Biomod/2015/StJohns:Results
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Results
Figure 11: Optimization methods for NClaw
Test # | DNA on Claw | DNA on MS2 Capsid | Capsid: Claw ratio | Mg2+ conc. (mM) | Temperature (°C) |
1 | A21 | T21 | 2:1 | ~10 | Room temp (RT) |
2 | A21 | T21 | 8:1 | ~10 | RT |
3 | A21 | T21 | 44:1 | ~24 | RT |
4 | A21 | T21 | 10:1 | ~24 | RT |
5 | A21 | T21 | 1:1 | ~24 | RT |
6 | A21 | T21 | 1:10 | ~24 | RT |
7 | A21 | T21 | 10:1 | ~24 | 4 |
8 | A21 | T21 | 1:1 | ~24 | 4 |
9 | A21 | T21 | 1:10 | ~24 | 4 |
10 | A21 | T21 | 10:1 | ~24 | 15 |
11 | A21 | T21 | 1:1 | ~24 | 15 |
12 | A21 | T21 | 1:10 | ~24 | 15 |
13 | A21 | T21 | 44:1 | ~24 | 15 |
14 | A21 | T21 | 10:1 | ~48 | RT |
15 | A21 | T21 | 40:1 | ~24 | RT |
16 | A21 | T21 | 30:1 | ~24 | RT |
17 | A21 | T21 | 20:1 | ~24 | RT |
18 | A21 | T21 | 40:1 | ~20 | RT |
19 | A21 | T21 | 40:1 | ~10 | RT |
20 | A21 | T21 | 40:1 | ~5 | RT |
21 | A21 | T21 | 40:1 | ~2.5 | RT |
22 | A21 | T21 | 40:1 | ~20 (with 1M urea) | RT |
23 | A21 | T21 | 40:1 | ~10 (with 1M urea) | RT |
24 | (TGA)7 | (TCA)7 | 10:1 | ~20 | RT |
25 | (TGA)7 | (TCA)7 | 10:1 | ~10 | RT |
26 | (TGA)7 | (TCA)7 | 10:1 | ~5 | RT |
27 | (TGA)7 | (TCA)7 | 10:1 | ~2.5 | RT |
28 | (TGA)7 | (TCA)7 | 20:1 | ~20 | RT |
29 | (TGA)7 | (TCA)7 | 20:1 | ~2.5 | RT |
30 | (TGA)7 | (TCA)7 | 30:1 | ~2.5 | RT |
31 | (TGA)7 | (TCA)7 | 50:1 | ~2.5 | RT |
NOTE:
Unless otherwise stated, the agarose gels run (Fig. 12-19) were 0.5X TBE ~10mM Mg2+ 1% denaturing, with 0.5X TBE 10mM Mg2+ buffer, run at 25°C and 450-700 volthours.
June 9 2015: Diff ratio MS2T21 gel
Lanes:
(Claw: 1 equivalent = 0.1 pmol)
- 1kb DNA marker
- Vanilla Claw Blunt (VB)
- Vanilla Claw Sticky (VS)
- T21 modified MS2 virus capsids (MS2T21)
- MS2T21:VB, 1:1
- MS2T21:VS, 1:1
- MS2T21:VB, 2:1
- MS2T21:VS, 2:1
- MS2T21:VB, 5:1
- MS2T21:VS, 5:1
- MS2T21:VB, 10:1
- MS2T21:VS, 10:1
June 30 2015: Triangle binding gel
Lanes:
(Triangle: 1 equivalent = 0.05 pmol)
- 1kb DNA marker
- Triangle Blunt
- Triangle Sticky
- MS2T21
- MS2T21:Triangle Blunt, 4:1
- MS2T21:Triangle Sticky, 4:1
July 9 2015: NClaw 5S and 4S binding
Lanes:
(Claw: 1 equivalent = 0.05 pmol)
- 1kb DNA marker
- NClaw Blunt
- MS2T21:NClaw Blunt, 4:1
- NClaw 5 Sticky/arm (NClaw 5S)
- Blunt MS2:NClaw 5S, 4:1
- MS2T21:NClaw 5S, 4:1
- NClaw 4 Sticky/arm (NClaw 4S)
- Blunt MS2:NClaw 4S, 4:1
- MS2T21:NClaw 4S, 4:1
July 16 2015: TClaw Synthesis and polymerization Gel
Date: 07.16.15;
Voltage: 70 Volts/Hour; Length of Run: 4 H 31M
Concentration of Sample: 19.23nanoM: Blunt T22 Claw and Sticky T22 Claw; 20.00nanoM Blunt T31 Claw; 20.83nanoM: Sticky T31 Claw; 18.87nanoM: Blunt T40 Claw and Sticky T40 Claw; 18.52nanoM: Blunt T49 Claw; 17.86nanoM: Sticky T49 Claw
Name of each sample:
- Blunt T40 Claw
- Sticky T40 Claw
- Blunt T49 Claw
- Sticky T49 Claw
- Blunt T22 Claw
- Sticky T22 Claw
- Blunt T31 Claw
- Sticky T31 Claw
- Blank
- Plasmid
- 1KB Ladder
Quantity of DNA in each sample: 0.05 picomoles
July 30 2015: TClaw Synthesis and polymerization Gel
Date: 06.30.15;
Voltage: 70 Volts/Hour; Length of Run: 5 H 20M
Concentration of Sample: 10.20nanoM: Blunt T Claw; 10.64nanoM Sticky T Claw; 0.3uM Tether
Name of each sample:
- Blunt Claw
- Sticky Claw
- Blunt Claw, 22nm Tether
- Sticky Claw, 22nm Tether
- Blunt Claw, 31 nm Tether
- Sticky Claw, 31 nm Tether
- Blunt Claw, 40nm Tether
- Sticky Claw, 40nm Tether
- Blunt Claw, 49nm Tether
- Sticky Claw 49nm Tether
- Blank
- 1 KB Marker
Quantity of DNA in each sample: 0.05 picomoles Claw; 0.25 picomoles of Tether; 9 picomoles Hex
August 12 2015: Post SYBR stain
Date: 08.12.15; Voltage: 70 Volts/Hour; Length of Run: 4 H 12M Concentration of Sample: 18.18nanoM: Blunt T Claw; 19.05nanoM Sticky T Claw; 0.2uM Tether; 4.5uMHex Name of each sample:
- Blunt T31 Claw+ Hex
- Sticky T31 Claw + Hex
- Blunt T40 Claw + Hex
- Sticky T40 Claw + Hex
- Blunt T 49 Claw + Hex
- Sticky T49 Claw +Hex
- Blunt NClaw (No tether) + Hex
- Sticky NClaw (No tether) + Hex
- 1KB
Quantity of DNA in each sample: 0.05 picomoles Claw; 0.25 picomoles of Tether; 9 picomoles Hex
Figure 18: Pre-SYBR staining using Fluorescent Hex Strand
Date: 08.31.15; Voltage: 70 Volts/Hour; Length of Run: 4 H 0M. Concentration of Sample: 18.18nanoM: Blunt T Claw; 19.05nanoM Sticky T Claw; 0.2uM Tether; 0.2uM CY3 Name of each sample:
- Blunt T31 Claw+ Hex
- Sticky T31 Claw + Hex
- Blunt T40 Claw + Hex
- Sticky T40 Claw + Hex
- Blunt T 49 Claw + Hex
- Sticky T49 Claw +Hex
- Blunt NClaw (No tether) + Hex
- Sticky NClaw (No tether) + Hex
- 1KB
Quantity of DNA in each sample: 0.120 picomoles Claw; 3 picomoles of Tether; 10.8 picomoles Cy3
Figure 18: Post SYBR stain
Figure 19: Pre-SYBR image using CY3 Fluorescent Strand
Figure 20: AFM image of NClaw
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Figure 20: AFM image of NClaw. AFM images of NClaw showing good formation.
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Zoomed in NClaw
Fab’ Results and Discussion
Unless otherwise stated for Figures 21-26, the Gel Type: SDS PAGE; Percentage: 12% Gel; Buffer Type: 1X SDS Buffer; Temperature: 25C. Stained in Oriole Stain and ran at 300V.
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Figure 21: Shows 3 different conditions for reducing Fab2’ to Fab’. Lane 3 (50mM 37°C 1hr) had the fewest amount of by-products and was continually used from then on instead of lane 4(50mM RT 1hr) and 5(1.6mM, 37°C, 1hr)
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Figure 22-Shows smearing upward of where Fab’ is usually shown, indicative of reactivity. PageRuler, IgG2a, Fab’, Fab’ and PEG maleimide rxn.
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Figure 23-Shows the smearing further up on the gel from where the DNA-Maleimide alone is shown, which proves the reactivity of the DNA. This gel is 20% Denaturing Acrylamide gel with 1X TBE Buffer at 65°C ran at 500V for 100min. Stained in SYBR for 15min.
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Figure 24-Shows the digestion and reduction of IgG1 to Fab’ and surrounding the Fab’ band on the gel, there are visibly more by-products.
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Figure 26-Shows the first visible conjugation on a gel.
Figure 25:Trials with Pepsin and IgG2a shown, not including other enzyme attempts or antibodies
Test # | Duration of conjugation | Humidity | Concentration | Temperature | Ratio of DNA to Fab’ | AAA or TCA | Conjugation? |
1 | 3hr | - | 8ug/ul | RT | 5:1 | both | No |
2 | 19hr | - | 1ug/ul | RT | 4:1 | both | No |
3 | 3hr | less | 10ug/ul | RT | 150:1 | both | No |
4 | 3hr | - | 10ug/ul | RT | 188:1 | both | No |
5 | 3hr 30min | less | 10ug/ul | RT | 250:1 | AAA | Yes |