Team:Stanford-Brown/SB16 Notebooks CE


Stanford-Brown 2016

Collagen/Elastin/rAIP · Benchling

Collagen/Elastin/rAIP

Made with Benchling
Project: iGEM 2016
Authors: Anna Le, Charles Gleason
Dates: 2016-06-27 to 2016-10-15
Monday, 6/27
-Performed a Gibson for rAIP and elastin fragments, followed standard protocol and used the same timing for both (5 minutes, 15 minutes, 5 minutes) and transformed the result into NEB5α cells and plated
Tuesday, 6/28
-Made liquid cultures for 4 colonies from the elastin and rAIP 4X dilution plates. 40X plates had no useful colonies for both, even after they were left for the morning in the incubator. rAIP plate is relatively streaky, so the 40X plate might have produced replacement rAIP colonies
-Performed colony PCR, ran a gel, looked confusing under UV and technical difficulties prevented a good scan, so gel was saved for the next day with the hopes of more success visualizing it
Wednesday, 6/29
-Tried a gel extraction on the presumed location of amplified DNA from colony 3; NanoDrop showed no DNA so the extraction product was discarded.
- The elastin 4X dilution plate from the day before was left out on the lab bench and appears to have bacteria expressing RFP contaminating it? Weird.
-Miniprepped and made new dilutions of the 4 liquid cultures from the day before
-Nanodropped the miniprep product and determined the high DNA concentration and purity in c1 for elastin and c4 for rAIP
-Transformed c1 and c4 DNA into T7 cells for eventual protein expression
-Ran PCR on the c1/c4 miniprep for gel extraction and hopefully sequencing tomorrow
Thursday, 6/30
- Ran colony PCR on 2 colonies from elastin 40x and and 2 from rAIP plates
Friday, 7/1
-Found out the T7 liquid cultures weren't inoculated
-Prepared 5-alpha elastin and rAIP for sequencing
Tuesday, 7/5
- Checked protein expression in elastin circular mRNA expressing T7 cells; took 1 mL of each colony's 50 mL liquid culture preinduction after 2 hours of growth at 37°C and used 20 μL to measure OD600 at 100x dilution (colony 1 corresponded to 0.5 and colony 2 to 0.4). Induced remaining 49 mL with 49 μL of 1000x IPTG solution (40 mg/mL) and waited 2.5 hrs. In the meantime, centrifuged remaining 980 μL of the pre-induction samples, removed supernatant, and added 50 μL CellLytic buffer to pellet. Resuspended and froze to perform extraction for comparison together. After 2.5 hrs, tested 20 μL of each culture; found both to be 0.6. Used 833 μL of colony 1 and 667 μL of colony 2 to get equivalent cell concentration, centrifuged, added CellLytic, and performed small-scale protein extraction on both. Additionally solubilized the insoluble fraction. In addition, anticipating existence of product, performed same procedures at large scale for the remaining 48 mL of each culture. Froze the soluble and insoluble fractions.
Wednesday, 7/6
- Lysyl oxidase plate showed growth, ran colony PCR and gel electrophoresis to see band sizes (CG/EL/AL)
- Made liquid cultures
-Ran SDS-PAGE on 10 μL samples from the small scale extraction products for comparison. Used 20 μL of the ~40 μL made for each so as not to overfill the wells, and put in 10 μL of ladder on either side. Stained with CYPRO Ruby, technology prevented rapid staining procedure from being effective so the gel was stored in stain overnight with shaking.
Thursday, 7/7
- Lysyl oxidase band sizes were approximately 1.5kb-2kb which is what we anticipated (CG/EL/AL)
- Made 2 cryostocks from 2 colonies on lysyl oxidase plate labeled "NEB5-alpha rAIP Chlor AL 7/7/16"
- Performed gel extraction on the lysyl oxidase for sequencing
-Technology continues to be a struggle, but:
(Charlie plz insert picture)
Friday, 7/8
- Sequencing looks rather accurate
- Transformed rAIP into T7 cells at 4x and 40x dilution, plated and left in room temperature for the weekend. (CG/EL/AL)
Monday, 7/11
- Plate for rAIP transformation from 7/7 did not work. No growth present
- Transformed rAIP plates. Used 100 uL without dilution when plating.
Tuesday, 7/12
rAIP plates worked; made two 5 mL liquid cultures
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Redid Gibson for elastin; fragment 2 probably didn't actually add
Wednesday, 7/13
rAIP
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Ran PAGE gel with Lumio Green detecon to verify protein product; colony 1 looked better
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Made a ~400 mL culture of colony 1 for large scale protein extraction
Elastin
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Transform
Thursday, 7/14
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Spun down the culture in 2 sections; got 1 gram of wet mass of cells; performed large scale extraction procedure and solubilized the insoluble fraction
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Made a cryostock
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Miniprepped 5 mL of culture
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Tried transforming onto 3x Chlor plates due to mistakes, saved some liquid culture in anticipation of failure
Friday, 7/15
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3x plates failed at all 3 possible dilutions, Eric made a 1x plate with 100 μL of undiluted culture
Monday, 7/18
rAIP
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Full density culture made a lawn pretty much everywhere; took what was hopefully a single colony on the edge and inoculated a liquid culture with it for the day, after 5 hours in a 1.5 mL tube this was used to replate
Tuesday, 7/19
rAIP
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Transformation plates worked; we put them in the fridge
Thursday, 7/21
Elastin
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Ran PCR on the two fragments and the plasmid backbone with Q5 (pSB1C3 for 2:15 elongation at 64.9°C, Elastin fragment 1 and 2 in the same thermal cycler for 1 minute extension with fragment 1 at 71.5°C and fragment 2 at 72.0°C
Friday, 7/22
Elastin
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Ran a recycled 1% gel of the PCR products from yesterday
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Gibson with 1 μL of pSB1C3, 1 μL of fragment 1, and 1.5 μL of fragment 2 for 10 minutes at 52°C and 50 minutes at 50°C trying to account for potential hairpin at fragment 1/2 overlap site
Monday, 7/25
Elastin
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Picked 5 colonies from 7/22 transformation. Grew liquid cultures from these 5 colonies for 5 hours and ran PCR.
Tuesday, 7/26
Elastin
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Ran gel on PCR, performed PCR cleanup
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Miniprepped the 5 colonies for plasmids.
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Continued liquid cultures of the 5 colonies.
Wednesday, 7/27
Elastin
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Sent 10 samples in for sequencing: miniprepped elastin plasmid samples 1-3, and Gibson PCR products 4 and 5
Thursday, 7/28
Elastin
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Ran SDS PAGE gel to determine if elastin polymer was expressed.
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Stained with SYPRO Red protein stain overnight
Friday, 7/29
Tuesday, 8/2
Elastin
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Performed gel extraction/gel clean up on the elastin fragment 1. Band on the gel was between 600-700 bp. Concentration was 13.6 ng/uL, purity 260/280 2.16.
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elastin fragment 1 most likely ligated incorrectly originally in the previous attempt, off by 1 bp.
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Performed Gibson assembly and transformed T7 cells (overall 6th try).
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Made a 40x plate using elastin colony #5 liquid culture.
rAIP
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Induced reaction between purified protein samples with racemic lysine. H2O2 is a side product of the reaction between lysine and lysyl oxidase. We want to characterize the performance of lysyl oxidase.
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Performed the Amplex Red hydrogen peroxide assay on protein purification fractions. We had a standard curve of H2O2. Initial concentrations were 0, 1, 2, 3... etc to 10 uM H2O2 (before adding Amplex red working solution).
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We predicted that the Elution buffer fraction would contain lysyl oxidase. The plate data showed that this prediction was correct, because the other fractions displayed less H2O2 activity.
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Plate reader data saved under file name: rAIP_h2o2assay
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Forgot to take picture of plate
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Stock solutions of horseradish peroxidase (HRP) are in the -20C freezer (Elastin/collagen box).
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All remaining solutions (purified protein samples, 1X Rxn buffer, 1M lysine) left in the fridge.
H2O2 working concentrations (total volume = 50 uL)
A
B
C
1
Concentration20 uM H2O2 stock1X Rxn buffer
2
0050
3
12.547.5
4
2545
5
37.542.5
6
41040
7
512.537.5
8
61535
9
717.532.5
10
82030
11
922.527.5
12
102525
Table1
Wednesday, 8/3
Elastin
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40X colony 5 plate was a lawn. Had to replate using 1:1000 dilution.
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Gibson assembly/transformation worked. Plate showed ~7 colonies. woo
Monday, 8/8
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Elastin colonies #8-16 sent out for sequencing after performing colony pcr and gel electrophoresis
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Plated collagen (c1-3, CN) 1x 100x 1000x, made liquid cultures
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Collagen/collagen sap1 digested fragments had pcr cl