Biosensing (IRES, FQ)
Made with Benchling
Project: iGEM 2016
Authors: Michael Becich, Amy Weissenbach, Julia Gross
Dates: 2016-06-24 to 2016-10-03
-Transformed NEB5a cells with BBa_E0040, the GFP plasmid to use as the backbone for the IRES construct (Julia + Amy)
-GFP plates had no colonies. Realized we had the wrong antibiotic, so we re-plated with the correct plates. (Julia + Amy)
-Checked GFP plates, they had non fluorescent colonies on them. (Julia + Amy)
-Debugging Heidelberg MAWS Software Issues
-Designing Initial Fluorophore-Quencher Aptamer System
-Intramolecular quenching with PEG linker? Fluorophore on 5' end, Quencher on 3' end
-Tau Day--Considering Biosignatures (ATP)/Toxins (Pb 2+)/Minerals for Biomining to sense
-Pivoting to other systems: Quantum Dots, Augmenting Atomic Force Microscopy, Mechanisms of attachment
-Did colony PCR on colonies from the GFP plates, and ran the products on a gel confirming that they had transformed with the correct insert.
-Ordered primers for linearizing the GFP backbone with ends compatible with Gibson assembly of the IRES construct.
-Started liquid cultures of GFP colonies, and of the Heidelberg BioBricks that came in today: BBa_K1614019, BBa_K1614002, and BBa_K1614016. (Julia + Amy)
-Plan to talk with Christina Smolke for ideas about aptamer applications and members of Stanley Qi's Lab for mechanisms of attachment (dCas9, Gal4/UAS)
-Cryostocked the Heidelberg BioBricks, as well as the GFP colonies.
-Minipreped both the Heidelberg BioBricks and the GFP plasmids.
-PCR amplified the GFP plasmids with the primers that have IRES overhangs, in preparation for doing a Gibson assembly of the IRES fragment into the GFP backbone. (Julia + Amy)
-Planned FQ Constructs too expensive--> Need to pivot to cheaper, 2-piece system
-Use freeze-drying as a positive control for testing attachment of biosensors
-Discussed idea of BioFoundry
-Flexible linkers (GGSGGS) superior to rigid ones
-Engineered cheaper solution to FQ design using 2 oligonucleotides rather than 1 (drawback--sensor must be in solution initially for mass action)
-Linearized GFP plasmid with forward and reverse IRES overlap primers through PCR. (JLG/AW)
-FQ Design tradeoff=sterically decreasing non-specific interactions vs. flexibility/separation
-Don't use high GC content
-A/T are susceptible to UV radiation
-No Guanine Quadruplexes
-Previous GFP plasmid linearizing PCR failed, tried again with a higher annealing temp and using Q5 instead of One-Taq (TP/AW)
-Dilution Assay with 5' Biotin Fluorescein to guage brightness of fluorophores-->how will our sensing platform be sensed once glowing?
-Detectable at mM with naked eye, uM-nM with fluorometers
-Idea: denature aptamer once bound to recover target in as high of a concentration possible, measure with Nanodrop
Higher annealing PCR of 7/7 was successful: 394ng/ul of fairly pure DNA. Ran the product out on a gel, and observed a large band at 3kb, with two smaller bands further forward (probably primer-dimer/non specific amplification). Cut the 3kb band out, and ran it through the Epoch gel extraction kit according to manufacturer instructions. The extraction kit did not work (yield was 6ng/ul of highly impure DNA). Set up new linearizing PCR with the same DNA, primers, Q5, and thermocycler settings as 7/7. (JLG/AW)
Tested various concentrations of ATP (100mM - .001nM) on the spectrophotometer. Determined that at concentrations that low, comparing absorption spectra was not a viable way to distinguish between different concentrations. Decided as a result to order the fluorescently labeled ATP DNA aptamer construct, rather than ordering ATP aptamer alone and attempting to gauge efficacy via comparing before and after incubation ATP concentrations.
Put in the order for biotin-ATP aptamer-flourophore oligo, and hybridizing sequence-quencher oligo, both of which should arrive on 7/12. (MB)
-Consider radio-labeled ATP to differentiate (cheaper assays include dNTP's vs. NTP's)
-Preliminary Plate Layout for 1:1 Fluorophore-Quencher Ratio (1:1 biotinylated fluorophore to Streptavidin-coated plate)
-IRES linearizing PCR of 7/8 was successful. Preformed PCR extraction on the results, . Preformed Gibson assembly with the backbone, and the linearized construct. Transformed the Gibson plasmids into NE5a chemical competent E. coli cells, plated at 1:10 and 1:100 dilution factors on Amp plates. (AW/JLG)
1..5uL of Plasmids obtained from Dave Dingal/Tony Gao of Stanley Qi Lab for possible attachment mechanisms:
pSLQ3604 --> pcDNA3-hNECD1-Gal4
When oligo's arrive, suspend both to 100 uM Stock
Prepare Wash Buffer: Tris-Buffered Saline (25mM Tris, 150 mM NaCL) and 0.1% BSA, 0.05% Tween -20
Adjusted concentration of biotinylated FQ to 10 ug/mL in wash buffer
Excitation/Emission 495/520 for FAM-6
Prepare 5x SSCT, 2x SSCT, and PBST Buffers according to https://tools.thermofisher.com/content/sfs/manuals/MAN0011250_Pierce_StreptavidinCoat_96Well_UG.pdf, https://www.thermofisher.co.nz/Uploads/file/Scientific/Applications/Lab-Plasticware-Glassware-Supplies/Thermo-Scientific-Nunc-Immobilizer-Streptavidin-Application-example-PCR-ELISA.PDF (ELISA Protocol Nunc immbolizer)
Wash wells 3-times with 5x SSCT Buffer
Dilute fluorophore and quencher to 100uM in EB
Dilute to 2 uM Stocks in 5x SSCT
Add 100uL to each well: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/s6940bul.pdf
Incubate for 1 hour (room temp/shaking/dark) to let binding of biotin/streptavidin to occur and let F/Q hybridize
Add ATP gradually and measure fluorescence over time period
Strong signal observed in solution. Focus on proof of concept for first iteration
Weak signal for wells that are not attached-->troubleshoot?