unsuccessful in reducing bacteria growth and therefore cannot be use to create a xenobiological organism. The next step to try was a full knock out using the crispr technique
CRISPR Knock Outs
The CRISPR technique is based on the bacterial ability to cut out or replace viral DNA that has been inserted into it’s plasmids. We now can now utilise the Cas9 protein with a target sequence to do cut out of any gene within a plasmid.
The Cas9 protein is expressed with gRNA that is complimentary for the target sequence within the DNA you wish to cut out with a PAM (Protospacer Adjacent Motif) sequence downstream of the target. The PAM sequence in e-coli must be an NGG sequence (N representing any base)
To design our gRNA we used the target sequence online design tool DNA 2.0: https://www.dna20.com/eCommerce/cas9/input
Our gRNA target sequences were as follows:
Despite our plans there was not sufficient time to complete a knockout using CRISPR and that will be our next step beyond the competition.
References:
- Wright, O., Stan, G.-B., and Ellis, T. (2013). Building-in biosafety for synthetic biology. (Review) Microbiology, 159, 1221-1235. http://www.ncbi.nlm.nih.gov/pubmed/23519158
- Okada, K., Minehira, M., and Zhu, X. (1997). The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. Journal of Bacteriology, 179, 3058–3060. http://www.ncbi.nlm.nih.gov/pubmed/9139929
- Søballe, B. , Poole, K. R. (1999). Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. (Review) Microbiology, 145, 1817-1830. http://www.ncbi.nlm.nih.gov/pubmed/10463148
- Schmidt, M (2010). Xenobiology: A new form of life as the ultimate biosafety tool Bioessays, 32, 322-331. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909387/
- Malyshev, D.A., Dhami, K., Lavergne, T. et al. (2014). A semi-synthetic organism with an expanded genetic alphabet Nature, 509, 385-388. http://www.nature.com/nature/journal/v509/n7500/full/nature13314.html
