-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)

Source: http://www.sciencedirect.com/science/article/pii/S1046202305001039

-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

-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

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

pSLQ3829--> pEV-UAS-hNECD-dCas9-VPR

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?

Test different buffers? F/Q combinations? Incubation times?

Signal spike seems to be instantaneous, so how to better measure live?

Results from first FQ fluorescence measurements inconclusive.

Back to drawing board. Rather than simulating ELISA conditions, research better conditions for FQ System: http://onlinelibrary.wiley.com/doi/10.1002/anie.200501214/full, http://www.sciencedirect.com/science/article/pii/S1046202305001039

Refined experiment included a hybridization buffer (Tris, NaH2PO4, Na2HPO4) to first let F and Q bind.

Crucial improvement was 1x Rxn Buffer for ATP sensing (300mM NaCl, 5mM MgCl2, 25mM HEPES)

Concession--must accept 30 second delay between introduction of ATP and Plate Reader (limited by equipment imaging capabilities)

ATP vs. GTP focused study (triplicate of wells)

Performed stepwise concentration plate reader assay to confirm fluorophore-quencher system

Incubated 20nM fluorophore and 20nM quencher in hybridization buffer for 1 hour at room temp. with gentle agitation

Took 11 readings across 5 minute intervals, gradually increasing ATP/NTP concentration according to (limited by stocks of NTP's):

Troubleshooting: Photobleaching, Further Buffer Optimization, Attached Sensor Still not as successful-->how to capture inital jump?

Analyzed Fluorescent Plate Reader Data for Sensing Capabilities (observed 9-fold change in fluorescence at 30mM and linear trend with ATP target concentration)--Confirms Literature

Reperformed assay with dATP, dGTP, ATP, GTP

Attempt to video tape fluoresence peak as added

Video unsuccessful

Contacted author Yingfu Li on responsiveness of aptamer signaling in nanoengine paper to get insights on other imaging techniques

Design a more useful FQ Assay-->Utilize plate reader at Stanford that can measure at certain heights in well

Use two identical plates (one Streptavidin-coated)

Achieve following concentrations in each well: 1 uL F, 1 uL Q in 2x SSCT buffer

Incubate gently while configuring plate reader to optimal fluorescence settings (Gain=65, Height=6.50mm), readjusted before each round of measurment

Took baseline at 12:50pm and every 5 minutes recorded fluorescence. NTP was added to each well to appropriately achieved 10-fold serial dilutions from 1 mM to 1 nM (target total volume=150uL)

Inconclusive results were achieved for both the attached and unattached variations.

Next steps: troubleshoot binding with ITC, fix buffers (1x rxn buffer has lower signal, but better performance)

Considerations for Biosensing: FQ Improvement, IRES Characerizaion, SELEX for Perchlorate/p-ABA Aptamer pool enrichment, PQQ DNAzyme

Building a biodevice for better characterization of FQ Activity:

FQ Final Validation:

92 uL Rxn Buffer + 4 uL F/ 4 uLQ (1:1 F:Q)

94 uL Rxn Buffer + 2 uL F/ 4 uLQ (1:2 F:Q)

Incubate for 2 hours.

Pipette into plates and dilute with 100uL 2x Rxn Buffer

Incubate in plate gentl shaking for 30 minutes before preparing to image

Settings (Height=6.25mm, Gain=70)

Stepwise increase of ATP/GTP/dATP/dGTP concentrations

1.5 uL of 1 mM, 15uL of 1 mM, 1.5 u of 100mM, 15uL of 100mM, 15uL of 100mM, 15uL of 100mM, 15uL of 100mM (*ran out in some trials)

Final Data:

Took extracted CBD-Streptavidin Part (purified earlier in August/September by Julia) and incubated (shaking, room temp, 2 hours) in 2x B&W Buffer with equimolar amounts of biotinylated fluorophore-quencher

1190ng/uL starting concentration, 100uM Biotinylated Fluorophore and Quencher (Recipe: 100 uL Protein, 31.7 uL Fluorophore)

Pipetted 25 uL onto 9 wax-based wells on cellulose filter paper. Let sit over night (CBD takes 2-3 days to set in)

Re-imaged Plate on reader and Typhoon scanner (Abmax 495 nm Emmax 520 nm) with Trevor's help

Took picture with iPhone under gel scanner

(Stanford School resumes)

Imaged in morning

Washed each square with 5x 1mL MilliQ Water (w/ 1mM ATP)

Imaged again on Typhoon Scanner and took picture:

Washed again 5x 1mL MilliQ Water (w/ 1mM ATP) and re-imaged

Low signal traces were still apparent on day after 4 washes

After repeating wash each morning for a week, the signal had all but disappeard from even the experimental square: