Project description
Motivation
As long as mankind remembers, different diseases struck from time to time and demanded millions of lives.
Maybe the most fatal of these epidemic outbreaks was the 1918 flu pandemic, which killed between 50-100 million people worldwide.
Derived from a simple influenza virus only a few mutations were necessary to change this virus into one of the deadliest threats
that ever existed. Pandemic viral infections like this were the reason for our project selection. Our highest
motivation was to create a system to counteract these
extremely high risk potential slumbering in commonly known and seemingly not to dangerous viruses. Of course the influenza virus
stated 1918 a much higher risk than it does today. But still viruses are very hard to treat and especially members of the family flaviviridae
like Zika or Dengue virus, show a high risk potential because of their high mutation rate.
We found a way to create in a short period of time antibody-like proteins.
Evobodies are binding
proteins that are able to quickly adapt to altered targets like viral hull proteins and
re-establish binding properties in extremely short periods of time
The limiting factors in this process are the rate at which mutations happen in the gene of the Evobody
and screening of different proteins. This is the reason why we wanted to build a mutation inducing system
which is not only able to change basepairs in vivo at a very high frequency,
but is also specific enough to provide stability in culture
and does not interfere too much with growth properties of the individual cell.
The revolutionary part of Evobody generation is the combination of an in vivo mutagenesis
and a selection system which is also capable of screening our mutants during the process of
cell cultivation. Due to our constant interaction of altered binding proteins and the in vivo
selection, we were eager to increase the mutation rate while retaining normal growth conditions. That is
why we did not only calculate the mutation rate of our two different mutagenesis approaches,
but also determined the growth rate under
different conditions. We compared mutating strains to strains without any amplified mutagenesis whatsoever to
quantify any influences regarding the growth rate and used the data in (eventually only compared to
)our modeling to further analyse and predict
our mutations.
Generation of binding proteins by directed evolution
![](https://static.igem.org/mediawiki/2016/4/44/Bielefeld_CeBiTec_2016_10_15_mutation_UNGUGIDNA.png)
The starting point - synthetic binding protein library
The randomization strategy as well as the choice of the protein scaffold is a key part of library generation. We identified amino acids, which are present in most protein-protein interaction areas and created a randomization scheme so that only these amino acids are encoded in the antibody-mimetics binding region. Read more about our library design here.
![](https://static.igem.org/mediawiki/2016/4/44/Bielefeld_CeBiTec_2016_10_15_mutation_UNGUGIDNA.png)
Survival of the fittest - bacterial two-hybrid
![](https://static.igem.org/mediawiki/2016/9/9e/Bielefeld_CeBiTec_2016_10_14_project_description_selection.png)
Accessing the sequence space - in vivo mutagenesis
![](https://static.igem.org/mediawiki/2016/d/d4/Bielefeld_CeBiTec_2016_10_14_project_description_mutation.png)
In detail, we will compare two different possibilities to diversify our binding proteins. The first approach is the use of an error-prone polymerase I in an otherwise Pol I temperature-sensitive E. coli strain. (Camps et al. 2003) Growth at a non-permissive temperature should result in accumulation of mutations in the part of the genom maintained by DNA polymerase I. The interesting idea behind this approach is the fact that large parts of plasmids carrying an origin of replication from the ColE1-familiy are replicated by the polymerase I. (Camps et al. 2003; Camps 2010) Because of this, the usage of the error-prone polymerase I should mutant mainly our Evobody sequence on a plasmid. Thereby off-target mutations, which are a major obstacle of in vivo mutagenesis, should be minimized. (Camps et al. 2003)
Our other approach is based on creating a plasmid borne hypermutator system by modulating the E. coli DNA fidelity systems. (Badran und Liu 2015) We will express known mutator genes under tight regulation from a plasmid. By using a plasmid borne mutator system, in contrast to the more classical approaches of incorporating the mutator genes directly inside the genom. (Agilent Technologies; Greener et al. 1997) Thereby we want to circumvent the known problems with globally increased mutation rate, which are genetic instability or general unviability.
Over the course of our project we want to find out which mutagenesis system is most suitable to our directed evolution approach. Therefore we will compare both possibilities in terms of mutagenesis rate, -spectrum, -controllability and -specifity. How will we do this? Find out on our mutation mainpage.
Improve a part
Mutator gene dnaQ926 - BBa_K1333108
Introduction
Literature
- Agilent Technologies: XL1-Red Competent Cells. Instruction Manual 2015.
- Badran, Ahmed H.; Liu, David R. (2015): Development of potent in vivo mutagenesis plasmids with broad mutational spectra. In: Nature communications 6, S. 8425. DOI: 10.1038/ncomms9425.
- Camps, Manel (2010): Modulation of ColE1-like plasmid replication for recombinant gene expression. In: Recent patents on DNA & gene sequences 4 (1), S. 58–73.
- Camps, Manel; Naukkarinen, Jussi; Johnson, Ben P.; Loeb, Lawrence A. (2003): Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I. In: Proceedings of the National Academy of Sciences of the United States of America 100 (17), S. 9727–9732. DOI: 10.1073/pnas.1333928100.
- Fijalkowska, I. J.; Schaaper, R. M. (1996): Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe. In: Proceedings of the National Academy of Sciences of the United States of America 93 (7), S. 2856–2861.
- Greener, A.; Callahan, M.; Jerpseth, B. (1997): An efficient random mutagenesis technique using an E. coli mutator strain. In: Molecular biotechnology 7 (2), S. 189–195. DOI: 10.1007/BF02761755.
- Teng, Grace; Papavasiliou, F. Nina (2007): Immunoglobulin somatic hypermutation. In: Annual review of genetics 41, S. 107–120. DOI: 10.1146/annurev.genet.41.110306.130340.