Team:CUNY Kingsborough/Collaborations
Collaborations
Improving on DNA Quantification Through Standardizing EtBr Spot Test
Last year, our project involved diluting 1 uL of DNA at varying concentrations in 10 uL EtBr and fluorescing the spot of diluted EtBr using a UV lamp. Afterwards, we photographed the spots and using ImageJ, an imaging program that can measure pixel intensity, we analyzed each spot. From there we created a standard curve using known concentrations of DNA – which we can then use to approximate how much DNA there is in a sample of an unknown concentration – based on its pixel intensity.
Which takes us to…
This year! We asked several teams to make dilutions of DNA – 1 uL of DNA of an unknown concentration into 9 uL EtBr (1 ng/uL), hit the samples with UV light, and send the images to us. We will compare the pixel intensity of the sample with a known DNA concentration to the predicted pixel intensity based on our standard curve from last year. We hope to see how accurate our standard curve is, with the more samples the better. Special thanks to teams HD Resolution in NYC, USA and Tec de Monterrey_Gdl in India!
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.
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Predicting Collateral Cleavage Activity of Cas13a
This year, we collaborated with the Columbia University iGEM Team to help them model the collateral cleavage activity of Cas13a, an enzyme used to cleave RNA in CRISPR technology. Our team provided the system of equations and resulting graphs.
What is a CRISPR?
CRISPRs, or Clustered Regularly Interspaced Short Palindromic Repeats, are DNA sequences commonly found in bacteria and archaea that serve as a defense mechanism. They originate from pathogens that previously infected the host are are used to detect said pathogens when they try to re-infect the host.
A Nuclease Defends - Its Mechanism of Action
When pathogens that have previously infected the host try to re-invade, host nucleases use CRISPR sequences to detect the RNA sequences of the pathogen. A nuclease is an enzyme the cleaves nucleic acids in DNA or RNA. In CRISPR derived technology, the nuclease is lead by a guid RNA (gRNA) - a sequence of nucleotides complementary to a target strand area - to the CRISPR originating from the pathogen, and cleaves it. Here, Cas13a is the nuclease.
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Exchanging Ideas
Thanks to the HD Resolution Team in NYC, USA, we collaborated to host an event to present our projects to each other. They learned about the EtBr Spot Protocol and the light operon and we learned about their goal to cure Huntington's Disease. It was a great experience!