A brief overview of the motivation and the features of our project
Background
Currently, labs use various techniques for directed evolution such as chemical mutagens, error prone PCR and bacterial hypermutator strains [1].
Chemical mutagens are a set of chemicals that interact with DNA and result in mutations [2].
Fig1(a): Working of hypermutator strains
Fig1(b): Mechanism of error-prone PCR
Error-prone PCR is used to create mutant versions of a gene by performing PCR with a low fidelity polymerase [2]. Hypermutator strains contain mutations in the DNA repair genes resulting in a very high mutation rate [3].
These techniques, however, have a variety of drawbacks making their use tedious and unsuitable for certain scenarios [2]. Chemical mutagens are generally harmful for the user and the system on which it is being used. Error-prone PCR and hypermutator strains require the knowledge of the gene or gene network. Hypermutator strains end up accumulating a lot of deleterious mutations in vital genes leading to the crashing (death) of the system. This means that they need to be constantly screened and once the mutation of interest occurs, the genes must be immediately removed and cloned into a stable strain.
Keeping in mind all these drawbacks, we decided to design a system that can regulate the mutation rate of E.coli (the genetic workhorse) and thus can be used as a useful tool for directed evolution.
Previous work on creating a tunable mutator system
Mutagenesis plasmids with various defective DNA repair system genes under the same inducer based operon have been shown to produce mutation rates which can be controlled externally [4]. In this system, variation in the levels of arabinose (a chemical inducer) in the external media has been shown to produce small variations in the mutation rates using a single plasmid. However, in such systems, the resultant mutation rates are too high and the cell viability is not maintained, making the system impractical for use.
Fig2(a): This figure taken from [4], shows the various mutagenesis plasmids (MP) created, the set of genes contained, the DNA repair pathways affected by different genes on the MPs and the resultant variation in mutation rates.
Temporary mutator strains with temperature-sensitive plasmids have been created using the gene mutD5 [5]. Alas, with this system the mutation rate can only be increased once. As to decrease the mutation rate, the replication of the temperature-sensitive plasmid is stopped by increasing the temperature and subsequent generations do not have the plasmid.
Fig2(b): This figure was taken from [5], shows the various steps involved in the usage of the mutator plasmid for accelerated evolution.
References
[1]https://www.sciencemag.org/collections/mutagenesis-techniques
[2]Packer, M. S., & Liu, D. R. (2015). Methods for the directed evolution of proteins. Nature Reviews Genetics, 16(7), 379.
[3]Greener, A., Callahan, M., & Jerpseth, B. (1997). An efficient random mutagenesis technique using an E. coli mutator strain. Molecular biotechnology, 7(2), 189-195
[4]Selifonova, O., Valle, F., & Schellenberger, V. (2001). Rapid evolution of novel traits in microorganisms. Applied and environmental microbiology, 67(8), 3645–3649. doi:10.1128/AEM.67.8.3645-3649.2001
[5]Schaaper, R. M., & Radman, M. (1989). The extreme mutator effect of Escherichia coli mutD5 results from saturation of mismatch repair by excessive DNA replication errors. The EMBO journal, 8(11), 3511-3516.
Our Approach
We aim to create a plasmid containing the gene mutD5. This mutant gene codes for a faulty ε subunit of the DNA polymerase III - the primary enzyme involved in DNA replication in E. coli, resulting in error-prone DNA mismatch repair and hence elevated mutation rates [4]. It has been shown that MutD5 outcompetes MutD in binding to DNA (dominant-negative) when introduced on a plasmid and overexpressed [4].
To achieve tunability of mutation rates using this system we decided to put it under the control of a blue light-inducible system developed by the 2014 iGEM team from USTC-China [6]. It has been shown that based on the intensity of the blue light provided, protein expression levels can be varied. Hence a correlation may be drawn between the input intensity and the number of MutD5 molecules and ultimately the mutation rate of the organism.
However, one common problem which is encountered by bacterial systems with increased mutation rate is the crashing of the bacterial system due to deleterious mutations in vital genes [7]. This is frequently observed when the mutation rate crosses a particular threshold. Accumulation of MutD5 (produced by the mutator plasmid) over time and across generations can cause mutation rates to cross the threshold and affect the stability of the culture.
To overcome this difficulty, we introduced a negative feedback loop in the genetic circuit to keep the levels of MutD5 in check.
Fig 3: A schematic representation of the working of the mutator plasmid and its use as a tool for directed evolution.
To demonstrate the working and efficacy of the mutator plasmid, we decided to dedicate the second part of our project to addressing an environmental or societal concern.
And we didn’t have to look too far to find something that has plagued Pune (or rather its waters) for quite some time. (Link to Mindspark kids)
Heavy metal pollution is a serious issue in South and Southeast Asia and is majorly caused by industrial effluents being released untreated into the rivers. An important river in Pune, the Mula-Mutha is reported to have high concentrations of heavy metals, especially lead [8].
Children are the primary suffers of lead pollution as it causes severe health
hazards, such as permanent brain damage, learning disabilities, and behavioural
abnormalities. In adults, it causes hypertension and cardiovascular diseases.
To combat this dire issue, we came up with the idea of making a lead bioremediating construct and evolving it using the mutator plasmid for higher bioremediating efficiency.
Following along the lines of the 2015 Bielefield team’s work on a lead biosensor system [9] containing genes taken from the bacteria Cupriavidus metallidurans (found in regions of heavy metal stress), we developed the design for the lead bioremediation plasmid. To test the efficacy of the bioremediating strain, we needed to look for methods to test lead in the samples. The techniques currently used are too expensive and not easily accessible. Hence we decided to test the Bielefield team’s biosensor (BBa_K1758333) and use it to measure the lead concentrations in our experiments.
References
[6]Blue light induced pathway, designed by USTC team (2014). http://parts.igem.org/Part:BBa_K1363400
[7]Sprouffske, K., Aguílar-Rodríguez, J., Sniegowski, P., & Wagner, A. (2018). High mutation rates limit evolutionary adaptation in Escherichia coli. PLoS genetics, 14(4), e1007324.
[8]Shivaji Jadhav, and Mrunalini Jadhav (2015). Heavy Metal Pollution Study of Mula-Mutha River in Pune (Maharashtra). International Journal of Innovative and Emerging Research in Engineering, 2(8), e-ISSN: 2394 - 5494.
[9]https://2015.igem.org/Team:Bielefeld-CeBiTec
