Improvement of an Existing Part

TrigGate's Parts Improvement

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Introduction

One of the most basic principles upon which the field of Synthetic Biology is relied on is genetic stability – the ability of genetic constructs to preserve functionality over long periods of time by avoiding loss-of-functionality mutations. Expression of hetrological genes requires energy production, resulting in reduced fitness of the hosting organism, naturally, as it is forced to invest resources in expressing the forgein gene and not only in growing and dividing (as it normally does). In order to form a stable construct, which avoids mutations as much as possible, the tools of synthetic biology must be brought into action.

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Design of the Experiment and methods

As a part of their project, the iGEM TAU 2020 group has designed the Evolutionary Stability Optimizer (ESO- https://www.cs.tau.ac.il/~tamirtul/ESO/) tool, a software which enables the formation of evolutionary stable constructs in an automatic manner, while taking into account both mutational and recombinational sites that could be evolutionarily unstable. We have decided to use the ESO tool in order to improve the evolutionary stability of existing iGEM genetic constructs, available in the iGEM Parts Registry. In order to accomplish that, we have chosen a part out of the registry (BBa_K079050; a DNA damage reporter, composed of a GFP reporter which is controlled by a constitutive promoter (J23100) and an operator (LexA 2)), optimized it using the ESO software in two fashions, one with codon optimization and another without codon optimization. The basic role of the ESO tool is to change the DNA sequence, while preserving the amino acid sequence in order to avoid mutational and recombinational hotspots (for further reading please visit - https://pubs.acs.org/doi/10.1021/acssynbio.1c00426). When choosing to perform codon optimization, the software also optimizes the codon usage of the sequence according to the organism the user has chosen.

All the DNA constructs were sent then for synthesizing by TWIST bioscience , after receiving them we have designed an evolution experiment, through which we have examined the evolutionary stability of the standard iGEM part in comparison with the evolutionary stability of the same part after optimization taken out by the ESO tool (with and without codon usage optimization). We wanted to show that the optimized parts reserve their functionality throughout longer periods of time than the non-optimized one, so we have designed the experiment in a way that has enabled us to follow the process of losing functionality: the part has contained a sequence encoding for a fluorescent protein (a GFP reporter), so we have planned to witness the time it takes the optimized part to lose its fluorescence due to evolutionary instability, in comparison with the same time it takes the non-optimized one. Both optimized and non-optimized parts have been transformed to DH5-α Competent E. coli bacteria, which have been placed in a 37⁰C incubator for a night in order for them to grow. After that, a colony of each bacteria containing a specific part has been nurtured with 5 ml of LB + chloramphenicol media and has been placed in a 37⁰C roller. Each part has been tested as a triplicate, so for each one, three separate bacterial colonies have been grown in different tubes, and their fluorescence levels have been analyzed separately over time. During the experiment, the media of the part-containing bacteria has been replaced each day, so 5 μl of bacteria have been passed down to a new 5 ml of LB + chloramphenicol and have been placed again in the 37⁰C roller. After each such replacement, the fluorescence of the bacteria have been analyzed using a platereader device: 1 ml of the bacteria + media mixture have been centrifuged for 1 minute at 14,000 rounds-per-minutes, then the supernatant has been discarded so the pellet could be mixed with 1 ml of PBS X1. This mixture has been divided between a 200 μl-containing 96-well plate, and the fluorescence levels have been captured using the Gen5 software, while the fluorescence of each part has been analyzed using its specific major excitation and emission peaks.

It is important to mention that in order to evoke activity of the BBa_K079050, which is a DNA damage reporter, we have added to the bacteria trace amounts (enough to activate the part while not causing any major DNA damage - 0.5ul per 5ml tube) methyl methanesulfonate (MMS) before the measurements so a damage to the DNA will initiate the part's activity.

The sequences of the non-optimized, the merely-optimized (without codon optimization) and the fully-optimized (with codon optimization) are as described at the last section of this page.

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Results

The results of the experiment are conclusive: the expression levels of the genetic construct and its evolutionary stability have been improved following an optimization taken out by the ESO software: the expression levels of the non-optimized part were lower in a significant manner than those of the mild optimized one (p-value = 0.0004) and those of the full optimized one (p-value = 9.3x10^-7) (the p-values of the expression levels were calculated over the optical densities values of the bacteria, measured via a platereader device; the statistical groups were formed by averaging the technical and biological repeats of each version of the part – non-optimized, merely-optimized and fully-optimized); the stability has been measured as the time it took the construct to lose its fluorescence (resembling its functionality), and it could be seen that the original non-optimized construct has lost its fluorescence signal faster than both the optimized constructs (while the mild optimized construct has lost its fluorescence faster than the full optimized one).

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Conclusion

It could be inferred that the ESO tool bears the ability to improve the evolutionary stability and the expression levels of genetic constructs. This ability is an important one in light of the growing need of Synthetic Biology applications to preserve both functionality and fitness of genetic constructs.

We have uploaded the fully-optimized version of the part to the iGEM parts registry. By doing that, we have formed a new part. A link to the new part is attached:

http://parts.igem.org/Part:BBa_K4219000