Team:Stanford-Brown/SB16 Notebooks FQsensor
Biosensing (IRES, FQ)
Made with Benchling
Project: iGEM 2016
Authors: Michael Becich, Amy Weissenbach, Julia Gross
Dates: 2016-06-24 to 2016-10-03
Friday, 6/24
-Transformed NEB5a cells with BBa_E0040, the GFP plasmid to use as the backbone for the IRES construct (Julia + Amy)
Monday, 6/27
-GFP plates had no colonies. Realized we had the wrong antibiotic, so we re-plated with the correct plates. (Julia + Amy)
Tuesday, 6/28
-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
Wednesday, 6/29
-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)
Thursday, 6/30
-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)
Friday, 7/1
-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
Tuesday, 7/5
-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)
Various_FQ_Designs.jpg
Source: http://www.sciencedirect.com/science/article/pii/S1046202305001039
Wednesday, 7/6
-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
Thursday, 7/7
-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
IMG_2632.JPG
-Idea: denature aptamer once bound to recover target in as high of a concentration possible, measure with Nanodrop
Friday, 7/8
IRES
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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)
FQ Sensors
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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.
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Put in the order for biotin-ATP aptamer-flourophore oligo, and hybridizing sequence-quencher oligo, both of which should arrive on 7/12. (MB)
ATP_Fluorophore_Biotinylated A new nucleotide sequence entered manually
ATP_Quencher A new nucleotide sequence entered manually
-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)
A | B | C | D | E | F | G | H | I | J | |
1 | -Control (No Sensor) | 10mM ATP | 1mM ATP | 100uM ATP | 10uM ATP | 1uM ATP | 100M ATP | 10nM ATP | 1nM ATP | No ATP |
2 | 10uM Sensor-> | 10mM ATP | 1mM ATP | 100uM ATP | 10uM ATP | 1uM ATP | 100M ATP | 10nM ATP | 1nM ATP | No ATP |
3 | 1uM Sensor-> | 10mM ATP | 1mM ATP | 100uM ATP | 10uM ATP | 1uM ATP | 100M ATP | 10nM ATP | 1nM ATP | No ATP |
4 | 10uM Sensor-> | 10mM GTP | 1mM GTP | 100uM GTP | 10uM GTP | 1uM GTP | 100M GTP | 10nM GTP | 1nM GTP | No GTP |
5 | 1uM Sensor-> | 10mM GTP | 1mM GTP | 100uM GTP | 10uM GTP | 1uM GTP | 100M GTP | 10nM GTP | 1nM GTP | No GTP |
Table1
Monday, 7/11
-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)
Tuesday, 7/12
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1..5uL of Plasmids obtained from Dave Dingal/Tony Gao of Stanley Qi Lab for possible attachment mechanisms:
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pSLQ3604 --> pcDNA3-hNECD1-Gal4
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pSLQ3829--> pEV-UAS-hNECD-dCas9-VPR
IRES gel:
IRES_7_12_16_PNG.PNG
Wednesday, 7/13
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When oligo's arrive, suspend both to 100 uM Stock
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Prepare Wash Buffer: Tris-Buffered Saline (25mM Tris, 150 mM NaCL) and 0.1% BSA, 0.05% Tween -20
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Adjusted concentration of biotinylated FQ to 10 ug/mL in wash buffer
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Excitation/Emission 495/520 for FAM-6
IRES_7_13_16_PNG.PNG
Thursday, 7/14
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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)
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Wash wells 3-times with 5x SSCT Buffer
IRES Gel:
IRES_7_14_16_PNG.PNG
Friday, 7/15
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Dilute fluorophore and quencher to 100uM in EB
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Dilute to 2 uM Stocks in 5x SSCT
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Add 100uL to each well: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/s6940bul.pdf
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Incubate for 1 hour (room temp/shaking/dark) to let binding of biotin/streptavidin to occur and let F/Q hybridize
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Add ATP gradually and measure fluorescence over time period
IRES Gel:
IRES_7_15_16_PNG.PNG
Monday, 7/18
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Strong signal observed in solution. Focus on proof of concept for first iteration
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Weak signal for wells that are not attached-->troubleshoot?