Team:Groningen/MutationRates

CryptoGE®M
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Acknowledgements

Mutation rates during the CryptoGErM process

We present an estimate of the number of bases expected to mutate during the whole process of insertion, sporulation, sending, treatment, germination and reading, considering the mutation rates known to Bacillus subtillis.

Figure 1. A mutation can occur in the sequence during one of the steps that our system requires. In essence: message insertion, sporulation, physical sending, treatment of spores and reading.

Reported mutation rates of Bacillus subtilis

A mutation in the genome of B. subtilis can occur in a coding or noncoding DNA sequence. If it occurs in a coding part, it can be an evolutionary advantage or a disadvantage. When it is a disadvantage, the bacteria will have less chance of passing its mutated genes to the next generation. Thus, some reports that measure the mutation frequency under evolutionary pressure conditions may underestimate its real value. As our message is a noncoding sequence, it will not be an evolutionary disadvantage (or an advantage) if it mutates or gets lost.

Maughan et al. (2006) measured the mutation rates of B. subtilis in an antibiotic-resistance gene under conditions that did not require such a gene to survive [3]. They also found that the mutation rate itself may increase as generations pass by until it reaches a plateau. Nonetheless, the necessary number of generations to see a difference in mutation rates is higher than those our methodology requires. For that reason, the value of \(0.5 \cdot 10^{-8} \frac{mutations}{bp \cdot generation}\) will serve as a good estimate.

Number of generations

The probability that our message is changed by a random mutation also depends on the number of generations that have passed since the insertion until the sequencing. The doubling time of B. subtilis is estimated to be 120.1 min[1] but depending on the growth phase this value may vary. For instance, Maughan (2006) reports that the number of generations that passed in one week was 500 (doubling time = 201 min)[3].

StepTime (h)Generations required
Message insertion – sporulation7236
Sporulation – sending00
Sending – germination01
Germination – message reading24 – 4812 – 24
TOTAL:~50
Figure 2. Using our system we take approximately 50 generations of cell growth counting from the sequence insertion to sequencing.

DNA damage by UV radiation

We planned to use a photoswitchable antibiotic as one of our bio-layers of security. Due to some experimental issues explained in the respective section, we are not using that approach anymore. However, we had considered that it might increase the mutation frequency to a considerable extent.

First, our calculations (shown in the respective section) indicated that the spores would survive the required UV treatment required for antibiotic activation (325 nm, 30 min, 28.8 kJ/m2) and experimentally we did find them to survive it.

In general, spores are 10-50 times more resistant to UV damage than vegetative cells and spores during the germination phase undergo a transcient period in which resistance to UV damage is even higher than that of normal spores. Spores also have a very interesting way of avoiding UV damage that is advantageous for our system, having an almost free-error repair system[2].

Tanooka (1978) reports that upon irradiation of B. subtilis with UV (254 nm, 1-100 J/m2) the mutation frequency increases in a linearly on a log-log scale, being the spores approximately 10 times more resistant than vegetative cells[5].

Calculation

As the UV treatment is not used anymore, it is not taken into account in this calculation.

\[ 50 generations \cdot 0.5 \cdot 10^{-8} \frac{mutations}{bp \cdot generation} \cdot 4,215,000 \frac{bp}{genome} = 1.05 \frac{mutations}{genome} \]

Thus, each line of B. subtilis will have around one mutation by the time we want to read the message. This mutation does not have to be inside our message, and does not have to be the same bp mutated for each cell line as explained in the next diagram.

Figure 3. Around one mutation will occur somewhere in the bacterial genome. Such mutation does not have to be inside our message and it does not need to be the exact same mutation for every cell line.
References
  • [1] Burdett (1986). Growth kinetics of individual Bacillus subtilis cells and correlation with Nucleoid extension. Journal of Bacteriology, 167(1):219-230
  • [2] Lenhart (2012). DNA repair and genome maintenance in bacillus subtilis. Microbiology and Molecular Biology Reviews, 76(3):530-564
  • [3] Maughan (2006). The population genetics of phenotypic deterioration in experimental populations of Bacillus subtilis. Evolution 60(4): 686-695
  • [4] Moeller (2014). Resistance of Bacillus subtilis spore DNA to lethal Ionizing radiation relies primarily on spore core components and DNA repair, with minor effects of Oxygen Radical Detoxification. Applied and Environmental Microbiology, 80(1):104-109
  • [5] Tanooka (1978). Mutation induction with UV and X-Radiations in spores and vegetative cells of Bacillus subtilis. Mutation Research, 49:179-186
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