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:
○
pSLQ3604 --> pcDNA3-hNECD1-Gal4
○
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?
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Test different buffers? F/Q combinations? Incubation times?
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Signal spike seems to be instantaneous, so how to better measure live?
Tuesday, 7/19
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Results from first FQ fluorescence measurements inconclusive.
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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
Wednesday, 7/20
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Refined experiment included a hybridization buffer (Tris, NaH2PO4, Na2HPO4) to first let F and Q bind.
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Crucial improvement was 1x Rxn Buffer for ATP sensing (300mM NaCl, 5mM MgCl2, 25mM HEPES)
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Concession--must accept 30 second delay between introduction of ATP and Plate Reader (limited by equipment imaging capabilities)
Thursday, 7/21
FQ Sensor
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ATP vs. GTP focused study (triplicate of wells)
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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):
A | B | C | |
1 | Time (min.) | μL 100mM ATP Added | μL 100mM NTP Added |
2 | 0-5 | 1.5 | 1.5 GTP |
3 | 6-10 | 1.5 | 1.5 UTP |
4 | 11-15 | 1.5 | 1.5 CTP |
5 | 16-20 | 10 | 1.5 GTP |
6 | 21-25 | 10 | 1.5 UTP |
7 | 26-30 | 10 | 1.5 CTP |
8 | 31-35 | 15 | 15 UTP |
9 | 36-40 | 15 | 15 CTP |
Table2
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Troubleshooting: Photobleaching, Further Buffer Optimization, Attached Sensor Still not as successful-->how to capture inital jump?
Friday, 7/22
FQ Sensor
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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
FQ_Stepwise_Graph_FoldChange.JPG
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Reperformed assay with dATP, dGTP, ATP, GTP
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Attempt to video tape fluoresence peak as added
Sunday, 7/24
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Video unsuccessful
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Contacted author Yingfu Li on responsiveness of aptamer signaling in nanoengine paper to get insights on other imaging techniques
Tuesday, 7/26
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Design a more useful FQ Assay-->Utilize plate reader at Stanford that can measure at certain heights in well
Wednesday, 7/27
IRES gel:
IRES_7-27.PNG
IRES_rAIP_7-27.PNG
Friday, 7/29
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Use two identical plates (one Streptavidin-coated)
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Achieve following concentrations in each well: 1 uL F, 1 uL Q in 2x SSCT buffer
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Incubate gently while configuring plate reader to optimal fluorescence settings (Gain=65, Height=6.50mm), readjusted before each round of measurment
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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)
IMG_3245.JPG
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Inconclusive results were achieved for both the attached and unattached variations.
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Next steps: troubleshoot binding with ITC, fix buffers (1x rxn buffer has lower signal, but better performance)
Saturday, 7/30
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Considerations for Biosensing: FQ Improvement, IRES Characerizaion, SELEX for Perchlorate/p-ABA Aptamer pool enrichment, PQQ DNAzyme
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Building a biodevice for better characterization of FQ Activity:
IMG_4543[1].JPG
Tuesday, 8/2
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FQ Final Validation:
1.
92 uL Rxn Buffer + 4 uL F/ 4 uLQ (1:1 F:Q)
2.
92 uL Hybridization Buffer + 4 uL F/ 4 uLQ (1:1 F:Q)
3.
94 uL Rxn Buffer + 2 uL F/ 4 uLQ (1:2 F:Q)
4.
94 uL Hybridization Buffer + 2 uL F/ 4 uLQ (1:2 F:Q)
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Incubate for 2 hours.
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Pipette into plates and dilute with 100uL 2x Rxn Buffer
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Incubate in plate gentl shaking for 30 minutes before preparing to image
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Settings (Height=6.25mm, Gain=70)
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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:
FQ_FINAL_GRAPHS_UNATTACHED_ATTACHED_10-13-2016.xlsx
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
A | ||||||||||||
B | ||||||||||||
C | ||||||||||||
D | ||||||||||||
E | Control | ATP 1F:1Q | ATP 1F:2Q | |||||||||
F | dATP 1F:1Q | dATP 1F:2Q | ||||||||||
G | GTP 1F:1Q | GTP 1F:2Q | ||||||||||
H | dGTP 1F:1Q | dGTP 1F:2Q |
Plate Layout
A | B | C | D | E | |
1 | dATP Added | [dATP] | AVG_RFU (Gain 70) Normalized to first trial | F/F0 | SD (n=2) |
2 | 0 | 0 | 167 | 1 | 0 |
3 | 0.0000015 | 0.00990099 | 168 | 1.005988024 | 0.243938 |
4 | 0.0000165 | 0.0990991 | 173 | 1.035928144 | 0.654097 |
5 | 0.0001665 | 0.99107143 | 207 | 1.239520958 | 0.169981 |
6 | 0.0016665 | 9.10655738 | 383 | 2.293413174 | 0.410408 |
7 | 0.0031665 | 15.9924242 | 820 | 4.910179641 | 0.966051 |
8 | |||||
9 | |||||
10 | ATP Added | [ATP] | |||
11 | 0 | 0 | 33 | 1 | 0 |
12 | 0.0000015 | 0.00990099 | 33 | 1 | 0.388719 |
13 | 0.0000165 | 0.0990991 | 38 | 1.151515152 | 0.965383 |
14 | 0.0001665 | 0.99107143 | 230 | 6.96969697 | 1.894411 |
15 | 0.0016665 | 9.10655738 | 551 | 16.6969697 | 1.547684 |
16 | 0.0031665 | 15.9924242 | 808 | 24.48484848 | 2.436957 |
17 | |||||
18 | |||||
19 | dGTP Added | [dGTP] | |||
20 | 0 | 0 | 134 | 1 | 0 |
21 | 5 | 0.00990099 | 134 | 1 | 0.064052 |
22 | 0.0000165 | 0.0990991 | 132 | 0.985074627 | 0.108625 |
23 | 0.0001665 | 0.99107143 | 130 | 0.970149254 | 0.032115 |
24 | 0.0016665 | 9.10655738 | 139 | 1.037313433 | 0.114406 |
25 | 0.0031665 | 15.9924242 | 121 | 0.902985075 | 0.120107 |
26 | |||||
27 | |||||
28 | GTP Added | [GTP] | |||
29 | 0 | 0 | 135 | 1 | 0 |
30 | 5 | 0.00990099 | 134 | 1 | 0.097803 |
31 | 0.0000165 | 0.0990991 | 132 | 0.985074627 | 0.045836 |
32 | 0.0001665 | 0.99107143 | 138 | 0.970149254 | 0.011335 |
33 | 0.0016665 | 9.10655738 | 143 | 1.037313433 | 0.033559 |
34 | 0.0031665 | 15.9924242 | 163 | 0.902985075 | 0.095841 |
35 |
Unattached FQ Results
A | B | C | D | E | |
1 | dATP Added (mmoles) | [dATP] | AVG_RFU (Gain 70) Normalized to first trial | F/F0 | SD (n=2) |
2 | 0 | 0 | 45 | 1 | 0 |
3 | 0.0000015 | 0.00990099 | 43 | 0.955555556 | 0.243937944 |
4 | 0.0000165 | 0.099099099 | 43 | 0.955555556 | 0.654097158 |
5 | 0.0001665 | 0.991071429 | 66 | 1.466666667 | 0.169981119 |
6 | 0.0016665 | 9.106557377 | 255 | 5.666666667 | 0.410407614 |
7 | 0.0031665 | 15.99242424 | 460 | 10.22222222 | 0.96605097 |
8 | 0.0046665 | 21.9084507 | 579 | 12.86666667 | 1.109325242 |
9 | 0.0061665 | 27.04605263 | 602 | 13.37777778 | 0.670327385 |
10 | ATP Added | [ATP] | |||
11 | 0 | 0 | 33 | 1 | 0 |
12 | 0.0000015 | 0.00990099 | 33 | 1 | 0.388719421 |
13 | 0.0000165 | 0.099099099 | 38 | 1.151515152 | 0.96538261 |
14 | 0.0001665 | 0.991071429 | 230 | 6.96969697 | 1.894411086 |
15 | 0.0016665 | 9.106557377 | 551 | 16.6969697 | 1.5476843 |
16 | 0.0031665 | 15.99242424 | 808 | 24.48484848 | 2.436957161 |
17 | 0.0046665 | 21.9084507 | 923 | 27.96969697 | 1.990514531 |
18 | 0.0061665 | 27.04605263 | 1094 | 33.15151515 | 3.210473445 |
19 | dGTP Added | [dGTP] | |||
20 | 0 | 0 | 28 | 1 | 0 |
21 | 0.0000015 | 0.00990099 | 35 | 1.25 | 0.064051714 |
22 | 0.0000165 | 0.099099099 | 35 | 1.25 | 0.108624815 |
23 | 0.0001665 | 0.991071429 | 32 | 1.142857143 | 0.032114939 |
24 | 0.0016665 | 9.106557377 | 33 | 1.178571429 | 0.114405538 |
25 | 0.0031665 | 15.99242424 | 44 | 1.571428571 | 0.120106536 |
26 | 0.0046665 | 21.9084507 | 43 | 1.535714286 | 0.053652181 |
27 | 0.0061665 | 27.04605263 | 47 | 1.678571429 | 0.06508307 |
28 | GTP Added | [GTP] | |||
29 | 0 | 0 | 0 | 0 | |
30 | 0.0000015 | 0.00990099 | 31 | 1.107142857 | 0.097802637 |
31 | 0.0000165 | 0.099099099 | 33 | 1.178571429 | 0.045835897 |
32 | 0.0001665 | 0.991071429 | 35 | 1.25 | 0.011335169 |
33 | 0.0016665 | 9.106557377 | 37 | 1.321428571 | 0.033558944 |
34 | 0.0031665 | 15.99242424 | 44 | 1.571428571 | 0.095841188 |
35 | 0.0046665 | 21.9084507 | 50 | 1.785714286 | 0.157668473 |
36 | 0.0061665 | 27.04605263 | 61 | 2.178571429 | 0.126588149 |
37 |
Attached Sensor
Unattached_Sensor_Final_Image_Legend.PNG
Attached_Sensor_Final_Image_Legend.PNG
Wednesday, 8/3
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Transformed http://parts.igem.org/Part:BBa_K1499004 from previous year's distribution kit into NEB5-alpha and plated on Cm plate
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IRES PAGE gel:
IRES_PAGE_8-3.PNG
Thursday, 8/4
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Miniprepped 2 colonies from CBD-Streptavidin part
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Contacted Alaina Schumate from 2014 iGEM team to confirm that this part has a promoter, terminator, and a FlagLumioHisTag as ambiguously specifed-->Never confirmed
○
We told her that we would purify and confirm with our fluorescent assay we developed
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Sent in for sequencing to verify expected sequence on Wiki with VF2/VR
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Grew up miniprepped DNA in 2 cultures
IRES PAGE gel:
Friday, 8/5
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Induced cultures with IPTG in 50mL flasks for large scale protein extraction to begn next week
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Confirmed presence in soluble fraction with small protein extraction (see duplication of lanes in middle at desired length of 39 kDa protein):
IRES_8-5.PNG
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Protein from BBa_K1499 004 indeed has FlagLumioHisTag as shown by luminescent gel image after HisTag column purification.
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Thus, we have already improved this part by verifying unknown design aspects, now onto functional verification!
Monday, 8/8
IRES PAGE gel:
IRES_8-8.PNG
Wednesday, 8/10
IRES PAGE gel:
IRES_8-10.PNG
Friday, 8/12
IRES_8-12.PNG
Monday, 8/15
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Extracted proteins from soluble fraction, ran gel to confirm presence, and purified:
Thursday, 9/22
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Took extracted CBD-Streptavidin Part (purified earlier in August 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)
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Pipetted 25 uL onto 9 wax-based wells on cellulose filter paper. Let sit over night (CBD takes 2-3 days to set in)
Friday, 9/23
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Re-imaged Plate on reader and Typhoon scanner (Abmax 495 nm Emmax 520 nm) with Trevor's help
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Took picture with iPhone under gel scanner
Pre-Wash
Monday, 9/26
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(Stanford School resumes)
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Imaged in morning
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Washed each square with 5x 1mL MilliQ Water (w/ 1mM ATP)
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Imaged again on Typhoon Scanner and took picture:
Day 0
Tuesday, 9/27
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Washed again 5x 1mL MilliQ Water (w/ 1mM ATP) and re-imaged
1 Day
Friday, 9/30
Low signal traces were still apparent on day after 4 washes
4 Days
Monday, 10/3
After repeating wash each morning for a week, the signal had all but disappeard from even the experimental square:
1 Week
