This year, we focused on improving two main components in our 2016-2017 iGEM projects. We sought to improve results for a lab protocol to quantify DNA without the use of a NanoDrop machine nor a spectrophotometer. We also looked to improve our modeling of the BBa K1616019 pDawn promoter and characterize production at low starting amounts which, to our knowledge, was not previously done. We had also explored the construction of a biological Turing system one of our initial project ideas. In the end, we left it as an academic exercise due to the complexity of the construction and limited lab access. Our proposed Turing system and related notes can be found under the Modeling tab.
Ethidium Bromide Spot Test
Being able to accurately quantify DNA is essential to many experiments in biology. For those without access to a spectrophotometer or NanoDrop machine, the process to quantify DNA becomes time-consuming and produces inaccurate results. One promising alternative is the Ethidium Bromide Spot technique. This recently developed protocol only requires a small amount of EtBr and DNA to measure DNA concentration. Last year, our team collected images of DNA diluted in EtBr to create a standard curve which predicts concentration based on pixel intensity. The paper detailing our full results can be found here: Quantification of DNA samples by Ethidium Bromide Spot Technique. This year, we provided a more rigorous measure of our curve’s accuracy and collected more data by collaborating with iGEM teams. Our end goal is to create a standardized protocol which any resource-challenged researcher or team can utilize in their experiments.
How is DNA Quantified?
DNA can be quantified through gel electrophoresis, a process that separates proteins in a sample by charge and molecular weight - with the lighter proteins traveling further down a gel and the heavier ones staying on the top. Since DNA is negatively charged, the more nucleotides in a sample (meaning the more DNA) the slower it will migrate to the end of the gel. These proteins are seen as bands on the gel - however, to truly visualize them the gel must be dyed with an agent such as EtBr.
How does EtBr Work?
Ethidium Bromide is an intercalating agent - this means that it inserts itself between the nucleotides of a nucleic acid such as DNA or RNA. It has been shown that the amount of EtBr intercalating throughout a sample is proportional to its concentration.
Once the agarose gel is stained with EtBr, it is run and imaged. During imaging, the gel is hit with UV light to visualize the bands. Fluorescence occurs because EtBr is an aromatic compound, meaning it contains many double bonds. When EtBr is hit with UV light, these double bonds absorb energy from the visible light at a certain wavelength and reflect light at others. The orange color we commonly associate with EtBr is the result of reflected light of a particular wavelength.
Developed by Ohlendorf et al., pDawn/pDusk or the light operon is a convenient system used to produce proteins and commonly used since it does not have limitations other optogenetic systems do. Last year, we chose to use the light operon to produce MazF, a cell–killing protein. This year, we used a stochastic algorithm to model protein production at low starting concentration and compared it to a deterministic model of the same system. See our model here.
In 1952, Alan Turing proposed a mechanism that explained pattern formation in developing embryos which are initially patternless in appearance. In the following years, Turing’s theory was shown to be valid as well as capable of explaining a much broader class of patterns. Despite its success as a mathematical model, the actual construction of a Turing system is extremely difficult. Read more about Turing patterns on our wiki.
However, in a recently published paper, Karig et al. and team were able to produce and prove the formation of Turing patterns in conditions that were originally thought to be incapable of doing so. We were inspired by their work to attempt to produce our own simplified version, utilizing their design with the Las/ Rhl quorum sensing system to induce the desired behavior but inserting the light operon in order to tune spot sizes.
Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J P Pearson, E C Pesci, B H Iglewski Journal of Bacteriology Sep 1997, 179 (18) 5756-5767; DOI: 10.1128/jb.179.18.5756-5767.1997
The Chemical Basis of Morphogenesis A. M. Turing Phiilosophical Transactions of the Royal Society of London. Series B, Biological Sciences, Vol. 237, No. 641. (Aug. 14, 1952), pp. 37-72.