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Strand Displacement

May 22

Activity

Preparation of two groups of gel electrophoresis samples and electrophoresis detection

Purpose

Prepare gel electrophoresis samples of specific groups, conduct electrophoresis detection, and obtain sample electrophoresis images.

Methods

  1. Prepare two groups of gel electrophoresis samples: the first group has labels: Marker, LBO1, SRO1, LBO1+SRO1, LBO2, SRO2, LBO2+SRO2, Marker; the second group has labels: Marker, LBO3, SRO3, LBO3+SRO3, LBO4, SRO4, LBO4+SRO4, Marker.
  2. Transfer the prepared gel to the electrophoresis tank, insert the comb and wait for solidification; add 10× Loading Buffer to the samples (final concentration 1×), centrifuge briefly after mixing; add 10μL sample to each well and equal amount of Marker to the wells at both ends; add PBS to the electrophoresis tank (outer liquid level is half the inner level), apply 120V voltage (max current 600mA) and electrophorese for about 45 minutes.

Results

Two corresponding gel electrophoresis images were obtained

Gel electrophoresis group 1 - May 22

Figure 1: Gel electrophoresis (May 22)

Observations & Notes

The binding rate of LBO and SRO those numbered 1, 2, 3 is high, but for those numbered 4 is relatively low.

May 26

Activity

Preparation of two groups of gel electrophoresis samples and electrophoresis detection

Purpose

Prepare gel electrophoresis samples of specific groups, conduct electrophoresis detection, and obtain sample electrophoresis images.

Methods

  1. Prepare two groups of gel electrophoresis samples: the first group has labels: Marker, LBO5, SRO5, LBO5+SRO5, LBO6, SRO6, LBO6+SRO6, IT, Marker; the second group has labels: Marker, LBO7, SRO7, LBO7+SRO7, LBO8, SRO8, LBO8+SRO8, E, Marker.
  2. Transfer the prepared gel to the electrophoresis tank, insert the comb and wait for solidification; add 10× Loading Buffer to the samples (final concentration 1×), centrifuge briefly after mixing; add 10μL sample to each well and equal amount of Marker to the wells at both ends; add PBS to the electrophoresis tank (outer liquid level is half the inner level), apply 120V voltage (max current 600mA) and electrophorese for about 45 minutes.

Results

Two corresponding gel electrophoresis images were obtained

Gel electrophoresis group 1

Figure 2: Gel electrophoresis (May 26)

Observations & Notes

The binding rate of LBO and SRO those numbered 5, 6, 7, and 8 is relatively low.

May 28

Activity

Initial Exploration of Strand Displacement: Vancomycin Concentration and Aptamer Binding Pre-experiment

Purpose

Conduct preliminary exploration of the strand displacement reaction and study the relationship between vancomycin concentration and aptamer binding.

Methods

1. Aptamer & Probe Preparation

Use LBO1, SRO1, LBO3, SRO3 with known nucleotide sequences.

Table 1: Aptamer Sequences
Name Sequence
LBO1 5'-CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA-3'
SRO1 5'-AGCCGAACTGAG-3'
LBO3 5'-TCTCTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG-3'
SRO3 5'-AGCCGGAGAGAGAGAGA-3'

2. Reaction System Preparation

Table 2: Reaction System Composition
Group Reactants LBO1 SRO1 LBO1+SRO1 LBO1+SRO1+ 5μM VAN LBO1+SRO1+ 10μM VAN LBO1+SRO1+ 20μM VAN LBO1+SRO1+ 50μM VAN LBO1+SRO1+ 100μM VAN
LBO1(μL) 1 0 1 1 1 1 1 1
SRO1(μL) 0 1 1 1 1 1 1 1
PBS(μL) 9 9 8 7 7 7 7 7
VAN(μL) 0 0 0 1(50μM VAN) 1(100μM VAN) 1(200μM VAN) 1(500μM VAN) 1(1mM VAN)
Overall system 10μL

3. Vancomycin (VAN) Solution Preparation

Weigh 5.5mg VAN (MW=1449.25), dissolve in 3.8mL PBS to make 1mM stock solution; dilute gradiently to 50μM, 100μM, 200μM, 500μM, 1mM working solutions; add 1μL corresponding VAN solution to annealed tubes and mix.

4. Gel Preparation (1mm thickness)

Table 3: Gel Composition
Reagent Volumn
H₂O 3.93mL
30%ACS 4mL
5x TBE 2mL
10%AP 70μL
TEMED 7μL

5. Electrophoresis & Staining

Transfer gel to tank and solidify; add 10× Loading Buffer to samples (1:10 dilution), centrifuge; add 10μL sample and Marker to wells; add PBS, electrophorese at 120V (600mA max) for 45min; shake gel with color developer in dark for 30min.

Results

1. Gel Electrophoresis Images

LBO1/SRO1 system gel electrophoresis

Figure 3: LBO1/SRO1 system gel electrophoresis

LBO3/SRO3 system gel electrophoresis

Figure 4: LBO3/SRO3 system gel electrophoresis

2. Interpretation

LBO1/SRO1 system: single strands show no obvious bands, hybrid chain has obvious band at ~50bp; VAN concentration increase (5μM-100μM) weakens band brightness (almost disappears at 50μM/100μM). LBO3/SRO3 system: band brightness weakens with VAN increase but remains visible at 100μM (weaker competitive effect than LBO1/SRO1).

3. Conclusion

VAN inhibits both hybrid structures in a concentration-dependent manner; stronger inhibition on LBO1/SRO1; high VAN (50μM/100μM) shows higher competitive binding.

Observations & Notes

  1. Experimental improvement: Incubate SRO and LBO at 37℃ overnight before adding VAN to optimize annealing.
  2. Reaction system (LBO1/SRO1 as example): total volume 10μL, with fixed volumes of LBO1 (1μL), SRO1 (1μL for mixed groups), PBS and VAN.
  3. Unified electrophoresis conditions: 120V, 600mA max, 45min electrophoresis, 30min dark staining.

June 16

Activity

Investigating the Competitive Matching Degree After Vancomycin and Hybridization Chain Binding at Different Concentrations

Purpose

  1. Simple Experiment: Verify the effect of adding VAN first then annealing on nucleic acid hybridization in LBO1/SRO1 and LBO3/SRO3 systems.
  2. Detailed Experiment: Explore the competitive matching degree between VAN (different concentrations) and hybrid chains after binding.

Methods

1. Simple Experiment

Add VAN to LBO1/SRO1 and LBO3/SRO3 systems first, then anneal to promote hybridization; conduct gel electrophoresis (samples include Marker, single probes, hybrid chains, hybrid chains + different VAN concentrations).

2. Detailed Experiment

① Aptamers & Probes
Table 4: Aptamer Sequences
Name Sequence
LBO1 5'-CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA-3'
SRO1 5'-AGCCGAACTGAG-3'
LBO3 5'-TCTCTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG-3'
SRO3 5'-AGCCGGAGAGAGAGAGA-3'
② Reaction System
Table 5: Reaction System Composition
Group Reactants LBO1 SRO1 LBO1+SRO1 LBO1+SRO1+ 5μM VAN LBO1+SRO1+ 10μM VAN LBO1+SRO1+ 20μM VAN LBO1+SRO1+ 50μM VAN LBO1+SRO1+ 100μM VAN
LBO1(μL) 1 0 1 1 1 1 1 1
SRO1(μL) 0 1 1 1 1 1 1 1
PBS(μL) 9 9 8 7 7 7 7 7
VAN(μL) 0 0 0 1(50μM VAN) 1(100μM VAN) 1(200μM VAN) 1(500μM VAN) 1(1mM VAN)
Overall system 10μL
③ VAN Solution

Weigh 5.5mg VAN (MW=1449.25), dissolve in 3.8mL PBS to make 1mM stock solution; dilute gradiently to 50μM, 100μM, 200μM, 500μM, 1mM working solutions; add 1μL corresponding VAN solution to annealed tubes and mix.

④ Electrophoresis Preparation

Centrifuge annealed samples, add 1μL 10× Loading Buffer, mix; gel preparation, electrophoresis and staining same as May 28.

Results

1. Simple Experiment

Simple experiment gel electrophoresis 1

Figure 5: Simple experiment gel electrophoresis 1

Simple experiment gel electrophoresis 2

Figure 6: Simple experiment gel electrophoresis 2

3. Conclusion

LBO1/SRO1 hybrid chain has good binding performance; LBO1 is more sensitive to VAN, suitable for subsequent fluorescence quantification.

Observations & Notes

  1. Key difference from May 28: VAN added before annealing (vs. after annealing on May 28) to compare VAN addition sequence effect.
  2. Reaction system and electrophoresis conditions same as May 28 for comparability.

June 19

Activity

Test LBO1/SRO1 & LBO2/SRO2 Systems (Add VAN First, Then Anneal)

Purpose

Explore the effect of VAN on nucleic acid hybridization and strand displacement in LBO1/SRO1 and LBO2/SRO2 systems under the condition of adding VAN first then annealing.

Methods

1. Sample Preparation

Take LBO1/SRO1 and LBO2/SRO2 systems; add VAN to each system first, then anneal to promote hybridization.

2. Electrophoresis

Prepare samples including Marker, single probes (LBO1, SRO1, LBO2, SRO2), hybrid chains (LBO1+SRO1, LBO2+SRO2), hybrid chains + VAN (5μM, 10μM, 20μM, 50μM, 100μM) and IHEL group; conduct gel electrophoresis (conditions same as June 16 simple experiment: 120V, 600mA max, 45min; dark staining for 30min).

Results

Two gel electrophoresis images obtained:

LBO1/SRO1 system gel electrophoresis

Figure 7: LBO1/SRO1 system gel electrophoresis

LBO2/SRO2 system gel electrophoresis

Figure 8: LBO2/SRO2 system gel electrophoresis

Observations & Notes

  1. LBO2/SRO2 system introduced for the first time to compare VAN response with LBO1/SRO1.
  2. IHEL group added (presumably a control) but its role was not explained.
  3. Experimental procedures (VAN addition sequence, annealing, electrophoresis) consistent with June 16 simple experiment.

June 22

Activity

Explore Hybridization Strength of LBO1 (Long Chain) and SRO1 (Short Chain) at Different Ratios

Purpose

Explore the difference in hybridization strength of hybrid chains formed by LBO1 (long chain) and SRO1 (short chain) at different ratios.

Methods

1. Aptamer & Probe

Use LBO1 and SRO1 with known nucleotide sequences.

2. Reaction System

Table 6: Reaction System Composition
Group Reactants LBO1 SRO1 LBO1:SRO1 1:1 LBO1:SRO1 1:1.5 LBO1:SRO1 1:2 LBO1:SRO1 1:2.5 LBO1:SRO1 1:3 LBO1:SRO1 1:3.5
LBO1(μL) 1 0 1 1.5 2 2.5 3 3.5
SRO1(μL) 0 1 1 1 1 1 1 1
PBS(μL) 9 9 8 7.5 7 6.5 6 5.5
Overall system 10μL

3. Electrophoresis

Centrifuge samples, add 1μL 10× Loading Buffer; gel preparation (same as May 28: 6%-20% gels), electrophoresis (120V, 600mA max, 45min) and dark staining (30min) same as previous experiments.

Results

1. Gel Electrophoresis Image

LBO1:SRO1 ratio hybridization gel electrophoresis

Figure 9: LBO1:SRO1 ratio hybridization gel electrophoresis

2. Conclusion

  1. Ratio 1:1 to 3.5:1: hybrid chain band intensity and position change, but all bands remain clear.
  2. Ratios 1:2 and 1:2.5: bands are prominent with moderate intensity (optimal long-short chain ratios).
  3. Recommend ratios 1:2 or 1:2.5 for subsequent experiments.

Observations & Notes

  1. Single variable design: Only long-short chain ratio changes; SRO1 volume fixed at 1μL, LBO1 volume adjusted by ratio, total volume 10μL.
  2. Gel and electrophoresis conditions consistent with previous experiments to ensure result reliability.

June 25

Activity

Explore VAN Strand Displacement Effect in High (1μM) and Low (100nM) Concentration Hybrid Chain Systems of LBO1/SRO1

Purpose

Prepare high (1μM) and low (100nM) concentration hybrid chain systems of LBO1/SRO1, and explore the strand displacement effect of VAN in different concentration systems.

Methods

1. Materials

LBO1, SRO1 (unmodified), LBO1' (with BHQ quencher), SRO1' (with FAM fluorophore) (initial concentration 10μM); PBS.

2. Hybrid Chain Preparation

① High concentration (1μM)
Table 7: High Concentration Hybrid Chain Preparation
Component FAM+BHQ (NEG) FAM (POS) No groups
SRO1 0 0 10
LBO1 0 20 20
SRO1' 5 10 0
LBO1' 10 0 0
PBS 35 70 70
② Low concentration (100nM)
Table 8: Low Concentration Hybrid Chain Preparation
Van concentration 0 0 0 0 50μM 100μM 200μM 500μM 1mM
Van volume 0 0 0 0 5 5 5 5 5
Hybrid chain volume 0 5 5 5 5 5 5 5 5
PBS 50 45 45 45 40 40 40 40 40
Types of hybrid chains - N P No groups N

3. Strand Displacement Reaction

① High concentration group

Set VAN concentrations (0μM, 50μM, 100μM, 200μM, 500μM, 1mM); add 5μL VAN, 5μL hybrid chain, 40μL PBS (total 50μL).

② Low concentration group

Same as high concentration group (only hybrid chain concentration differs).

4. Fluorescence Detection

Detect fluorescence signals of both systems.

Results

Two sets of fluorescence images obtained: one for high-concentration strand displacement reaction, the other for low-concentration.

High-concentration group fluorescence

Figure 10: High-concentration group fluorescence

Low-concentration group fluorescence

Figure 11: Low-concentration group fluorescence

Analysis

  • High-concentration group: Too strong background fluorescence, insensitive to VAN concentration (unsuitable for subsequent experiments).
  • Low-concentration group: Low background fluorescence, good response to VAN concentration (suitable for subsequent experiments).

Observations & Notes

  1. Hybrid chain groups clearly classified (NEG/POS/Unmodified) to compare modification effects on fluorescence.
  2. Reaction system total volume 50μL (fixed VAN volume 5μL) for consistent conditions.
  3. Low-concentration system diluted from high-concentration (1:10 ratio) to control concentration gradient.

July 4

Activity

Fluorescence Quantification Experiment of LBO1 and SRO1 with T7 Promoter

Purpose

Conduct fluorescence quantification of LBO1 and SRO1 containing T7 promoter, and explore the effects of T7 promoter and VAN concentration on hybrid chain fluorescence signals.

Methods

1. Materials

10μM LBO1, SRO1, LBO1-BHQ1 (LBO1'), SRO1-6-FAM (SRO1'), SRO1-T7, SRO1-T7-6-FAM (SRO1'-T7); PBS, ddH₂O.

2. Hybrid Chain Preparation (long:short=2:1)

Table 9: Hybrid Chain Preparation
Group Volume(μL) NEG POS No Groups NEG-T7 POS-T7 No Groups-T7
LBO1 0 20 20 0 20 20
LBO1' 20 0 0 20 0 0
SRO1 0 0 10 0 0 0
SRO1' 10 10 0 0 0 0
SRO1-T7 0 0 0 0 0 10
SRO1'-T7 0 0 0 10 10 0
PBS 70
Total 100μL

3. 384-Well Plate Reaction

Reaction setup with various hybrid chain types and VAN concentration gradients.

4. Fluorescence Detection

Detect hybrid chain fluorescence and VAN concentration gradient (L1'+S1'-T7 group).

Results

1. Hybrid Chain Fluorescence Data

Table 10: Fluorescence Intensity Measurements
Group 1 PBS 2 van 3 L1'+S1' 4 L1+S1' 5 L1+S1 6 L1'+S1'T7 7 L1+S1'T7 8 L1+S1T7
ReplicateⅠ 67 55 91114 OVRFLW 51 17583 OVRFLW 57
ReplicateⅡ 59 52 86309 OVRFLW 40 14767 OVRFLW 61
ReplicateⅢ 44 55 OVRFLW OVRFLW 45 26036 OVRFLW 57
Mean 57 54 - - 45 19462 - 58

2. VAN Gradient Experiment

VAN gradient fluorescence

Figure 12: VAN gradient fluorescence experiment

3. Analysis

PBS/VAN have no fluorescence interference; some groups have low hybridization efficiency or high concentration; T7 may promote L1'-S1' binding.

Observations & Notes

  1. Reduce chain concentration in subsequent experiments (some groups exceed detection range).
  2. 384-well system covers multiple modified groups for multi-dimensional comparison.
  3. VAN final concentration controlled by dilution for accurate gradient.

July 5

Activity

Fluorescence Quantification Experiment of LBO1 and SRO1 with T7 Promoter (Optimized Hybrid Chain Concentration)

Purpose

Continue the July 4 experiment; after optimizing hybrid chain concentration, re-conduct fluorescence quantification of LBO1/SRO1 with T7 promoter to verify effects of T7 and VAN concentration.

Methods

1. Hybrid Chain Preparation (long:short=2:1)

Optimized concentrations with reduced volumes.

2. 384-Well Reaction

Same setup as July 4 but with optimized concentrations.

3. Fluorescence Detection

Detect 30min fluorescence intensity, plot curve and bar chart.

Results

1. Fluorescence Analysis

Optimized fluorescence quantification

Figure 13: Optimized fluorescence quantification results

2. Analysis

  1. PBS/VAN have low fluorescence (no interference).
  2. Fluorescence intensity of L1'+S1' < L1'+S1'T7 (T7 may reduce hybridization efficiency or weaken BHQ quenching).
  3. Endpoint fluorescence does not increase strictly with VAN concentration (system response needs optimization).

Observations & Notes

  1. Hybrid chain volume halved to reduce concentration.
  2. Annealing changed from "rapid cooling" to "slow cooling" to optimize hybridization.
  3. 30min detection to observe signal dynamics.

July 6

Activity

Fluorescence Quantification of Hybrid Chains with Different Modified SRO2 and LBO2

Purpose

Conduct fluorescence quantification (RFU detection) of SRO2-LBO2 hybrid chains with different modifications (BHQ/FAM, unmodified) and analyze their fluorescence characteristics.

Methods

1. Materials

10μM LBO2 (L2), SRO2 (S2), LBO2-BHQ1 (L2'), SRO2-6-FAM (S2'); PBS, ddH₂O.

2. Hybrid Chain Preparation (LBO:SRO=2:1)

Table 11: LBO2/SRO2 Hybrid Chain Preparation
Group Volume(μL) L2'+S2' L2+S2' L2+S2
LBO2 0 20 20
LBO2' 20 0 0
SRO2 0 0 10
SRO2' 10 10 0
PBS 70
Total 100μL

3. RFU Detection

Standard fluorescence detection protocol.

Results

LBO2/SRO2 fluorescence quantification

Figure 14: LBO2/SRO2 fluorescence quantification

Observations & Notes

  1. Probe combination changed to LBO2/SRO2 (vs. LBO1/SRO1 on July 4/5) to compare fluorescence response differences.
  2. Unified detection parameters (490nm/520nm) for data comparability.
  3. Step-by-step dilution (10μM→1μM→100nM) to reduce concentration error.

Activity

Fluorescence Quantification of Hybrid Chains with Different Modified SRO1 and LBO1

Purpose

Conduct fluorescence quantification (RFU detection) of SRO1-LBO1 hybrid chains with different modifications (BHQ/FAM, T7 promoter, unmodified) and explore T7's effect on hybrid chain stability and binding efficiency.

Methods

1. Materials

10μM LBO1 (L1), SRO1 (S1), LBO1-BHQ1 (L1'), SRO1-6-FAM (S1'), SRO1-T7 (S1T7), SRO1-T7-6-FAM (S1'T7); PBS, ddH₂O.

2. Hybrid Chain Preparation (long:short=2:1)

Table 12: LBO1/SRO1 Hybrid Chain Preparation
Group Volume(μL) L1'+S1' L1+S1' L1+S1 L1'+S1'T7 L1+S1'T7 L1+S1T7
LBO1 0 20 20 0 20 20
LBO1' 20 0 0 20 0 0
SRO1 0 0 10 0 0 0
SRO1' 10 10 0 0 0 0
SRO1-T7 0 0 0 0 0 10
SRO1'-T7 0 0 0 10 10 0
PBS 70
Total 100μL

3. RFU Detection

Standard fluorescence detection protocol.

Results

LBO1/SRO1 fluorescence quantification 1

Figure 15: LBO1/SRO1 fluorescence quantification 1

LBO1/SRO1 fluorescence quantification 2

Figure 16: LBO1/SRO1 fluorescence quantification 2

Key Conclusion

T7-modified chains have lower stability; binding efficiency between long and short chains is lower than unmodified chains.

Observations & Notes

  1. T7-modified groups added (vs. Experiment 1) to focus on T7's role.
  2. Hybrid chain ratio, dilution and detection parameters consistent with Experiment 1 (only probe modification differs) for rigorous design.

July 16

Activity

Explore VAN Strand Displacement Effect in High (1μM) and Low (100nM) Concentration Hybrid Chain Systems of LBO1/SRO1 and LBO2/SRO2

Purpose

Prepare high (1μM) and low (100nM) concentration hybrid chain systems for LBO1/SRO1 and LBO2/SRO2 respectively, and compare VAN's strand displacement effect in the two probe systems.

Methods

1. Nucleotide Sequences

Table 13: Aptamer Sequences
Name Sequence
sro AGCCGAACTGAG
lbo1 CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA
sro2 AGCCGGAGAGAGAAA
lbo2 TTTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG

2. Materials

LBO1, SRO1, LBO1' (BHQ), SRO1' (FAM), LBO2, SRO2, LBO2' (BHQ), SRO2' (FAM) (10μM, unmodified for LBO1/SRO1/LBO2/SRO2); PBS.

3. Hybrid Chain Preparation

① High concentration (1μM)

Detailed preparation protocols for both LBO1/SRO1 and LBO2/SRO2 systems.

② Low concentration (100nM)

Diluted systems for both probe combinations.

4. Strand Displacement Reaction

Reaction setup with VAN concentration gradients for both systems.

5. Fluorescence Detection

Detect fluorescence of both systems.

Results

Two fluorescence quantification images of strand displacement reaction obtained:

LBO1/SRO1 system fluorescence

Figure 17: LBO1/SRO1 system fluorescence

LBO2/SRO2 system fluorescence

Figure 18: LBO2/SRO2 system fluorescence

Observations & Notes

  1. High-concentration system results not provided (inferred to have high background interference per June 25 conclusion).
  2. LBO1/SRO1 and LBO2/SRO2 systems have identical preparation methods (only probes differ) for direct comparison.
  3. Reaction system (50μL) and VAN gradient (5μM-100μM) consistent with previous experiments.

July 27

Activity

Explore Hybridization Efficiency of LBO1/SRO1, LBO1/SRO2, LBO2/SRO2 and VAN Strand Displacement Effect on LBO1-SRO2 System

Purpose

  1. Explore hybridization efficiency differences among LBO1/SRO1, LBO1/SRO2, LBO2/SRO2 systems.
  2. Study VAN's strand displacement effect on the LBO1-SRO2 system.

Methods

1. Nucleotide Sequences

Table 14: Aptamer Sequences
Name Sequence
LBO1 GACGTATCGACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACT
SRO1 GGAGACGCCGAACTGAGTCGATACGTCTAGATTG
LBO2 GACGTATCGACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTCGAT
SRO2 GGAGACCACTGAGCCGAACTGAGTCGCTAGATTG

2. VAN Solution Preparation

Weigh 6.0mg VAN (MW=1449.25), dissolve in 4.14mL PBS to make 1mM stock solution; dilute to 50μM, 100μM, 200μM, 500μM, 1mM; add 1μL to annealed tubes.

3. Nucleic Acid Hybridization System

① Gel1 (hybridization efficiency)

Compare LBO1/SRO1, LBO1/SRO2, LBO2/SRO2 hybridization efficiency.

② Gel2 (LBO1-SRO2 displacement)

Study VAN strand displacement effect on LBO1-SRO2 system.

4. Gel Preparation & Electrophoresis

Optimized gel configuration for better band separation.

Results

1. Gel Images

Hybridization efficiency gel

Figure 19: Hybridization efficiency comparison

LBO1-SRO2 displacement gel

Figure 20: LBO1-SRO2 strand displacement

2. Conclusion

  1. LBO1 hybridizes almost completely with SRO1/SRO2 at 1:1 ratio.
  2. LBO2-SRO2 hybridization efficiency is poor.
  3. LBO1-SRO2 system: Strand displacement hardly proceeds with 5μM-100μM VAN.

Observations & Notes

  1. Gel configuration adjusted (more ACS, less H₂O, 30min electrophoresis) to optimize band separation.
  2. Marker volume reduced to 3μL (avoid signal interference).
  3. First study LBO1-SRO2 cross-system hybridization to expand probe exploration.

August 7

Activity

Quantify displacement events by labeling nucleic acid strands with fluorescent probes and quenching agents

Purpose

The aim of this experiment was to quantitatively analyze SRO1-Cas displaced by vancomycin.

Methods

1. Strand Hybridization

Table 3: Strand Hybridization Components
Reagent Experimental Group Positive Control
10μM LBO1-Cas-BHQ (μL) 30 0
10μM SRO1-Cas-FAM (μL) 30 3
PBS (μL) 240 27
Overall (μL) 300 30

2. Construct Strand Displacement System

Table 4: Strand Displacement System
Component No RNA 100 nM RNA Positive Control
0 1 2 3 4 5 0 1 2 3 4 5
1 μM crRNA (μL) 0 0 0 0 0 0 5 5 5 5 5 5 0
VAN (μL) 0 5 5 5 5 5 0 5 5 5 5 5 0
Hybridized strand (μL) 5 5 5 5 5 5 5 5 5 5 5 5 5
PBS (μL) 45 40 40 40 40 40 40 35 35 35 35 35 45

Note: The concentrations of added vancomycin in group 1-5 were 50 μM, 100 μM, 200 μM, 500 μM, and 1 mM respectively, and are diluted ten times in the reaction system, and the final concentrations are respectively 5μM, 10μM, 20μM, 50μM, 100μM.

Results

SRO1-Cas displacement quantification

Figure 21: SRO1-Cas displacement quantification

Observations & Notes

The results show that the addition of crRNA1 to facilitate vancomycin-mediated displacement of SRO1-Cas did not significantly improve the outcome.

August 11

Activity

Increase the concentration of crRNA to investigate whether this strategy promotes SRO shedding

Purpose

In this experiment, the crRNA added to the system had a concentration of 5 µM, meaning the crRNA concentration was increased fivefold.

Methods

In this experiment, only the group with crRNA added and the positive control group were tested, while other procedures were the same as those in the experiment on August 7.

Results

High crRNA concentration effect

Figure 22: High crRNA concentration effect

Observations & Notes

The results demonstrate that even with elevated crRNA concentrations, SRO-Cas dissociation was not enhanced.

August 13

Activity

Perform polyacrylamide gel electrophoresis and obtain images

Purpose

Verify the assembly of LBO1-Cas and SRO2-Cas and determine whether vancomycin can displace SRO2-Cas.

Methods

1. Nucleotide Sequences

Table 1: Nucleotide Sequences
Component Sequence (5'-3')
LBO1-Cas GACGTATCGACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACT
SRO2-Cas GGAGACCACTGAGCCGAACTGAGTCGCTAGATTG

2. Construction of Nucleic Acid Hybridization System

Table 2: Hybridization System Components
Group Reactants LBO1-Cas SRO2-Cas L+S L+S+5μM VAN L+S+10μM VAN L+S+20μM VAN L+S+50μM VAN L+S+100μM VAN
10 μM LBO1-Cas (μL) 1 0 1 1 1 1 1 1
10 μM SRO2-Cas (μL) 0 1 1 1 1 1 1 1
PBS (μL) 9 9 8 7 7 7 7 7
VAN (μL) 0 0 0 1 1 1 1 1
Overall system 10μL

Results

LBO1-Cas and SRO2-Cas gel electrophoresis

Figure 23: LBO1-Cas and SRO2-Cas gel electrophoresis

Observations & Notes

  1. LBO1-Cas and SRO2-Cas hybridize almost completely at a 1:1 ratio.
  2. In the LBO1-SRO2-Cas system, the displacement reaction could hardly proceed with the addition of 5-100 μM vancomycin.

August 15

Activity

Repeat 8.13' experiment

Results

LBO1+SRO2 repeat experiment

Figure 24: LBO1+SRO2 repeat experiment

Observations & Notes

The results of this experiment demonstrate a concentration-dependent relationship between SRO2-Cas and vancomycin.

August 24

Activity

Quantify displacement events by labeling nucleic acid strands with fluorescent probes and quenching agents

Purpose

The aim of this experiment was to quantitatively analyze SRO2-Cas displaced by vancomycin. Investigating whether the newly designed SRO strand is more prone to detachment.

Methods

Except for the use of a different nucleic acid strand (SRO2-Cas instead of SRO1-Cas), the experimental procedures and group settings in this experiment are the same as those conducted on August 7.

Results

SRO2-Cas displacement quantification

Figure 25: SRO2-Cas displacement quantification

Observations & Notes

  1. The results of this experiment differed considerably from those obtained on August 15.
  2. Fluorescence intensity did not show a significant correlation with vancomycin concentration.
  3. Samples with added RNA exhibited higher fluorescence intensity compared to those without RNA, suggesting that RNA may facilitate the displacement of SRO2-Cas by vancomycin.
  4. The displacement occurred very rapidly, and fluorescence intensity was not significantly related to reaction time.
  5. There are two possible sources for the observed differences: first, vancomycin was not co-annealed with the sensor in this experiment; second, there was a difference in concentration, as the final sensor concentration here was 100 nM.

August 25

Activity

Strand displacement experiments incorporating an additional step of co-annealing vancomycin with the sensor

Purpose

Given the substantial discrepancies between the results of the August 24 and August 15 experiments, we modified the reaction conditions. Vancomycin was co-annealed with the sensor at unchanged concentrations to examine the impact of co-annealing on the reaction.

Results

Co-annealing vancomycin effect

Figure 26: Co-annealing vancomycin effect

Note: This graph illustrates the strand displacement reaction of LBO1-SRO2-Cas. Column 1 represents the positive control (SRO2-Cas-FAM). Columns 2-7 and 8-13 correspond to the normalized fluorescence intensity at antibiotic concentrations of 0, 5, 10, 20, 50, and 100 µM under crRNA-absent and crRNA-present conditions, respectively.

Observations & Notes

  1. In the current experiment, fluorescence intensity exhibited a clear concentration-dependent gradient with respect to vancomycin.
  2. Validated the August 24 hypothesis that co-annealing promotes displacement and that RNA facilitates this process.
  3. Additionally, sensor concentration influenced displacement, as the maximum fluorescence intensity nearly matched that of the positive control, indicating near-complete displacement.
  4. In contrast, the previous gel results did not display such high-intensity SRO2-Cas bands.

August 26

Activity

Seeking a method that provides better sealing

Purpose

Due to the suboptimal inhibition observed previously, a higher concentration of LBO was employed to enhance suppression.

Methods

Table 5: LBO:SRO Ratio Optimization
Component No RNA 100 nM RNA
1:1 1.5:1 2:1 2.5:1 3:1 1:1 1.5:1 2:1 2.5:1 3:1 1:1
1 μM crRNA (μL) 0 0 0 0 0 5 5 5 5 5 5
Hybridized strands (μL) 5 5 5 5 5 5 5 5 5 5 5
PBS (μL) 45 45 45 45 45 40 40 40 40 40 40

Results

LBO concentration optimization

Figure 27: LBO concentration optimization

Observations & Notes

The strategy of increasing the LBO proportion did not result in a notable improvement in inhibition efficiency.

August 27-30

Activity

Quantify displacement events by labeling nucleic acid strands with fluorescent probes and quenching agents

Purpose

Due to the poor sealing effect of the negative control group in the 8.25 experiment, a repeat experiment was conducted. Attempt to reproduce the experimental results from 8.25.

Methods

In this experiment, only the group with crRNA added and the positive control group were tested, while other procedures were the same as those in the experiment on August 25.

Results

Displacement experiment repetition 1

Figure 28: Displacement experiment repetition 1

Displacement experiment repetition 2

Figure 29: Displacement experiment repetition 2

Observations & Notes

  1. Between August 27 and 29, multiple repetitions of the displacement experiment were performed, yet the results remained suboptimal.
  2. Literature review suggested that improper storage conditions, specifically repeated freeze-thaw cycles, may have led to nucleic acid strand breakage, thereby impacting the experimental results.

September 11

Activity

Quantify displacement events by labeling nucleic acid strands with fluorescent probes and quenching agents

Purpose

To test our hypothesis, LBO1-Cas and SRO2-Cas were reordered, and the strand displacement experiment was repeated.

Methods

In this experiment, only the group with crRNA added and the positive control group were tested, while other procedures were the same as those in the experiment on August 25.

Results

Fresh strands displacement experiment

Figure 30: Fresh strands displacement experiment

Note: This graph illustrates the strand displacement reaction of LBO1-SRO2-Cas. Column 1 represents the positive control (SRO2-Cas-FAM). Columns 2-7 and 8-13 correspond to the normalized fluorescence intensity at antibiotic concentrations of 0, 5, 10, 20, 50, and 100 µM under crRNA-absent and crRNA-present conditions, respectively.

Observations & Notes

A concentration-dependent trend was observed, potentially confirming that strand storage issues were a critical factor underlying the previous experimental failures.

T7 Transcription Amplification

May 16

Activity

Attempt to construct a T7 transcription system

Purpose

We used T7 promoter sequences and template to test the T7 in vitro transcription system, investigating the appropriate amounts of substances such as NTPs, T7 enzyme, and dyes for the system.

Methods

1. Sequences

Table 1: T7 Promoter and Template Sequences
Component Sequence (5'-3')
T7 promoter TAATACGACTCACTATAGG
T7 template GGTGCTCACACTCTACTCAACAGTAGCGAACTACTGGACCCGTCCTTCACCTATAGTGAGTCGTATTAGACTC

2. DNA Annealing

Table 2: DNA Annealing Components
Component Volume
10 μM T7 template strand 10 μL
10 μM T7 promoter 0.5 μL

Incubate in hot water bath for 30 minutes (above 95℃)

3. Construction of in vitro transcription system (100 μL)

Table 3: Transcription System Composition
Component Final Concentration Volume
5x transcription buffer - 20 μL
10 μM T7 Template strand 1μM 10 μL
10 μM T7 promoter 50nM 0.5 μL
25 mM NTP mix 2 mM 8 μL
38.3μM T7 polymerase 50 nM 5 μL
100 μM DFHBI 1.9 μM 1 μL
DEPC water - 55.5 μL
Total Volume - 100 μL

Results

The kinetics of transcription were measured by monitoring fluorescence intensity within the first two hours of transcription initiation.

Transcription kinetics fluorescence measurement

Figure 1: Transcription kinetics fluorescence measurement

Observations & Notes

  1. The initial fluorescence intensity of one experimental group reached approximately 1,600 RFU, significantly higher than the previous result (<50 RFU), confirming successful transcription.
  2. Fluorescence intensity decreased over time due to instrument parameter optimization delay (30 minutes).
  3. Premature transcription termination may be attributed to magnesium pyrophosphate precipitation inhibition.

May 27-June 18

Activity

Test on the appropriate enzyme amount for adding pyrophosphatase

Purpose

Pyrophosphatase was added to investigate whether it affects correct RNA transcription and whether it can improve transcription efficiency.

Methods

1. Sequences

Table 4: T7 Sequences
Component Sequence (5'-3')
T7 promoter TAATACGACTCACTATAGG
T7 template GGTGCTCACACTCTACTCAACAGTAGCGAACTACTGGACCCGTCCTTCACCTATAGTGAGTCGTATTAGACTC

2. Experimental Groups

Table 5: Pyrophosphatase Test Groups
Component Group 1 (G8) Group 2 (G9) Group 3 (G10) Group 4 (G11)
T7 promoter 0 50nM 50nM 50nM
T7 template 1μM 1μM 1μM 1μM
NTP mix 2 mM 2 mM 2 mM 2 mM
T7 polymerase 1.9 μM 1.9 μM 1.9 μM 1.9 μM
DFHBI 10 μM 10 μM 10 μM 10μM
PPase 0 0 0.001U 0.1 U

3. Construct 100 μL in vitro transcription system

Results

Pyrophosphatase effect on transcription

Figure 2: Pyrophosphatase effect on transcription

Observations & Notes

Results show the effect of pyrophosphatase on transcription efficiency and RNA product quality.

June 19-June 30

Activity

Test on transcriptional activation of T7 promoter at different concentrations

Purpose

Depending on the concentration of the displaced promoter strand in the sensor's upstream region, this experiment investigated the transcriptional activation efficiency under gradient promoter strand concentrations. Since the maximum concentration of the displaced promoter strand was 100 nM (100% displacement), the promoter concentration gradient in this experiment was set to 10 nM, 25 nM, 50 nM, 75 nM, and 100 nM.

Methods

1. Experimental Groups

Table 6: T7 Promoter Concentration Gradient
Component Group 0 Group 1 Group 2 Group 3 Group 4 Group 5
T7 promoter 0 10 nM 25 nM 50nM 75 nM 100 nM
T7 Template strand 1 μM 1 μM 1 μM 1 μM 1 μM 1 μM
NTP mix 2 mM 2 mM 2 mM 2 mM 2 mM 2 mM
T7 polymerase 1.9 μM 1.9 μM 1.9 μM 1.9 μM 1.9 μM 1.9 μM
DFHBI 20 μM 20 μM 20 μM 20 μM 20 μM 20 μM
Pyrophosphatase 0.001 U 0.001 U 0.001U 0.001 U 0.001 U 0.001 U

2. Construct 50 μL in vitro transcription system

Results

T7 promoter concentration gradient effect

Figure 3: T7 promoter concentration gradient effect

Observations & Notes

  1. The fluorescence intensity ratios between the experimental groups and the control group were calculated across different T7 promoter concentration gradients.
  2. The differences in fluorescence ratios among various T7 promoter concentrations were not statistically significant.
  3. The relative ranking of these ratios continually changed over time, indicating unstable results.
  4. Further optimization of experimental conditions is required before repeating the gradient concentration experiments.

July 1-July 15

Activity

Test on transcriptional activation of SRO1-T7

Purpose

Test whether SRO1-T7 could activate transcription.

Methods

1. Sequences

Table 7: SRO1-T7 and Template Sequences
Component Sequence (5'-3')
SRO1-T7 AGCCGAACTGAGTAATACGACTCACTATAGG
T7 template GGTGCTCACACTCTACTCAACAGTAGCGAACTACTGGACCCGTCCTTCACCTATAGTGAGTCGTATTAGACTC

Except for replacing the SRO1 sequence with SRO1-T7, all other operations and group settings are the same as in 5.27' experiment.

Results

SRO1-T7 transcription activation

Figure 4: SRO1-T7 transcription activation

Average slopes of 20-40 minutes

Figure 5: Average slopes of 20-40 minutes

Observations & Notes

  1. A concentration-dependent relationship with SRO-T7 exists for 0, 10 nM, and 20 nM fluorescence intensities.
  2. A pronounced downward trend appears at 50 nM, 75 nM, and 100 nM.
  3. The value at 100 nM even approaches that of the negative control.

July 16-August 3

Activity

Couple strand displacement to T7 in vitro transcription

Purpose

Couple strand displacement to T7 in vitro transcription to test whether the two modules could work together.

Methods

1. Conduct strand displacement

2. Construct 50 μL in vitro transcription system

After strand displacement, we added reaction mixture from the strand displacement reaction to T7 in vitro system.

Results

Strand displacement coupled transcription

Figure 6: Strand displacement coupled transcription

Observations & Notes

We observed signal leakage in the system, where even when the antibiotic did not bind to lbo1 and therefore did not release SRO1-T7, the promoter sequence on SRO1-T7 was still able to bind to the template and activate transcription.

August 17

Activity

Aptamer sequence design (SSS-S, SSS-M, SSS-L)

Purpose

Design a single-strand aptamer sensor to prevent T7 promoter leakage in antibiotic-free conditions.

Methods & Procedures

  1. Added reverse complementary T7 sequence and forward sequence to the 3′ end of LBO strand to form a hairpin.
  2. Designed three variants: SSS-S, SSS-M, SSS-L.
  3. Inserted bulges in antisense region to reduce nonspecific T7 polymerase binding.
  4. DNA sequences were ordered from the company.

Results

Table 8: SSS Family Sequences
Variant Sequence (5'-3')
SSS-S CTCAAACTTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTATAGTTTGAGTCGTATTTTATTTTTAATACGACTCACTATAGG
SSS-M CGACTCAAACTTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTATAGTTTGAGTCGTATTTTATTTTTAATACGACTCACTATAGG
SSS-L ATACGACTCAAACTTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTATAGTTTGAGTCGTATTTTATTTTTAATACGACTCACTATAGG

Observations & Notes

This stage focused only on theoretical design; wet-lab validation scheduled after synthesis.

August 25

Activity

Experimental validation of SSS-M prototype

Purpose

Test whether the single-strand design yields a vancomycin-dependent transcription response.

Methods

1. Experimental Setup

  • Selected SSS-M as representative for initial validation.
  • Annealed 1 μM SSS-M in Grade I water, added directly to transcription system.
Table 9: SSS-M Validation Conditions
Component Concentration
Template strand 1 μM
SSS-M 50 nM
Vancomycin (VAN) 0, 5, 20, 100 μM
T7 polymerase 2.5 μM

Results

SSS-M prototype validation

Figure 7: SSS-M prototype validation

Observations & Notes

  1. Fluorescence intensity showed no significant difference across vancomycin concentrations.
  2. Single-strand hairpin formed correctly but did not generate expected concentration-dependent output.
  3. Possible issues: template-to-sensor ratio, annealing method, or polymerase efficiency.

August 26

Activity

Template concentration gradient test

Purpose

To evaluate effect of template DNA concentration on response.

Methods

  • Concentrations: 10 nM, 100 nM, 1 μM template
  • Groups: negative (0 μM VAN) vs positive (100 μM VAN)
  • Other components fixed: SSS-M 50 nM, T7 polymerase 2.5 μM

Results

Template concentration gradient

Figure 8: Template concentration gradient effect

Observations & Notes

  1. At 10 nM template: some difference between groups but high noise.
  2. At higher concentrations: difference disappeared.
  3. Low template concentration may help discrimination, but reproducibility is poor.

August 27

Activity

Antibiotic gradient under low template concentration

Purpose

Verify if dose-response curve could be obtained.

Methods

  • Template: 10 nM
  • SSS-M: 50 nM
  • VAN: 0, 5, 20, 100 μM
  • T7 polymerase: 2.5 μM

Results

Antibiotic gradient test

Figure 9: Antibiotic gradient test

Observations & Notes

No clear correlation between vancomycin concentration and fluorescence intensity. Suggests that transcription system not yet optimized.

August 28

Activity

Adjusted annealing conditions and template-to-sensor ratio

Purpose

Optimize signal kinetics.

Methods

  • Template : SSS-M = 1:1 (0.5 μM : 0.5 μM)
  • Increased DFHBI-1T to 10 μM
  • Annealed with template + sensor + VAN simultaneously

Results

Optimized annealing conditions

Figure 10: Optimized annealing conditions

Observations & Notes

  1. Faster transcription (peak ~10 min)
  2. No VAN-dependent response
  3. Overly fast reaction masked possible differences.

August 29

Activity

Polymerase gradient experiments

Purpose

Test transcription kinetics at different T7 polymerase concentrations.

Methods

  • Polymerase: 0.025-2.5 μM
  • Template : sensor = 0.5 μM : 0.5 μM
  • VAN: 0 μM, 100 μM

Results

Polymerase gradient kinetics

Figure 11: Polymerase gradient kinetics

Polymerase concentration effect

Figure 12: Polymerase concentration effect

Observations & Notes

  1. High polymerase: strong transcription but no VAN effect
  2. Low polymerase: minimal transcription
  3. 1.25 μM polymerase: anomalous results (later confirmed experimental error)

August 30

Activity

Confirmation experiment for polymerase anomaly

Purpose

Verify whether the unusual result at 1.25 μM T7 polymerase was due to actual VAN response or experimental error.

Methods

  • Fixed template concentration: 0.5 μM
  • SSS-M: 0.5 μM
  • T7 polymerase: 1.25 μM
  • VAN gradient: 0, 5, 20, 100 μM

Results

Polymerase anomaly confirmation

Figure 13: Polymerase anomaly confirmation

Observations & Notes

  1. Fluorescence remained insensitive to vancomycin.
  2. No dose-response trend detected.
  3. The previous anomaly (0 μM > 100 μM VAN) was confirmed as an experimental error.
  4. SSS-M still failed to produce vancomycin-dependent responses under multiple enzyme concentrations.

September 1

Activity

Variant comparison (SSS-S, SSS-M, SSS-L)

Purpose

Determine effect of sequence length on promoter sealing efficiency.

Results

SSS variants kinetics

Figure 14: SSS variants kinetics comparison

SSS variants bar plot

Figure 15: SSS variants 10h results

Observations & Notes

  1. SSS-S: ~16% higher signal at 100 μM VAN compared to 0 μM
  2. Still lacked strong dose-response
  3. NUPACK showed aptamer binding region disrupted

September 3

Activity

Redesign of TS families (5TS & 3TS) - Design and Secondary Structure Prediction

Purpose

To address promoter leakage and preserve the aptamer recognition domain by redesigning the aptamer sensors.

Design

5TS Family

  • T7 promoter at the 5′ end, followed by reverse complementary segment.
  • Four variants (5TS-1 ~ 5TS-4) with 0-3 bulges in the complementary strand.
  • Goal: hairpin closure in absence of vancomycin; hairpin disruption upon vancomycin binding.

3TS Family

  • Reverse complementary sequence + T7 promoter appended to the 3′ end.
  • Variants (3TS-1 ~ 3TS-8) designed with different mismatch numbers/positions and altered 5′ bases.
  • Goal: test how mismatch placement and induced hairpin pairing affect recognition and leakage.

Prediction

  • Used NUPACK to simulate secondary structures.
  • Ensured aptamer recognition domain retained its native conformation.
  • Compared bulge numbers (5TS) and mismatch positions (3TS).

Results

5TS Family Sequences

Table 10: 5TS Family Sequences
Variant Sequence (5'-3')
5TS-1 AGTAATACGACTCACTATAGGTTTTCCTTTATAGTGTTAGTCGTTTATTACTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGTAATA
5TS-2 AGTAATACGACTCACTATAGGTTTTCCTATAGTGTTAGTCGTTTATTACTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGTAATA
5TS-3 AGTAATACGACTCACTATAGGTTTTCCTATAGTGTTAGTCGTATTACTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGTAATA
5TS-4 AGTAATACGACTCACTATAGGTTTTCCTATAGTGAGTCGTATTACTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGTAATA

3TS Family Sequences

Table 11: 3TS Family Sequences
Variant Sequence (5'-3')
3TS-1 AGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTATAGTGAGTCGTATTATTTTTAATACGACTCACTATAGG
3TS-2 AGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTCGCGTGAGTCGTATTATTTTTAATACGACTCACTATAGG
3TS-3 AGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTATAGTGCTGCGTATTATTTTTAATACGACTCACTATAGG
3TS-4 AGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTCGCGTGCTGCGTATTATTTTTAATACGACTCACTATAGG
3TS-5 TATAGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTATAGTGAGTCGTATTATTTTTAATACGACTCACTATAGG
3TS-6 GCGAGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTCGCGTGAGTCGTATTATTTTTAATACGACTCACTATAGG
3TS-7 TATAGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTATAGTGCTGCGTATTATTTTTAATACGACTCACTATAGG
3TS-8 GCGAGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTCGCGTGCTGCGTATTATTTTTAATACGACTCACTATAGG

Observations & Notes

  1. 5TS: predicted hairpin sealing maintained, promoter locked in silico.
  2. 3TS: mismatches close to hairpin core predicted to destabilize sealing, allowing easier promoter release.

September 6

Activity

Initial double-strand extension design (L1-L6 + S1)

Purpose

To prevent leakage from the T7 promoter by extending the 5′ end of the LBO strand to complement the SRO's T7 promoter, thereby blocking transcription in absence of vancomycin.

Design

  • Constructed SRO (S1) carrying T7 promoter (19 bases).
  • Designed six LBO variants (L1-L6) with increasing complementary bases (0, 4, 8, 12, 16, 19 bases).
  • Principle: In presence of vancomycin, conformational change in LBO forms hairpin, promoting displacement of SRO and transcription initiation.

Results

Table 12: S1 and LBO Variant Sequences
Component Sequence (5'-3')
S1 AGCCGAACTGAGTAATACGACTCACTATAGG
L1 CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA
L2 ATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA
L3 TCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA
L4 TGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA
L5 ATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA
L6 CCTATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA

Observations & Notes

Sequences designed for systematic testing of complementary base length effects on transcription regulation.

September 14-16

Activity

Experimental validation of TS families (5TS & 3TS)

Purpose

To experimentally test the redesigned aptamer sensors for vancomycin-dependent responses.

Methods

1. Experimental Setup

  • Ordered DNA sequences for all 5TS and 3TS variants.
Table 13: Annealing System
Component Concentration
3TS-n, 5TS-n 5μM
Binding Buffer 1x
Grade I water /
Table 14: Transcription System
Component Concentration
T7 Template strand 0.25μM
3TS-n, 5TS-n 0.25μM
VAN 0,100μM
T7 polymerase 2.5μM

Results

5TS Family Results

5TS family 5h results

Figure 16: 5TS family 5h results

3TS Family Results

3TS family results

Figure 17: 3TS family results

Observations & Notes

  1. 5TS family: bulge number influenced fluorescence intensity, but no significant vancomycin response.
  2. 3TS family: mismatch number/position strongly affected fluorescence.
  3. Variants with mismatches closer to hairpin interior + longer 5′ complement showed modest vancomycin responses (~11-13% increase), but with high background fluorescence.
  4. Structural design improved vancomycin sensitivity slightly (3TS), but background leakage remains problematic.
  5. Future optimization needed: balance mismatch design with complementary strand length.

September 17

Activity

Experimental validation of S1+L1~L6

Methods

  • Annealing: L-n (2 μM) + S1 (1 μM), heat 95 °C → slow cooling.
  • Transcription reaction: Template strand 100 nM, L-n 200 nM, S1 100 nM
  • NTP mix 2 mM, DFHBSI 1 μM, pyrophosphatase, T7 polymerase 2.5 μM
  • VAN: 0 μM vs 100 μM

Results

S1+L1-L6 validation

Figure 18: S1+L1-L6 validation results

Observations & Notes

  1. Best-performing: S1+L2 (fluorescence ratio ~1.21:1 at 100 μM vs 0 μM VAN).
  2. From L1 → L6: background fluorescence decreased as sealing strength increased.
  3. Strategy preliminarily validated by S1+L2, but background fluorescence high and signal-to-background ratio low.

September 18

Activity

Redesign of L4 & L5 extensions

Purpose

To reduce background fluorescence and improve signal-to-background ratio by modifying L4 and L5.

Design

  • Extended the 3′ ends of L4 and L5 by 5-17 bases to destabilize LBO-SRO duplex and promote vancomycin-induced hairpin.
  • Generated L4_1, L4_2, L4_3 and L5_1, L5_2, L5_3, L5_4 variants.

Results

Table 15: L4 and L5 Extension Variants
Variant Sequence (5'-3')
L4_1 TGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAG
L4_2 TGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTAAT
L4_3 TGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTAATACGA
L5_1 ATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAG
L5_2 ATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTAAT
L5_3 ATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTAATACGA
L5_4 ATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTAATACGACTCA

Observations & Notes

Ordered sequences from company for experimental validation.

September 24-26

Activity

Experimental testing of S1+L4_n and S1+L5_n

Methods

  • Dye switched from DFHBSI → DFHBI-1T (higher fluorescence contrast).
  • Tested S1+L4, L4_1, L4_2, L4_3 and S1+L5, L5_1, L5_2, L5_3, L5_4 under 0 μM vs 100 μM VAN.

Results

S1+L4 variants

Figure 19: S1+L4 variants 10h results

S1+L5 variants

Figure 20: S1+L5 variants 10h results

45min results

Figure 21: 45min results comparison

Observations & Notes

  1. L4_n group: insensitive to vancomycin.
  2. S1+L5_3: strongest improvement, fluorescence ratio ~1.35:1 (100 μM vs 0 μM VAN).
  3. L5_n designs followed "over-sealed → optimal → unsealed" trend.
  4. Best balance at L5_3 (13 bases added).
  5. L4_n increments were too large, missing intermediate "optimal" state (likely between +9 and +13 bases).

September 27

Activity

Template concentration gradient optimization (S1+L5_3)

Purpose

To explore whether varying template concentration can optimize signal-to-background ratio.

Methods

  • Tested template:S1 ratios: 0.5:1, 1:1, 5:1, 10:1.
  • S1+L5_3 sequences ordered and annealed as before.

Results

Template concentration optimization

Figure 22: Template concentration optimization

Observations & Notes

  1. Best responsiveness observed at 1:1 ratio.
  2. Too low template: signal weak, differences masked by noise.
  3. Too high template: template displaces S1 directly, overriding vancomycin effect.
  4. Optimal transcriptional performance lies in narrow template concentration range.
  5. Confirms importance of tuning template-to-sensor ratio.

Cas12a Amplification

August 29

Activity

Amplify the displaced signal using the Cas12a system

Purpose

Test whether the coupling of the complete signal generation and signal amplification modules can be successfully achieved.

Methods

1. Strands Hybridization

Table 1: Strand Hybridization Components
Component Group 0 Group 1 Group 2 Group 3 Group 4 Group 5
LBO1-Cas (μL) 2 2 2 2 2 2
SRO1-Cas (μL) 2 2 2 2 2 2
VAN (μL) 0 2 2 2 2 2
PBS (μL) 16 0 0 0 0 0
Overall (μL) 20 20 20 20 20 20

2. Final Concentrations in Hybridization System

Table 2: Final Concentrations
Component Group 0 Group 1 Group 2 Group 3 Group 4 Group 5
LBO1-Cas (μM) 1 1 1 1 1 1
SRO1-Cas (μM) 1 1 1 1 1 1
VAN (μM) 0 5 10 20 50 100

3. Cas System Construction

After cooling to room temperature in hot water and annealing, construct a 20 μL Cas system according to the table below.

Table 3: Cas System Components
Component Volume Final Concentrations
10× Reaction buffer 2 μL -
1μM reporter 2 μL 200 nM
20× Cas12a 2 μL 100 nM
1μM crRNA 2 μL 100 nM
Displaced solution 2 μL -
DEPC water 10 μL -
Total Volume 20 μL -

4. Master Mix Preparation

6 target concentrations total, each concentration in quintuplicate (5 repeats).

Table 4: Master Mix Composition
Component Volume (μL)
10× Reaction buffer 72
1μM reporter 14.4
20× Cas12a 36
crRNA 72
DEPC water 453.6
Overall (μL) 648

After preparation, add 18 μL of master mix and 2 μL of target to each tube. Perform the experiment in triplicate for each target concentration. Measure the fluorescence change of the system using a microplate reader.

Results

Cas12a amplification results August 29

Figure 1: Cas12a amplification results - August 29

Observations & Notes

The results suggest that the rate of fluorescence increase shows a concentration-dependent relationship with vancomycin. Nevertheless, additional experiments are needed to confirm this observation.

September 4

Activity

Amplify the displaced signal using the Cas12a system

Purpose

Standardize experimental procedures and attempt to replicate the experiment from 8.29 to obtain better results.

Methods

This experiment was performed in the same way as 8.29.

Results

Cas12a amplification results September 4

Figure 2: Cas12a amplification results - September 4

Observations & Notes

The outcomes of this experiment were suboptimal, necessitating further repetitions.

September 13

Activity

Amplify the displaced signal using the Cas12a system

Purpose

Standardize experimental procedures and attempt to replicate the experiment from 9.4 to obtain better results. The concentration of vancomycin was reduced to determine the minimum detectable limit.

Methods

This experiment was performed in the same way as 8.29.

Results

Cas12a amplification results September 13

Figure 3: Cas12a amplification results - September 13

Observations & Notes

The outcomes of this experiment were suboptimal, necessitating further repetitions. However, compared to the experimental results from 9.4, there has been a significant improvement.

September 14

Activity

Amplify the displaced signal using the Cas12a system

Purpose

Standardize experimental procedures and attempt to replicate the experiment from 9.13 to obtain better results. The concentration of vancomycin was reduced to determine the minimum detectable limit.

Methods

This experiment was performed in the same way as 9.13.

Results

Cas12a amplification results September 14

Figure 4: Cas12a amplification results - September 14

Observations & Notes

The results of this experiment were excellent except for the negative control group. This may be due to experimental procedure issues, necessitating a repeat of the experiment.

September 18

Activity

Amplify the displaced signal using the Cas12a system

Purpose

To enhance the sealing effect of the sensor, we have appropriately increased the amount of LBO.

Methods

1. Strand Hybridization

Table 5: Strand Hybridization Components (Optimized)
Component Group 0 Group 1 Group 2 Group 3 Group 4 Group 5
LBO1-Cas (μL) 2.6 2.6 2.6 2.6 2.6 2.6
SRO1-Cas (μL) 2 2 2 2 2 2
VAN (μL) 0 10 10 10 10 10
PBS (μL) 15.4 5.4 5.4 5.4 5.4 5.4
Overall (μL) 20 20 20 20 20 20

2. Final Concentrations in Hybridization System

Table 6: Final Concentrations (Optimized)
Component Group 0 Group 1 Group 2 Group 3 Group 4 Group 5
LBO1-Cas (μM) 1.3 1.3 1.3 1.3 1.3 1.3
SRO1-Cas (μM) 1 1 1 1 1 1
VAN (μM) 0 25 50 100 250 500

3. Cas System Construction

Table 7: Cas System Components
Component Volume Final Concentrations
10× Reaction buffer 2 μL -
1μM reporter 2 μL 200 nM
20× Cas12a 2 μL 100 nM
1μM crRNA 2 μL 100 nM
DEPC water 10 μL -
Total Volume 20 μL -

4. Master Mix Preparation

6 target concentrations total, each concentration in quintuplicate (5 repeats). Scale the master mix volume from the 9.13 experiment by 2 times.

Table 8: Master Mix Composition
Component Volume (μL)
10× Reaction buffer 72
1μM reporter 14.4
20× Cas12a 36
crRNA 72
DEPC water 453.6
Overall (μL) 648

After preparation, add 18 μL of master mix and 2 μL of target to each tube. Perform the experiment in triplicate for each target concentration. Measure the fluorescence change of the system using a microplate reader.

Results

Cas12a amplification results September 18

Figure 5: Cas12a amplification results - September 18

Observations & Notes

The experimental results were exceptionally outstanding. Even the fluorescence intensity growth rate of the negative control group exceeded that of the 2.5μM group. Compared to the results of Experiment 9.14, this effectively reduced leakage in the negative control group, demonstrating the significant efficacy of our strategy to rationally increase the LBO concentration.

September 21

Activity

Amplify the displaced signal using the Cas12a system

Purpose

To investigate the reproducibility of the experiment, we repeated the experiment described in 9.18.

Methods

Same as September 18 experiment.

Results

Cas12a amplification results September 21

Figure 6: Cas12a amplification results - September 21

Observations & Notes

The experimental results remain favorable, clearly demonstrating the positive correlation between vancomycin concentration and the rate of increase in fluorescence intensity.

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