Team:Cadets2Vets/Notebook June

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<!DOCTYPE html> Notebook-June - USE THIS ONE!!!


Notebook



Date: June 21, 2017





Experiments Performed:

Testing a circuit for biological competency within transformed E.coli.


Why We Performed them:


These experiments were performed to test for the presents of GFP within E. coli cells after exposure to Arsenate and arsenite.



Results:

The resulting cultures were inconclusive while consistently negative (no growth) at the 500uM concentration across all three types. The remaining cultures after the initial concentration were 50/50 in terms of E. coli growth. When checked for fluorescence all samples below 100uM showed possible signal from GFP. However further analysis showed an odd mitigating variable, being that the culture media has an auto-fluorescence within the same spectra as GFP. To prove GFP signal each culture was pelleted out via centrifugation, washed with PBS twice and suspended to eliminate any chance of background signal. The results showed little to no GFP signal. However due to culture variability, this will be replicated. Glycerol stocks have also been created of the stock circuit.




Moving Forward:

Due to the results above due to many mitigating factors the cell may not be biologically competent. However, because of the variability between replicates of the varying culture concentrations that will need to be repeated. In parallel to the replication of the culture study, a miniprep and paper based assay will be looked at as well utilizing the Promega S30 kit.



Date: June 26, 2017





Experiments Performed:

Validate the use of LC 480 II utilizing T7 constitutive GFP construct.


QC/ QA of Ars. 1.1 plasmid cel


Culture of Ars 1.1 transformed E. coli were exposed to 25 uM Aresnate + Aresnite at 37 C in the LC 480 II for 24 hours


Why We Performed them:

We need a way to properly quantify GFP production and QC/QA the plasmid


Results:

Validating the use of LC 480 II utilizing T7 constitutive GFP construct

The LC 480 II was successfully able to monitor the increase GFP over the incubation time

QC/QA the plasmid

Circularized plasmid gels proved inconclusive however M13 amplification did provide strong indication of proper fragment. The fragment size is 1461 bp, with the second bright band in the ladder being equal to 1650 bp. All minipreps exhibit a band slight below the 1625 bp band of the ladder, which is evident of proper fragment size.

Moving Forward:

TBD



Image Gallery



This is the test of T7 constitutiveGFP on a paper based ticket. From left-to-right it is positive control, negative control, water, and another water



This is a M13 confirmation fragment insert. of arsenic circuit. Top band is confirmation of presence of insert at approximately 1.4 kb




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