Description

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 not only be correct, but also capable of explaining a much broader class of patterns. Everything from leopard spots to zebra stripes to leaf veins are classified as Turing Patterns and can be easily recreated in computer simulations. Despite its success as a mathematical model, the actual construction of a Turing system is extremely difficult due to the un-physical requirements for a “Turing capable” biological system. Read more about Turing patterns on our wiki page.

However, in a recently published paper, Karig et al. and team were able to produce and prove the formation of Turing patterns outside the original regime. We were inspired by their work to attempt to produce our own simplified version, still utilizing the Las and Rhl quorum sensing system to induce the desired behavior but using our past experience of working with the light operon to tune spot sizes. Due to a lack of access to a lab we were not able to test our hypothesis as desired. However, we recognized the benefit of further characterizing the light operon so that we or future teams could get a head start. We also wanted to make an effort to support resource limited teams so we worked on improving the standardization of last year’s Ethidium Bromide Spot test. This would enable teams and researchers a method of quantifying DNA without the use of a spectrophotometer or nanodrop machine within a reasonable threshold of the true quantity.

Light Operon

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 – pDawn/pDusk – to produce MazF, a cell – killing protein. This year, we planned on implementing the light operon in a Turing system – however, due to resource limitations, as a first step we modeled it stochastically. Learn more about the light operon here.

Ethidium Bromide Spot Test

Quantifying DNA may be time – consuming, especially if results are not as expected. One protocol that may help speed up the process of DNA quantification is the Ethidium Bromide Spot technique. The protocol, developed recently, requires a minimal amount of EtBr and DNA to measure DNA concentration. It also decreases contact time with harmful EtBr and does not require the use of a spectrophotometer or nanodrop machine. Last year, our team collected data from the images of diluted DNA in EtBr to create a standard curve varying pixel intensity of the DNA sample and its concentration. This year, we are improving our standard curve by finding its accuracy and adding more data – in hopes of measuring DNA as accurately as a spectrophotometer or nanodrop machine may. We hope to further standardize the EtBr Spot protocol so future beginner or funding-limited researchers and teams working with DNA can accurately perform 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.

Citations