iGEM 2025 — Team hunanu

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* [Strand Displacement](#experiment-records)
  + [May 22](#may-22)
  + [May 26](#may-26)
  + [May 28](#may-28)
  + [June 16](#june-16)
  + [June 19](#june-19)
  + [June 22](#june-22)
  + [June 25](#june-25)
  + [July 4](#july-4)
  + [July 5](#july-5)
  + [July 6](#july-6)
  + [July 16](#july-16)
  + [July 27](#july-27)
  + [August 7](#august-7)
  + [August 11](#august-11)
  + [August 13](#august-13)
  + [August 15](#august-15-replacement)
  + [August 24](#august-24)
  + [August 25](#august-25-replacement)
  + [August 26](#august-26-replacement)
  + [August 27-30](#august-27-30)
  + [September 11](#september-11-replacement)
* [T7 Transcription Amplification](#transcription-records)
  + [May 16](#may-16)
  + [May 27-June 18](#may-27-june-18)
  + [June 19-June 30](#june-19-june-30)
  + [July 1-July 15](#july-1-july-15)
  + [July 16-August 3](#july-16-august-3)
  + [August 17](#august-17)
  + [August 25](#august-25)
  + [August 26](#august-26)
  + [August 27](#august-27)
  + [August 28](#august-28)
  + [August 29](#august-29)
  + [August 30](#august-30)
  + [September 1](#september-1)
  + [September 3](#september-3)
  + [September 6](#september-6)
  + [September 14-16](#september-14-16)
  + [September 17](#september-17)
  + [September 18](#september-18)
  + [September 24-26](#september-24-26)
  + [September 27](#september-27)
* [Cas12a Amplification](#cas-records)
  + [August 29](#august-29-cas)
  + [September 4](#september-4)
  + [September 13](#september-13)
  + [September 14](#september-14-cas)
  + [September 18](#september-18-cas)
  + [September 21](#september-21)

# 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](https://static.igem.wiki/teams/5772/wiki/note1.avif)

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](https://static.igem.wiki/teams/5772/wiki/note2.avif)

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](https://static.igem.wiki/teams/5772/wiki/zh3.webp)

Figure 3: LBO1/SRO1 system gel electrophoresis

![LBO3/SRO3 system gel electrophoresis](https://static.igem.wiki/teams/5772/wiki/xzh4.avif)

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](https://static.igem.wiki/teams/5772/wiki/nzh5.avif)

Figure 5: Simple experiment gel electrophoresis 1

![Simple experiment gel electrophoresis 2](https://static.igem.wiki/teams/5772/wiki/zh6.webp)

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](https://static.igem.wiki/teams/5772/wiki/zh7.webp)

Figure 7: LBO1/SRO1 system gel electrophoresis

![LBO2/SRO2 system gel electrophoresis](https://static.igem.wiki/teams/5772/wiki/zh8.webp)

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](https://static.igem.wiki/teams/5772/wiki/zh9.webp)

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](https://static.igem.wiki/teams/5772/wiki/zh10.webp)

Figure 10: High-concentration group fluorescence

![Low-concentration group fluorescence](https://static.igem.wiki/teams/5772/wiki/zh11.webp)

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](https://static.igem.wiki/teams/5772/wiki/zh12.webp)

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](https://static.igem.wiki/teams/5772/wiki/zh13.webp)

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](https://static.igem.wiki/teams/5772/wiki/zh14.webp)

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](https://static.igem.wiki/teams/5772/wiki/zh15.webp)

Figure 15: LBO1/SRO1 fluorescence quantification 1

![LBO1/SRO1 fluorescence quantification 2](https://static.igem.wiki/teams/5772/wiki/zh16.webp)

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](https://static.igem.wiki/teams/5772/wiki/zh17.webp)

Figure 17: LBO1/SRO1 system fluorescence

![LBO2/SRO2 system fluorescence](https://static.igem.wiki/teams/5772/wiki/zh18.webp)

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](https://static.igem.wiki/teams/5772/wiki/zh19.webp)

Figure 19: Hybridization efficiency comparison

![LBO1-SRO2 displacement gel](https://static.igem.wiki/teams/5772/wiki/zh20.webp)

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](https://static.igem.wiki/teams/5772/wiki/zhn2.webp)

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](https://static.igem.wiki/teams/5772/wiki/zhn3.webp)

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](https://static.igem.wiki/teams/5772/wiki/zhn1.webp)

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](https://static.igem.wiki/teams/5772/wiki/zhn4.webp)

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](https://static.igem.wiki/teams/5772/wiki/zhn5.webp)

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](https://static.igem.wiki/teams/5772/wiki/zhn6.webp)

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](https://static.igem.wiki/teams/5772/wiki/zhn7.webp)

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](https://static.igem.wiki/teams/5772/wiki/zhn8.webp)

Figure 28: Displacement experiment repetition 1

![Displacement experiment repetition 2](https://static.igem.wiki/teams/5772/wiki/zhn9.webp)

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](https://static.igem.wiki/teams/5772/wiki/zhn10.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl1.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl2.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl3.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl4.webp)

Figure 4: SRO1-T7 transcription activation

![Average slopes of 20-40 minutes](https://static.igem.wiki/teams/5772/wiki/zl5.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl6.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl7.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl8.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl9.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl10.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl11.webp)

Figure 11: Polymerase gradient kinetics

![Polymerase concentration effect](https://static.igem.wiki/teams/5772/wiki/zl12.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl13.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl14.webp)

Figure 14: SSS variants kinetics comparison

![SSS variants bar plot](https://static.igem.wiki/teams/5772/wiki/zl15.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl16.webp)

Figure 16: 5TS family 5h results

#### 3TS Family Results

![3TS family results](https://static.igem.wiki/teams/5772/wiki/zl17.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl18.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl19.webp)

Figure 19: S1+L4 variants 10h results

![S1+L5 variants](https://static.igem.wiki/teams/5772/wiki/zl20.webp)

Figure 20: S1+L5 variants 10h results

![45min results](https://static.igem.wiki/teams/5772/wiki/zl21.webp)

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](https://static.igem.wiki/teams/5772/wiki/zl22.webp)

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](https://static.igem.wiki/teams/5772/wiki/cas1.webp)

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](https://static.igem.wiki/teams/5772/wiki/cas2.webp)

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](https://static.igem.wiki/teams/5772/wiki/cas3.webp)

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](https://static.igem.wiki/teams/5772/wiki/cas4.webp)

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](https://static.igem.wiki/teams/5772/wiki/cas5.webp)

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](https://static.igem.wiki/teams/5772/wiki/cas6.webp)

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.

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

* [Overview](#overview)
* [Cycle 1: Strand Displacement Module](#cycle1)
  + [1.1 Design and Implementation of SRO Non-T7 Sequence Strand Displacement System](#subsystem-1-1)
    - [Design](#design-1-1)
    - [Build](#build-1-1)
    - [Test](#test-1-1)
    - [Learn](#learn-1-1)
  + [1.2 Strand Displacement System with SRO Incorporating a T7 Sequence](#subsystem-1-2)
    - [Design](#design-1-2)
    - [Build](#build-1-2)
    - [Test](#test-1-2)
    - [Learn](#learn-1-2)
  + [1.3 Cas Strand Displacement](#cycle1-3)
    - [1.3.1 Design](#design-1-3-1)
    - [1.3.1 Build](#build-1-3-1)
    - [1.3.1 Test](#test-1-3-1)
    - [1.3.1 Learn](#learn-1-3-1)
    - [1.3.2 Design](#design-1-3-2)
    - [1.3.2 Build](#build-1-3-2)
    - [1.3.2 Test](#test-1-3-2)
    - [1.3.2 Learn](#learn-1-3-2)
* [Cycle 2: T7 in vitro transcription module](#cycle2)
  + [2.1 Transcription system based on the original LBO1 and SRO1-T7](#subsystem-2-1)
    - [Design](#design-2-1)
    - [Build](#build-2-1)
    - [Test](#test-2-1)
    - [Learn](#learn-2-1)
  + [2.2 Three-strand system to decrease the signal leakage](#subsystem-2-2)
    - [Design](#design-2-2)
    - [Build](#build-2-2)
    - [Test](#test-2-2)
    - [Learn](#learn-2-2)
  + [2.3 Double-strand system to decrease the signal leakage](#subsystem-2-3)
    - [2.3.1 Initial Design and Verification](#design-2-3-1)
    - [2.3.2 Parameter Optimization and Performance Improvement](#design-2-3-2)
    - [2.3.3 Transcription Experiment Verification](#design-2-3-3)
  + [2.4 Single-strand system to decrease the signal leakage](#subsystem-2-4)
    - [2.4.1 Design and Initial Test](#design-2-4-1)
    - [2.4.2 Template Concentration Test](#design-2-4-2)
    - [2.4.3 Antibiotic Gradient Test](#design-2-4-3)
    - [2.4.4 Ratio and Annealing Optimization](#design-2-4-4)
    - [2.4.5 Polymerase Concentration Test](#design-2-4-5)
    - [2.4.6 Sub-gradient VAN Test](#design-2-4-6)
    - [2.4.7 Evaluation of SSS-S and SSS-L](#design-2-4-7)
    - [2.4.8 Redesign: 5TS and 3TS Families](#design-2-4-8)
  + [2.5 LBO modification for reduced signal leakage](#subsystem-2-5)
    - [2.5.1 Design and Initial Test](#design-2-5-1)
    - [2.5.2 Optimized Design and Performance Evaluation](#design-2-5-2)
    - [2.5.3 Template Concentration Optimization](#design-2-5-3)
* [Cycle 3: CRISPR-Cas12a amplification module](#cycle3)
  + [Design](#design-3-1)
  + [Build](#build-3-1)
  + [Test](#test-3-1)
  + [Learn](#learn-3-1)

# Overview

Our experiment is divided into three cycles.

### Cycle 1: Strand Displacement Reaction

In Cycle 1, we primarily investigated the strand displacement reaction, which consists of two main parts:

1. **Strand displacement coupled with the T7 transcription amplification pathway:** Cycle 1.1 and Cycle 1.2.
   * The initial Cycle 1.1 used SRO without the T7 promoter, simplifying hybridization and displacement.
   * Cycle 1.2 incorporated the T7 promoter sequence to facilitate coupling with T7 transcription.
2. **Strand displacement coupled with the CRISPR-Cas12a amplification pathway:** Cycle 1.3.

### Cycle 2: T7 Transcription Amplification

In Cycle 2, we studied T7 transcription amplification. We observed signal leakage when applying the Cycle 1.2 sequence to T7 transcription (Cycle 2.1) and explored multiple solutions to address this issue (Cycles 2.2–2.5).

### Cycle 3: CRISPR-Cas12a Signal Amplification

In Cycle 3, we investigated the CRISPR-Cas12a system coupled with Cycle 1.3 for signal amplification and successfully achieved amplification.

# Cycle 1: Strand Displacement Module

## 1.1 Design and Implementation of SRO Non-T7 Sequence Strand Displacement System

## Design

The core principle of strand displacement reaction is competitive binding. In this system, vancomycin (Vancomycin, VAN) acts as a small molecule inducer, binding to the specific binding site on the long-strands aptamer LBO, thereby disrupting the pre-formed hybrid double-strand structure between LBO and the short-strands probe SRO. The SRO sequence is designed as short strands **without T7 promoter sequences**, focusing on the strand displacement function, and avoiding interference from the T7 promoter sequence.

![Schematic diagram of strand displacement system](https://static.igem.wiki/teams/5772/eng/figure1.webp)

Figure 1: Schematic of SRO non-T7 strand displacement system

## Build

We designed SRO1-8 and their corresponding LBO1-8, whose lengths and sequences were optimized to reduce nonspecific binding. The LBO long strand contains the vancomycin (VAN)-specific aptamer region, with aptamers of varying affinities. The key binding domains within the LBO sequence allow competitive access of VAN. The specific sequences of LBO and SRO, as well as the aptamer affinities [2][3], are shown in the table below. (Complementary base pairs are marked in red)

Table 1: LBO-SRO Pairs Designed for Initial Screening

| Name | Sequence | Number of complementary bases | Predicted hybridization rate(1:1) | aptamer affinity |
| --- | --- | --- | --- | --- |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA | 12 | 77.8% | 58± 12.2 μM |
| SRO1 | AGCCGAACTGAG |
| LBO2 | TTTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG | 15 | 91.4% | 58± 12.2 μM |
| SRO2 | AGCCGGAGAGAGAAA |
| LBO3 | TCTCTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG | 17 | 100% | 58± 12.2 μM |
| SRO3 | AGCCGGAGAGAGAGAGA |
| LBO4 | CCTGAACCCCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGGTT | 12 | 36.8% | 3.92±0.90 μM |
| SRO4 | TCGGGGTTCAGG |
| LBO5 | GCCTGAACCCCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGGTT | 13 | 79.9% | 3.92±0.90 μM |
| SRO5 | TCGGGGTTCAGGC |
| LBO6 | TAAGATCTCTCGGGACGACCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGTCGTCCC | 13 | 71.3% | 3.92±0.90 μM |
| SRO6 | GTCGTCCCGAGAGTA |
| LBO7 | TAAGATCTCTCAGGGACGACCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGTCGTCCCT | 15 | 86.4% | 3.92±0.90 μM |
| SRO7 | GTCGTCCCTGAGAGAA |
| LBO8 | TAAGATCTCTCAGGGGACGACCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGTCGTCCCCT | 14 | 90.9% | 3.92±0.90 μM |
| SRO8 | GTCGTCCCCTGAGAGAT |

**Complementary base pairs are marked in red in the original NUPACK simulation.**

## Test

### Step 1. Strand hybridization

Our first objective was to obtain a stable LBO–SRO duplex, ensuring that in the absence of antibiotics, the hybridized duplex does not spontaneously dissociate and that free SRO remains minimal in the system.

**Procedure**

Hybridization mixtures were prepared according to the table below, annealed by cooling from hot water to room temperature, and then analyzed by PAGE for preliminary validation.

Table 2: Strand Hybridization Procedure

| Group Reactants | 1 | 2 | 3 |
| --- | --- | --- | --- |
| LBO(μL) | 1 | 0 | 1 |
| SRO(μL) | 0 | 1 | 1 |
| PBS(μL) | 9 | 9 | 8 |

**Results**

![PAGE results for LBO1-SRO1 hybridization](https://static.igem.wiki/teams/5772/eng/figure2.webp)

Figure 2

![PAGE results for LBO2-SRO2 hybridization](https://static.igem.wiki/teams/5772/eng/figure3.webp)

Figure 3

![PAGE results for LBO3-SRO3 hybridization](https://static.igem.wiki/teams/5772/eng/figure4.webp)

Figure 4

![Control hybridization experiments](https://static.igem.wiki/teams/5772/eng/figure5.webp)

Figure 5

According to the electrophoresis results, LBO–SRO1–3 exhibited high hybridization efficiency.

### Step 2. Strand displacement upon antibiotic addition

Based on the results from Step 1, LBO-SRO1–3 were selected to perform strand displacement assays in the presence of antibiotics.

**Procedure**

1. Prepare the hybridization strands according to the table below.

Table 3: Strand Displacement Procedure

| Group Reactants | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| LBO (μL) | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| SRO (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(50 μM VAN) | 1(100 μM VAN) | 1(200 μM VAN) | 1(500 μM VAN) | 1(1 mM VAN) |
| Overall system (μL) | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |

**Note:** The concentrations of added vancomycin 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 5 μM, 10 μM, 20 μM, 50 μM, 100 μM.

2. Place the prepared hybridization strands in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used as a sample for polyacrylamide gel electrophoresis.

**Results**

![LBO1+SRO1 displacement with VAN](https://static.igem.wiki/teams/5772/eng/figure6.webp)

Figure 6

![LBO2+SRO1 displacement with VAN](https://static.igem.wiki/teams/5772/eng/figure7.webp)

Figure 7

![LBO3+SRO3 displacement with VAN](https://static.igem.wiki/teams/5772/eng/figure8.webp)

Figure 8

LBO1+SRO1 System and LBO2+SRO1 System: As the VAN concentration increased (5→100 μM), the brightness of the hybridization bands significantly decreased, with the bands almost disappearing at concentrations above 50 μM, indicating high disassembly efficiency and potential for sensor construction.

LBO3+SRO3 system: The strip attenuation is relatively small, only slightly weakened at 100 μM, indicating higher hybridization stability.

### Step 3. Strand proportional hybridization

In previous experiments, we observed a distinct SRO1 band when the LBO:SRO ratio was 1:1. Therefore, we increased the LBO:SRO ratio in an attempt to achieve higher SRO hybridization efficiency and to reduce the concentration of free SRO.

**Procedure**

1. Prepare the hybridization strands according to the table below.

Table 4: Proportional Hybridization Procedure

| Group Reactants | LBO | SRO | LBO:SRO 1:1 | LBO:SRO 1:1.5 | LBO:SRO 1:2 | LBO:SRO 1:2.5 | LBO:SRO 1:3 | LBO:SRO 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 (μL) | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |

2. Place the prepared hybridization strands in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used as a sample for polyacrylamide gel electrophoresis.

**Results**

![Proportional hybridization results](https://static.igem.wiki/teams/5772/eng/figure9.webp)

Figure 9

The experiment shows that the highest hybridization efficiency (with the best band clarity) is achieved when the molar ratio of LBO:SRO is **2:1 or 2.5:1**, and a higher ratio leads to an enhanced background signal.

## Learn

The LBO1–SRO1 and LBO2–SRO2 combinations exhibited higher hybridization efficiency and demonstrated superior concentration-dependent strand displacement upon vancomycin stimulation, making them suitable as the foundational framework for subsequent sensor design. Moving forward, the strand displacement system will be integrated with the transcriptional module to achieve signal amplification. Through systematic analysis of these modules, the SRO-based strand displacement system with the incorporated T7 sequence can be further optimized toward higher sensitivity and reduced background leakage, thereby providing a robust foundation for biosensor applications.

## 1.2 Strand Displacement System with SRO Incorporating a T7 Sequence

## Design

LBO1–SRO1 and LBO2–SRO2 exhibited superior stability and stronger concentration–reaction rate correlation in the strand displacement assays, with relatively higher reaction efficiencies. Therefore, LBO1–SRO1 and LBO2–SRO2 are recommended as the core components. Incorporating a T7 promoter sequence further extends their functionality and enables coupling with the transcriptional amplification system.

![SRO-T7 system design schematic](https://static.igem.wiki/teams/5772/eng/figure10.webp)

Figure 10

## Build

We added a T7 promoter sequence to the SRO, designated as SRO-T7. The LBO strand was labeled with the quencher group BHQ, while the SRO-T7 strand was labeled with the fluorophore FAM. This design allows fluorescence measurements to quantitatively investigate LBO–SRO hybridization and strand displacement.

**NUPACK simulation** (Complementary base pairs are marked in red)

![NUPACK simulation for LBO1-SRO1-T7](https://static.igem.wiki/teams/5772/eng/figure11.webp)

Figure 11

Table 1: Sequences for LBO1-SRO1-T7 Pair

| Component | Sequence | Number of complementary bases | Predicted hybridization rate |
| --- | --- | --- | --- |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA | 12 | 59.7% |
| SRO1-T7 | AGCCGAACTGAGTAATACGACTCACTATAGG |

![Additional simulation result 1](https://static.igem.wiki/teams/5772/eng/figure12.webp)

Figure 12

![Additional simulation result 2](https://static.igem.wiki/teams/5772/eng/figure13.webp)

Figure 13

Table 2: Sequences for LBO2-SRO2-T7 Pair

| Component | Sequence | Number of complementary bases | Predicted hybridization rate |
| --- | --- | --- | --- |
| LBO2 | TTTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG | 15 | 91.4% |
| SRO2-T7 | AGCCGGAGAGAGAAATAATACGACTCACTATAGG |

![NUPACK simulation for LBO2-SRO2-T7](https://static.igem.wiki/teams/5772/eng/figure14.webp)

Figure 14

![Additional simulation result 3](https://static.igem.wiki/teams/5772/eng/figure15.webp)

Figure 15

## Test

### Step 1. Strand hybridization

**Procedure**

1. Preparation of hybrid strands (long strands: short strands = 2:1)

Table 3: Preparation of Hybrid Strands

| Reagent | Group | | |
| --- | --- | --- | --- |
| L'+S' | L+S' | L+S |
| LBO | 0 | 0 | 20 |
| LBO' | 20 | 0 | 0 |
| SRO | 0 | 0 | 10 |
| SRO' | 10 | 10 | 0 |
| SRO-T7 | 0 | 0 | 0 |
| SRO'-T7 | 0 | 0 | 0 |
| PBS | 70 | 70 | 70 |
| Total | 100μL | 100μL | 100μL |

The hybrid strands system was prepared as above, with the concentration diluted from 10μM to 1μM, incubated at 95℃ for 5 min, and then slowly cooled to room temperature in boiling water.

2. RFU measurement of hybrid strands

The reaction system was added to a 384-well plate, with the hybrid strands concentration diluted from 1μM to 100nM. Triplicate measurements were performed for each group, and the microplate reader was set at an excitation wavelength of 490 nm and an emission wavelength of 520 nm.

Table 4: RFU Measurement Setup

|  | L2'+S2' | L2+S2' | L2+S2 |
| --- | --- | --- | --- |
| Hybrid strands(μL) | 5 | 5 | 5 |
| PBS(μL) | 45 | 45 | 45 |
| Total(μL) | 50 | 50 | 50 |

**Results**

![Hybridization fluorescence results 1](https://static.igem.wiki/teams/5772/eng/figure16.webp)

Figure 16

The T7-modified strands showed lower stability, and the binding efficiency between long and short strands was reduced.

As shown in the figure, the fluorescence intensity of the LBO1 and SRO1-T7 system is more stable; therefore, this system was selected for subsequent experiments.

### Step 2. Strand proportional hybridization

In the absence of antibiotics, hybridize SRO with LBO and measure the fluorescence of the system. The fluorescence value reflects the amount of free SRO in the system, which may activate downstream amplification pathways and generate background signals. Therefore, it is necessary to identify an appropriate SRO:LBO ratio that keeps the background signal within an acceptable range.

**Procedure**

1. Different SRO:LBO ratios were tested to evaluate the background fluorescence levels under each condition, enabling the selection of an optimal SRO:LBO ratio for sensor construction.

Table 5: Proportional Hybridization Testing

| Reagent | SRO:LBO Ratio | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1:1 | 1:2 | 1:3 | 1:5 | 1:7 | 1:9 | Positive control (0:1) | PBS |
| 10mM LBO (μL) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 10mM SRO (μL) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 10mM LBO-BHQ (μL) | 3 | 6 | 9 | 15 | 21 | 27 | 0 | 0 |
| 10mM SRO-FAM (μL) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 0 |
| PBS (μL) | 24 | 21 | 18 | 12 | 6 | 0 | 27 | 30 |
| Overall (μL) | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 |

2. RFU measurement of hybrid strands

The reaction system was added to a 384-well plate, with the hybrid strands concentration diluted from 1μM to 100nM. Each group was diluted according to the table below for fluorescence measurement. Five replicate experiments were performed for each group.

Table 6: RFU Measurement Dilution

| Reagent | Volume |
| --- | --- |
| Hybrid strands (μL) | 5 |
| PBS(μL) | 45 |

**Results**

![Hybridization fluorescence results 2](https://static.igem.wiki/teams/5772/eng/figure17.webp)

Figure 17

At hybridization ratios of 1:1, 2:1, and 3:1, the hybridization strands were unstable, with fluorescence values increasing over time, indicating strand separation at 37 °C. At ratios of 5:1, 7:1, and 9:1, hybridization was stable, and background fluorescence was low. To ensure sensor sensitivity, a ratio of 5:1 was selected for subsequent experiments.

### Step 3. Strand displacement reaction upon antibiotic addition

**Procedure**

1. Prior to the strand displacement reaction, we first prepare the required hybridization strands in a unified manner to ensure that all experimental groups utilize the same hybridization strands when constructing the reaction system. Prepare the hybridization strands according to the table below.

Table 7: Preparation of Unified Hybridization Strands

| Reagent | Experimental group(1-6) | Positive control(7) |
| --- | --- | --- |
| 10mM LBO (μL) | 0 | 0 |
| 10mM SRO (μL) | 0 | 0 |
| 10mM LBO-BHQ (μL) | 100 | 0 |
| 10mM SRO-FAM (μL) | 20 | 3 |
| PBS (μL) | 80 | 27 |
| Overall (μL) | 200 | 30 |

2. After preparation, vortex the mixture thoroughly and centrifuge for 10 seconds.

3. Place the prepared hybridization strands in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used for construct the strand displacement reaction system.

Table 8: Strand Displacement Reaction System

| Groups | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Van (μL) | 0 | 5 | 5 | 5 | 5 | 5 | 0 |
| Hybrid strands (μL) | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| PBS (μL) | 45 | 40 | 40 | 40 | 40 | 40 | 45 |
| Overall (μL) | 50 | 50 | 50 | 50 | 50 | 50 | 50 |

**Results**

![Proportional hybridization results](https://static.igem.wiki/teams/5772/eng/figure18.webp)

Figure 18

## Learn

The LBO1–SRO1-T7 system exhibited relatively stable hybridization and a concentration-dependent response to antibiotics, thereby preparing the system for integration with the transcriptional amplification module.

#### References

1. Tan, J.H., Fraser, A.G. Quantifying metabolites using structure-switching aptamers coupled to DNA sequencing. Nat Biotechnol (2025).
2. Alexander Shaver, J.D. Mahlum, Karen Scida, Melanie L. Johnston, Miguel Aller Pellitero, Yao Wu, Gregory V. Carr, and Netzahualcóyotl Arroyo-Currás. Optimization of Vancomycin Aptamer Sequence Length Increases the Sensitivity of Electrochemical, Aptamer-Based Sensors In Vivo ACS Sensors 2022 7 (12), 3895-3905
3. Xiaona Fang, Tian Gao , Zhaofeng Luo , Renjun Pei . Efficient selection of vancomycin-specific aptamers via particle display and development of a high-sensitivity fluorescent apta-sensor for vancomycin detection . Sensors and Actuators B: Chemical 2025 8(1),137681

## 1.3 Cas Strand Displacement

### Cycle 1.3.1

## Design

In the T7 transcription system of Cycle 2, detection of antibiotics within a certain concentration range was achieved, but it did not demonstrate significant amplification effects. We are still exploring alternative amplification approaches. Through literature review, we learned that in the CRISPR-Cas12a system, activated Cas12a exhibits trans-cleavage activity[1], enabling continuous and non-specific cleavage of single-stranded DNA reporter probes in the system, thereby achieving efficient fluorescence signal amplification.

For this purpose, we designed LBO1-Cas and SRO1-Cas, predicted binding efficiency with NUPACK, and experimentally verified whether vancomycin could displace SRO1-Cas.

Table 1: Sequences for LBO1-Cas and SRO1-Cas

| Component | Sequence |
| --- | --- |
| LBO1-Cas | GACGTATCGACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACT |
| SRO1-Cas | GGAGACGCCGAACTGAGTCGATACGTCTAGATTG |
| crRNA-1 | UAAUUUCUACUAAGUGUAGAUACGUAUCGACUCAGUUCGGC |
| Number of complementary bases | 20 |

## Build

**NUPACK Simulation**

![Strand displacement results](https://static.igem.wiki/teams/5772/eng/figure19.webp)

Figure 19

![LBO1-Cas secondary structure](https://static.igem.wiki/teams/5772/eng/figure20.webp)

Figure 20

![SRO1-Cas secondary structure](https://static.igem.wiki/teams/5772/eng/figure21.webp)

Figure 21

![crRNA-1 secondary structure](https://static.igem.wiki/teams/5772/eng/figure22.webp)

Figure 22

![LBO1-Cas and SRO1-Cas hybridization structure](https://static.igem.wiki/teams/5772/eng/figure23.webp)

Figure 23

## Test

BHQ group was added to LBO1-Cas, and 6-FAM group was added to SRO1-Cas. Fluorescence intensity was measured with a microplate reader to verify whether vancomycin could displace SRO1-Cas. Each group was tested in quintuplicate, with the highest and lowest values discarded before averaging.

![Fluorescence results for initial Cas design](https://static.igem.wiki/teams/5772/eng/figure24.webp)

Figure 24: Fluorescence intensity vs vancomycin concentration

Note: This graph illustrates the strand displacement reaction of LBO1-SRO1-Cas. Column 1 represents the positive control (SRO1-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.

The results showed no significant correlation between fluorescence intensity and vancomycin concentration. Even when the final concentration of vancomycin reached 100 μM, there was still no significant difference compared with 0 μM vancomycin.

## Learn

Our first sensor design bound too tightly, so even high doses of vancomycin could not displace SRO. Therefore, we shifted the binding site of SRO and LBO so that the hairpin-forming sequence of LBO was positioned in the middle of the LBO–SRO binding region, in an attempt to facilitate SRO release.

### Cycle 1.3.2

## Design

To address the overly tight binding between LBO and SRO in the previous design, we altered the binding positions of LBO and SRO so that SRO could be more easily displaced.

![Redesigned binding site schematic](https://static.igem.wiki/teams/5772/eng/figure25.webp)

Figure 25: Improved binding site design

## Build

For this purpose, we designed a new SRO, which we named SRO2-Cas.

Table 2: Sequences for the Redesigned System

| Component | Sequence |
| --- | --- |
| LBO1-Cas | GACGTATCGACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACT |
| SRO2-Cas | GGAGACCACTGAGCCGAACTGAGTCGCTAGATTG |
| crRNA-2 | UAAUUUCUACUAAGUGUAGAUCGACUCAGUUCGGCUCAGUG |
| Number of complementary bases | 20 |

**NUPACK Simulation**

![SRO2-Cas secondary structure](https://static.igem.wiki/teams/5772/eng/figure26.webp)

Figure 26: SRO2-Cas secondary structure

![crRNA-2 secondary structure](https://static.igem.wiki/teams/5772/eng/figure27.webp)

Figure 27: crRNA-2 secondary structure

![LBO1-Cas and SRO2-Cas hybridization structure](https://static.igem.wiki/teams/5772/eng/figure28.webp)

![LBO1-Cas and SRO2-Cas hybridization structure](https://static.igem.wiki/teams/5772/eng/figure29.webp)

Figure 29: LBO1-Cas and SRO2-Cas hybridization (1:1)

## Test

### Step 1. PAGE electrophoresis to determine strand displacement upon antibiotic addition

Learning from the previous experiment and to save costs, we first ordered LBO2-Cas and SRO2-Cas without fluorescent or quenching groups. A PAGE gel was used for qualitative analysis of whether vancomycin could displace SRO2-Cas.

**Note:** Except for the sequences of the strands used, the group settings and operating methods for PAGE electrophoresis are the same as in Cycle 1.1 step 2.

![PAGE electrophoresis results for strand displacement](https://static.igem.wiki/teams/5772/eng/figure30.webp)

Figure 30: PAGE analysis of SRO2-Cas displacement

The results showed that vancomycin was able to displace SRO, confirming that our design was effective.

### Step 2. Fluorescence quantitative to determine strand displacement upon antibiotic addition

We then ordered LBO1-Cas with a BHQ group and SRO2-Cas with a 6-FAM group. Fluorescence intensity was measured with a microplate reader to quantitatively analyze SRO displacement by vancomycin. Each group was tested in quintuplicate, with the highest and lowest values discarded before averaging.

**Procedure**

1. Prepare the hybridization chain of LBO1-Cas and SRO2-Cas at a 1:1 ratio, with both LBO1-Cas and SRO2-Cas at a concentration of 1 μM in the system.

2. Construct strand displacement system

Table 3: Strand Displacement Reaction System

|  | No RNA | | | | | | 100 nM RNA | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 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 |
| Van (μL) | 0 | 5 | 5 | 5 | 5 | 5 | 0 | 5 | 5 | 5 | 5 | 5 |
| 1 μM strands (μL) | 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 |

**Note:** The concentrations of added vancomycin 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 5 μM, 10 μM, 20 μM, 50 μM, 100 μM.

![Quantitative fluorescence displacement results](https://static.igem.wiki/teams/5772/eng/figure31.webp)

Figure 31: Fluorescence intensity vs vancomycin concentration for redesigned system

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.

## Learn

By shifting the LBO–SRO binding site into the middle of LBO's self-complementary sequence, we successfully enhanced vancomycin's ability to displace SRO. We ultimately selected **LBO1-Cas and SRO2-Cas** for subsequent signal amplification experiments.

#### References

1. Janice S. Chen et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436-439 (2018).

# Cycle 2: T7 in vitro transcription module

## 2.1 Transcription system based on the original LBO1 and SRO1-T7

## Design

Based on wide research and application, T7 in vitro transcription system is able to product certain RNA molecules with high efficiency, high yield and high fidelity, and exhibits strict specificity for the T7 promoter [1]. Therefore it has potential to be designed as a signal amplification platform.

In the T7 in vitro transcription system, SRO was designed to comprise T7 promoter sequence. When it released and bound with a downstream template, it would activate transcription. The transcription product was an aptamer RNA that enhanced the fluorescence of a specific dye DFBHSI [2], enabling the quantification of antibiotic residues through fluorescence intensity.

![Diagram of T7 transcription system](https://static.igem.wiki/teams/5772/eng2/figure1.webp)

Figure 1: Schematic of T7 transcription system design

## Build

We added T7 promoter sequence to SRO1, and named it as SRO1-T7.

Table 1: Component Sequences

| Component | Sequence |
| --- | --- |
| T7 promoter | TAATACGACTCACTATAGG |
| SRO1 | AGCCGAACTGAG |
| SRO1-T7 | AGCCGAACTGAGTAATACGACTCACTATAGG |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| SRO1-T7-FAM | AGCCGAACTGAGTAATACGACTCACTATAGG |
| LBO1-BHQ | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| T7 template | GGTGCTCACACTCTACTCAACAGTAGCGAACTACTGGACCCGTCCTTCACCTATAGTGAGTCGTATTAGACTC |

![NUPACK simulation result 1](https://static.igem.wiki/teams/5772/eng2/figure2.webp)

Figure 2: NUPACK simulation of hybridization

Table 2: Hybridization Parameters for LBO1 and SRO1-T7

| Component | Sequence | Number of complementary bases | Predicted hybridization rate |
| --- | --- | --- | --- |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA | 12 | 59.7% |
| SRO1-T7 | AGCCGAACTGAGTAATACGACTCACTATAGG | 12 | 59.7% |

![NUPACK simulation result 2](https://static.igem.wiki/teams/5772/eng2/figure3.webp)

Figure 3: Secondary structure prediction

![Hybridization analysis](https://static.igem.wiki/teams/5772/eng2/figure4.webp)

Figure 4: Hybridization rate analysis

## Test

### Step 1. Attempt to construct a T7 transcription system

First, 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 [3].

Table 3: DNA Annealing Components

| Components | 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℃)

Table 4: Construction of in vitro transcription system (100 μL)

| Components | Final concentration | Volume |
| --- | --- | --- |
| 5x transcription buffer | / | 20 μL |
| 10 μM T7 Template strand | 1 μM | 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 |

**Results**

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

![Transcription kinetics result](https://static.igem.wiki/teams/5772/eng2/figure5.webp)

Figure 5: Transcription kinetics monitoring

The initial fluorescence intensity of one experimental group reached approximately 1,600 RFU, significantly higher than the previous result (<50 RFU), confirming successful transcription. However, the fluorescence intensity decreased over time. This phenomenon occurred because after T7 RNA polymerase addition, approximately 30 minutes were spent for instrument parameter optimization before measurement began. By the time detection started, transcription had already ceased, and the fluorescence signal had passed its peak. The premature termination of transcription may be attributed to magnesium pyrophosphate precipitation, which could inhibit the reaction.

### Step 2. Test on the appropriate enzyme amount for adding pyrophosphatase

Based on previous experimental results, transcription could proceed in the system with a 50 nM T7 promoter, but it stopped within half an hour, with no further increase in fluorescence intensity—suggesting that magnesium pyrophosphate (PPase) deposition may have inhibited transcription [4]. In this experiment, factors such as the T7 promoter and T7 RNA polymerase remained consistent with the previous setup. On this basis, pyrophosphatase was added to investigate whether it affects correct RNA transcription and whether it can improve transcription efficiency.

Table 5: Experimental Groups for Pyrophosphatase Test

| Groups | G8 | G9 | G10 | G11 |
| --- | --- | --- | --- | --- |
| T7 promoter | 0 | 50nM | 50nM | 50nM |
| T7 template | 1 μM | 1 μM | 1 μM | 1 μM |
| 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 | 10 µM |
| PPase | 0 | 0 | 0.001 U | 0.1 U |

Table 6: Reaction Mixture Preparation (100 μL)

| Components | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| 5x transcription Buffer | 24 µL | 24 µL | 24 µL | 24 µL |
| 10 µM T7 Template strand | 12 µL | 12 µL | 12 µL | 12 µL |
| 10 µM T7 promoter | 0 | 0.6 µL | 0.6 µL | 0.6 µL |
| 25 mM NTP mix | 9.6 µL | 9.6 µL | 9.6 µL | 9.6 µL |
| 38.3µM T7 polymerase | 6 µL | 6 µL | 6 µL | 6 µL |
| 100 µM DFHBI | 12 µL | 12 µL | 12 µL | 12 µL |
| PPase | 0 | 0 | 1.2 µL | 1.2 µL |
| DEPC water | 56.4 µL | 55.8 µL | 54.6 µL | 54.6 µL |

**Results:**

![Pyrophosphatase test results](https://static.igem.wiki/teams/5772/eng2/figure6.webp)

Figure 6: Effect of pyrophosphatase on transcription

### Step 3. Test on transcriptional activation at different promoter concentrations

This experiment investigated the transcriptional activation efficiency under gradient promoter strand concentrations (10 nM, 25 nM, 50 nM, 75 nM, and 100 nM).

Table 7: Experimental Groups for Gradient Concentration Test

| Groups | 0 | 1 | 2 | 3 | 4 | 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 |

Table 8: Reaction Mixture Preparation (50 μL)

| Groups | 0 | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- | --- |
| 5x transcription buffer | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL |
| 10 μM T7 template | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL |
| 1 μM T7 promoter | 0 | 0.5 μL | 1.3 μL | 2.5 μL | 3.8 μL | 5 μL |
| 25 mM NTP mix | 4 μL | 4 μL | 4 μL | 4 μL | 4 μL | 4 μL |
| 38.3µM T7 polymerase | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL |
| 500 μM DFHBI | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL |
| pyrophosphatase | 0.0005U | 0.0005U | 0.0005U | 0.0005U | 0.0005U | 0.0005U |
| DEPC water | 21 μL | 20.5 μL | 19.7 μL | 18.5 μL | 17.2 μL | 15 μL |

**Results:**

![Gradient concentration test results](https://static.igem.wiki/teams/5772/eng2/figure7.webp)

Figure 7: Transcription activation at different promoter concentrations

The differences in fluorescence ratios among various T7 promoter concentrations were not statistically significant, and the relative ranking of these ratios continually changed over time, indicating unstable results. Further optimization is required.

### Step 4. Test on transcriptional activation of SRO1-T7

All operations are the same as in step 3, except replacing the promoter with SRO1-T7.

![SRO1-T7 activation test results](https://static.igem.wiki/teams/5772/eng2/figure8.webp)

Figure 8: SRO1-T7 transcription activation

![Average slopes analysis](https://static.igem.wiki/teams/5772/eng2/figure9.webp)

Figure 9: Average slopes analysis (20-40 minutes)

A concentration-dependent relationship exists for 0, 10 nM, and 20 nM fluorescence intensities. However, a pronounced downward trend appears at 50 nM, 75 nM, and 100 nM, with the value at 100 nM even approaching that of the negative control.

### Step 5. Couple strand displacement to T7 in vitro transcription

All operations about strand displacement are the same as in Cycle 1. After strand displacement, the reaction mixture was added to the T7 in vitro system.

Table 9: Coupling Reaction Mixture (50 μL)

| Groups | 0 | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- | --- |
| 5x transcription buffer | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL |
| 10 μM T7 template | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL |
| reaction mixture | 23 μL | 23 μL | 23 μL | 23 μL | 23 μL | 23 μL |
| 25 mM NTP mix | 4 μL | 4 μL | 4 μL | 4 μL | 4 μL | 4 μL |
| 38.3µM T7 polymerase | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL |
| 1 μM DFHBI | 2.5 μL | 2.5 μL | 2.5 μL | 2.5 μL | 2.5 μL | 2.5 μL |
| pyrophosphatase | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL |

![Coupled system results](https://static.igem.wiki/teams/5772/eng2/figure10.webp)

Figure 10: Coupled strand displacement and transcription system

We observed signal leakage in the system, where transcription was activated even without antibiotic binding.

## Learn

The in vitro transcription system possesses high sensitivity and has the potential for signal amplification. However, due to signal leakage, it cannot yet be utilized. Causes include:

1. The SRO1-T7 sequence formed a hairpin structure, reducing hybridization efficiency and leaving excess free promoter.
2. The promoter sequence was not effectively blocked, allowing activation without release.

![NUPACK simulation of SRO1-T7](https://static.igem.wiki/teams/5772/eng2/figure11.webp)

Figure 11: NUPACK simulation of SRO1-T7 hairpin structure

![Sequence design issue](https://static.igem.wiki/teams/5772/eng2/figure12.webp)

Figure 12: Sequence design issue analysis

In the next cycle, we will work on solutions to decrease signal leakage.

### References

1. Diane Imburgio, Minqing Rong, Kaiyu Ma et al. Studies of Promoter Recognition and Start Site Selection by T7 RNA Polymerase Using a Comprehensive Collection of Promoter Variants. Biochemistry 2000, 39, 34, 10419–10430
2. Dao, N.T., Haselsberger, R., Khuc, M.T. et al. Photophysics of DFHBI bound to RNA aptamer Baby Spinach. Sci Rep 11, 7356 (2021).
3. Tianshuo Liu, Shivali Patel, Anna Marie Pyle. Making RNA: Using T7 RNA polymerase to produce high yields of RNA from DNA templates. Methods in Enzymology. Volume 691, 2023
4. Milligan, J. F., & Uhlenbeck, O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods in Enzymology, 180(1989), 51-62.

## 2.2 Three-strand system to decrease the signal leakage

## Design

In the previous cycle, we observed significant transcriptional leakage caused by the exposed promoter sequence on sro1-T7. By consulting the literature, we learned that the hybridization and displacement between nucleic acid strands can be regulated by adding an additional regulatory strand to the system [1], or a catalytic strand can be used to open previously blocked domains [2].

This inspired us to consider adding an additional blocker strand to hybridize with the T7 promoter region and prevent premature transcription. Blocker strands with 0, 3, 6 and 9 base truncations at the 5' end were selected for further experiments.

![Three-strand system design](https://static.igem.wiki/teams/5772/eng2/figure13.webp)

Figure 13: Three-strand system design with blocker strands

## Build

Table 10: Component Sequences

| Component | Sequence |
| --- | --- |
| LBO1 | (BHQ)CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| SRO1 | AGCCGAACTGAGT(FAM)AATACGACTCACTATAGG |
| Blocker1 | CCTATAGTGAGTCGTATTA |
| Blocker2 | ATAGTGAGTCGTATTA |
| Blocker3 | GTGAGTCGTATTA |
| Blocker4 | AGTCGTATTA |
| Template | GGTGCTCACACTCTACTCAACAGTAGCGAACTACTGGACCCGTCCTTCACCTATAGTGAGTCGTATTAGACTC |

## Test

### Step 1. Strand displacement upon blocker 4 addition

Table 11: Strand Hybridization Components

| Group | Positive control | 0 | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| lbo (μL) | 0 | 9 | 9 | 9 | 9 | 9 | 9 |
| sro-T7 (μL) | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
| blocker4 (μL) | 0 | 9 | 9 | 9 | 9 | 9 | 9 |
| PBS (μL) | 27 | 9 | 9 | 9 | 9 | 9 | 9 |

Table 12: Strand Displacement Reaction System

| Group | Positive control | 0 | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Hybridized strand (μL) | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| VAN (μL) | 0 | 0 | 5 | 5 | 5 | 5 | 5 |
| PBS (μL) | 45 | 45 | 40 | 40 | 40 | 40 | 40 |

Each set of experiments was repeated five times, and the fluorescence changes in the system were measured over 30 minutes using a microplate reader at 37°C.

**Results**

![Strand displacement with blocker 4](https://static.igem.wiki/teams/5772/eng2/figure14.webp)

Figure 14: Strand displacement kinetics with Blocker4

Upon addition of the blocker strand, the system exhibited an antibiotic concentration-dependent strand displacement reaction. However, the presence of the blocker may facilitate hybridization between LBO and SRO, thereby reducing the reaction rate. After antibiotic addition, the strand displacement reaction reached equilibrium at approximately 30 minutes, and the fluorescence intensity of the system stabilized.

### Step 2. Test Blocker4 in transcription system

Table 13: Strand Hybridization for Blocker4 Test

| Group | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| Sro1-T7 | 2 | 2 | 2 | 2 |
| lbo1 | 0 | 0 | 6 | 6 |
| blocker4 | 6 | 0 | 6 | 0 |
| PBS | 12 | 18 | 6 | 12 |

Table 14: Transcription System Setup

| Group | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| 5x transcription buffer | 10 μL | 10 μL | 10 μL | 10 μL |
| 10 μM T7 Template | 5 μL | 5 μL | 5 μL | 5 μL |
| Hybridized strands | 5μL | 5 μL | 5 μL | 5 μL |
| 25 mM NTP mix | 4 μL | 4 μL | 4 μL | 4 μL |
| T7 polymerase | 4 μL | 4 μL | 4μL | 4 μL |
| 1 mM DFHBI | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL |
| Pyrophosphatase | 0.0005 U | 0.0005 U | 0.0005U | 0.0005U |
| DEPC water | 21.5 μL | 21.5 μL | 21.5 μL | 21.5 μL |

**Results**

![Blocker4 transcription test results](https://static.igem.wiki/teams/5772/eng2/figure15.webp)

Figure 15: Transcription test with Blocker4

Theoretically, Group 3 should exhibit the lowest fluorescence value, demonstrating that the blocker can effectively reduce signal leakage. However, in practice, the fluorescence intensity of Group 3 is higher than that of Group 4, and the large error bars within the group indicate that the results cannot conclusively prove the blocker's efficacy in reducing signal leakage.

### Step 3. Test Blocker2 in transcription system

Possibly due to the excessively short length of Blocker4, it failed to effectively reduce signal leakage. In this step, Blocker2 with only three truncated bases was used for testing.

Table 15: Strand Hybridization for Blocker2 Test

| Group | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| sro1-T7 | 2 | 2 | 2 | 2 |
| lbo1 | 0 | 0 | 6 | 6 |
| Blocker2 | 6 | 0 | 6 | 0 |
| PBS | 12 | 18 | 6 | 12 |

Table 16: Transcription System Setup with Blocker2

| Group | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| 5x transcription buffer | 10 μL | 10 μL | 10 μL | 10 μL |
| 10 μM T7 Template | 2.5 μL | 2.5 μL | 2.5 μL | 2.5 μL |
| Hybridized strands | 5μL | 5 μL | 5 μL | 5 μL |
| 25 mM NTP mix | 4 μL | 4 μL | 4 μL | 4 μL |
| T7 polymerase | 4 μL | 4 μL | 4μL | 4 μL |
| 1 mM DFHBI | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL |
| Pyrophosphatase | 0.0005 U | 0.0005 U | 0.0005U | 0.0005U |
| DEPC water | 24 μL | 24 μL | 24 μL | 24 μL |

**Results**

![Blocker2 transcription test results](https://static.igem.wiki/teams/5772/eng2/figure16.webp)

Figure 16: Transcription test with Blocker2

Theoretically, Group 3 should exhibit the lowest fluorescence value, demonstrating that the blocker can effectively reduce signal leakage. However, in practice, the fluorescence intensity of Group 3 is higher than that of Group 4, and the large error bars within the group indicate that the results cannot conclusively prove the blocker's efficacy in reducing signal leakage.

## Learn

The three-strand system did not successfully reduce signal leakage. The blocker strands either failed to bind stably or were displaced too easily. Future designs may require longer blocker sequences or alternative blocking strategies.

### References

1. Lee, H., Xie, T., Kang, B. et al. Plug-and-play protein biosensors using aptamer-regulated in vitro transcription. Nat Commun 15, 7973 (2024).
2. Soshu Yasuda, Kunihiko Morihiro, Shuichiro Koga. et al. Amplified Production of a DNA Decoy Catalyzed by Intracellular MicroRNA. Angewandte chemie 2025 4(64), e202424421

## 2.3 Double-strand system to decrease the signal leakage

## 2.3.1 Initial Design and Verification

With the core goals of sealing the T7 promoter on the closed SRO1-T7 and suppressing transcriptional leakage in the absence of vancomycin, the design scheme achieves self-sealing by extending the SRO1-T7-2 sequence: a 14-base extension is added to the 3' end of SRO1-T7-2, enabling it to form complementary base pairing with its own T7 promoter region (in strict accordance with the A-T and G-C base pairing rules). This ensures that the T7 promoter is sealed by a double-stranded structure when there is no vancomycin. Meanwhile, the original complementary region between SRO1-T7-2 and LBO1 is retained to ensure that after vancomycin binds to LBO1, SRO1-2 can dissociate normally, unfold the T7 promoter, and initiate transcription. Concurrently, the original unextended SRO1-T7-2 is designed as a negative control to evaluate the sealing effect; the template sequence (temp.1) is modified to include a binding region complementary to SRO1-T7-2, meeting the template requirements for transcription initiation.

![Diagram of double-strand sealing system](https://static.igem.wiki/teams/5772/eng2/figure17.webp)

Figure 17: Double-strand sealing system design

## Build

Feasible sequences were simulated using Nupack software:

Table 17: Component Sequences for Double-strand System

| Name | Sequence (5' to 3') |
| --- | --- |
| SRO1-T7-2 | AGCCGAACTGAGTAATACGACTCACTATAGGAGTGAGTCGTATTA |
| SRO1-T7-2-FAM | AGCCGAACTGAGTAATACGACTCACTATAGGAGTGAGTCGTATTA |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| LBO1-BHQ1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| temp.1 | GGTGCTCACACTCTACTCAACAGTAGCGAACTACTGGACCCGTCCTTCACCTATAGTGAGTCGTATTACA |

## Test

### Nupack Simulation Results:

![SRO1-T7-2 structure](https://static.igem.wiki/teams/5772/eng2/figure18.webp)

Figure 18: SRO1-T7-2 secondary structure

![LBO1 structure](https://static.igem.wiki/teams/5772/eng2/figure19.webp)

Figure 19: LBO1 secondary structure

![temp.1 structure](https://static.igem.wiki/teams/5772/eng2/figure20.webp)

Figure 20: temp.1 secondary structure

![Hybridization analysis](https://static.igem.wiki/teams/5772/eng2/figure21.webp)

Figure 21: SRO1-T7-2 and LBO1 hybridization

![Complex formation](https://static.igem.wiki/teams/5772/eng2/figure22.webp)

Figure 22: Complex formation analysis

![Binding analysis](https://static.igem.wiki/teams/5772/eng2/figure23.webp)

Figure 23: Binding analysis

![No vancomycin condition](https://static.igem.wiki/teams/5772/eng2/figure24.webp)

Figure 24: Behavior without vancomycin

![Dissociation analysis](https://static.igem.wiki/teams/5772/eng2/figure25.webp)

Figure 25: Dissociation analysis

## 2.3.2 Parameter Optimization and Performance Improvement

The hybridization ratio of SRO1-T7-2 to LBO1 directly affects the assembly efficiency and stability of their complex: a too low ratio may lead to free SRO1-T7-2 (failing to form effective sealing), while a too high ratio may interfere with vancomycin-induced dissociation due to excess LBO1. Both scenarios affect the system's sealing effect and signal response specificity. Therefore, the goal of this stage is to screen the optimal hybridization ratio through gradient ratio experiments, ensuring complete assembly of the complex without interference from free SRO1-T7-2 strands, and clear signal gradients under different vancomycin concentrations. This provides a stable reaction basis for subsequent transcription experiments.

### Build

To investigate the hybridization effect of different SRO1-T7-2:LBO1 ratios, the two strands were mixed at molar ratios of 1:1, 1:3, and 1:5, followed by slow annealing at 95℃. Each group was repeated 3 times.

### Test

#### 1. SRO1-T7-2:LBO1 = 1:1

![Fluorescence result for 1:1 ratio](https://static.igem.wiki/teams/5772/eng2/figure26.webp)

Figure 26: Hybridization at 1:1 ratio

#### 2. SRO1-T7-2:LBO1 = 1:3

![Fluorescence result for 1:3 ratio](https://static.igem.wiki/teams/5772/eng2/figure27.webp)

Figure 27: Hybridization at 1:3 ratio

#### 3. SRO1-T7-2:LBO1 = 1:5

![Fluorescence result for 1:5 ratio](https://static.igem.wiki/teams/5772/eng2/figure28.webp)

Figure 28: Hybridization at 1:5 ratio

### Learn

The optimal hybridization ratio is SRO1:LBO1 = 1:1. At this ratio, the two strands can fully assemble into a stable complex, and regular signal gradients can be observed under induction by different vancomycin concentrations, ensuring the sealing effect.

## 2.3.3 Transcription Experiment Verification

**(Subsequent experiments not conducted due to time limitation)**

The concentration of the template strand (temp.1) is a key parameter affecting transcription efficiency: a too low concentration leads to insufficient SRO1-T7-2 binding sites, limiting transcription signal amplification; a too high concentration may cause non-specific binding (e.g., cross-reaction between free template and LBO1), increasing background leakage. The design goal of this stage is to screen the optimal temp.1 concentration. Based on the SRO1:LBO1 hybridization ratio of 1:1, the following must be ensured:

1. No increase in background leakage in the absence of vancomycin;
2. Transcription signals increase clearly with the vancomycin concentration gradient (0 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1 mM) in the presence of vancomycin;
3. The template concentration matches the ratio of SRO1-LBO1 complex formation to avoid resource waste or reaction limitations.

The concentration gradient is set to 0.1 μM, 0.5 μM, 1 μM, and 2 μM (focusing on the low-to-medium concentration range based on the pre-experiment phenomenon that excess template increases background).

The concentration of the T7 promoter and the dosage of pyrophosphatase are also critical parameters for transcription initiation efficiency, signal stability, and persistence, and will be optimized under the same conditions.

## 2.4 Single-strand system to decrease the signal leakage

## 2.4.1 Design and Initial Test (SSS-M)

To prevent the T7 promoter from leaking transcription under antibiotic-free conditions, we designed a single-strand aptamer sensor. Based on the conformational change of the LBO strand induced by vancomycin, we added a T7 promoter reverse complementary sequence followed by the forward sequence to the 3′ end of the LBO strand, forming a closed hairpin structure. In the absence of vancomycin, the T7 promoter is sequestered; upon binding, the aptamer undergoes a conformational change that disrupts the hairpin, exposing the T7 promoter and initiating transcription to produce a fluorescence signal.

To reduce nonspecific binding by T7 polymerase, two bulges were introduced in the antisense complementary region to further minimize erroneous recognition.

![Single-strand sensor design diagram](https://static.igem.wiki/teams/5772/eng2/figure29.webp)

Figure 29: Schematic of the single-strand aptamer sensor design (Cycle 2.4.1)

### Build

We designed three variants differing in the length of the 5′ disruptive sequence:

* **SSS-S** (short)
* **SSS-M** (medium)
* **SSS-L** (long)

Table 18: Single-strand Sensor Sequences

| Sensor | Sequence (5' to 3') |
| --- | --- |
| SSS-S | CTCAAACTTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTATAGTTTGAGTCGTATTTTATTTTTAATACGACTCACTATAGG |
| SSS-M | CGACTCAAACTTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTATAGTTTGAGTCGTATTTTATTTTTAATACGACTCACTATAGG |
| SSS-L | ATACGACTCAAACTTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCTATAGTTTGAGTCGTATTTTATTTTTAATACGACTCACTATAGG |

DNA sequences were ordered from a synthesis company. We first tested **SSS-M** using an annealing protocol in Grade I water (1 μM), then added directly into the transcription reaction.

Table 19: Transcription System for SSS-M

| Component | Concentration |
| --- | --- |
| T7 Template strand | 1 μM |
| SSS-M | 50 nM |
| VAN | 0, 5, 20, 100 μM |
| T7 polymerase | 2.5 μM |

### Test

![Initial test results for SSS-M with vancomycin gradient](https://static.igem.wiki/teams/5772/eng2/figure30.webp)

Figure 30: Fluorescence response of SSS-M at varying VAN concentrations (Cycle 2.4.1)

### Learn

No significant change in fluorescence was observed with increasing vancomycin concentration. Although the hairpin formed, the system failed to show dose dependence. Possible issues include suboptimal template-sensor ratio, annealing method, or polymerase activity.

## 2.4.2 Template Concentration Optimization

We hypothesized that template concentration affects sensitivity and signal-to-noise ratio.

Table 20: Template Gradient Test Conditions

| Component | Concentration |
| --- | --- |
| T7 Template strand | 10 nM, 100 nM, 1 μM |
| SSS-M | 50 nM |
| VAN | 0 μM (−), 100 μM (+) |
| T7 polymerase | 2.5 μM |

### Test

![Fluorescence comparison across template concentrations](https://static.igem.wiki/teams/5772/eng2/figure31.webp)

Figure 31: Effect of template concentration on ON/OFF states (Cycle 2.4.2)

### Learn

A difference between negative and positive groups was only visible at 10 nM template, but with high standard deviation. Higher concentrations showed no distinction. Low template may improve contrast but lacks reliability.

## 2.4.3 Antibiotic Gradient Test

With template fixed at 10 nM, we tested whether a dose-dependent response could be achieved.

Table 21: Dose-Response Setup with 10nM Template

| Component | Concentration |
| --- | --- |
| T7 Template strand | 10 nM |
| SSS-M | 50 nM |
| VAN | 0, 5, 20, 100 μM |
| T7 polymerase | 2.5 μM |

### Test

![Dose-response curve at low template concentration](https://static.igem.wiki/teams/5772/eng2/figure32.webp)

Figure 32: Antibiotic gradient response at 10 nM template (Cycle 2.4.3)

### Learn

No clear dose-dependence was observed. Even at low template, the system remained unresponsive, suggesting deeper issues in design or kinetics.

## 2.4.4 Ratio and Annealing Optimization

The template-to-sensor ratio may deviate from conventional in vitro transcription conditions, so we adjusted the ratio and annealing conditions. Template:SSS-M ratio = 1:1 (0.5 μM : 0.5 μM). DFHBI concentration increased to 10 μM. Annealing was performed with sensor, template, and VAN (500 μM) together.

Table 22: Optimized Ratio and Annealing Setup

| Component | Concentration |
| --- | --- |
| T7 Template strand | 0.5 μM |
| SSS-S / SSS-L | 0.5 μM |
| VAN | 0, 100 μM |
| T7 polymerase | 2.5 μM |

### Test

![Fast kinetic response after ratio adjustment](https://static.igem.wiki/teams/5772/eng2/figure33.webp)

Figure 33: Rapid fluorescence increase with new annealing method (Cycle 2.4.4)

### Learn

Reaction speed significantly increased (peak at ~10 min), but no meaningful difference between +VAN and −VAN groups. Fast kinetics may mask subtle regulatory effects.

## 2.4.5 Polymerase Concentration Test

To better resolve differences, we reduced T7 polymerase concentration to slow down reactions. Tested: 0.025, 0.25, 1.25, 2, 2.5 μM.

Table 23: Polymerase Titration Conditions

| Component | Concentration |
| --- | --- |
| T7 polymerase | 0.025, 0.25, 1.25, 2, 2.5 μM |
| Template : SSS-M | 0.5 μM : 0.5 μM |
| VAN | 0, 100 μM |

### Test

![Low enzyme concentration test](https://static.igem.wiki/teams/5772/eng2/figure34.webp)

Figure 34: Transcription efficiency at low T7 pol concentrations

### Learn

No consistent vancomycin-dependent response across enzyme levels. At 1.25 μM, −VAN showed higher fluorescence than +VAN—likely due to experimental variability rather than biological effect.

## 2.4.6 Sub-gradient VAN Test

To verify if the anomaly at 1.25 μM T7 pol was reproducible, we repeated the test with finer VAN gradients.

Table 24: Sub-gradient VAN Test

| Component | Concentration |
| --- | --- |
| VAN | 0, 5, 20, 100 μM |
| Template | 0.5 μM |
| SSS-M | 0.5 μM |
| T7 polymerase | 1.25 μM |

### Test

![Fine gradient test shows no response](https://static.igem.wiki/teams/5772/eng2/figure35.webp)

Figure 35: No dose dependence observed even at optimized enzyme level (Cycle 2.4.6)

### Learn

The earlier anomaly was not reproducible. The SSS-M system does not generate a genuine antibiotic-dependent response under these conditions.

## 2.4.7 Evaluation of SSS-S and SSS-L

We tested SSS-S and SSS-L under the same conditions as Cycle 2.4.4 to assess impact of 5′ sequence length.

Table 25: SSS-S and SSS-L Testing Conditions

| Component | Concentration |
| --- | --- |
| T7 Template strand | 0.5 μM |
| SSS-S / SSS-L | 0.5 μM |
| VAN | 0, 100 μM |
| T7 polymerase | 2.5 μM |

### Test

![SSS-S and SSS-L performance comparison](https://static.igem.wiki/teams/5772/eng2/figure36.webp)

Figure 36: Comparative performance of SSS-S and SSS-L variants

### Learn

SSS-S showed the best performance (~16.2% higher signal at 10 h with VAN), likely due to more effective T7 promoter sealing. However, sensitivity remains low. NUPACK analysis revealed that the vancomycin-binding domain was structurally altered during design, impairing recognition.

## 2.4.8 Redesign: 5TS and 3TS Families

Upon analysis, poor performance stemmed from two factors: (1) incomplete sealing of the T7 promoter, causing leakage; (2) alteration of the aptamer binding area, preventing vancomycin recognition. To address this, we redesigned two families:

### 5TS Family (5′-T7 Hairpin)

The principle is similar to the SSS-n design, but in this case, the T7 promoter was linked to the 5′ end of the original LBO sequence, followed by a reverse complementary segment. The overall effect was to form a hairpin structure at the 5′ end of the sequence, such that in the absence of vancomycin, the T7 promoter is locked and transcription cannot be triggered; when vancomycin is present, it induces a conformational change in the aptamer, disrupting the hairpin and releasing the T7 promoter to initiate transcription.

When designing the 5TS family, we drew lessons from the shortcomings of the SSS-n scheme. We used NUPACK to simulate the secondary structures of the aptamer sequences, ensuring that the vancomycin recognition domain maintained its native conformation. In addition, we considered the effect of the number and position of bulges in the T7 complementary strand on promoter sealing. As a result, we designed four variants (5TS-1 to 5TS-4), with their T7 complementary strands containing 3, 2, 1, or 0 bulges, respectively.

![5TS family design schematic](https://static.igem.wiki/teams/5772/eng2/figure37.webp)

Figure 37: 5TS family design schematic

Table 26: 5TS Family Sequences

| Sensor | Sequence (5' to 3') |
| --- | --- |
| 5TS-1 | AGTAATACGACTCACTATAGGTTTTCCTTTATAGTGTTAGTCGTTTATTACTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGTAATA |
| 5TS-2 | AGTAATACGACTCACTATAGGTTTTCCTATAGTGTTAGTCGTTTATTACTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGTAATA |
| 5TS-3 | AGTAATACGACTCACTATAGGTTTTCCTATAGTGTTAGTCGTATTACTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGTAATA |
| 5TS-4 | AGTAATACGACTCACTATAGGTTTTCCTATAGTGAGTCGTATTACTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGTAATA |

![3TS family design schematic](https://static.igem.wiki/teams/5772/eng2/figure38.webp)

Figure 38: 3TS family design schematic

![5TS-1 test result](https://static.igem.wiki/teams/5772/eng2/figure39.webp)

Figure 39: 5TS-1 performance

![5TS-2 test result](https://static.igem.wiki/teams/5772/eng2/figure40.webp)

Figure 40: 5TS-2 performance

![5TS-3 test result](https://static.igem.wiki/teams/5772/eng2/figure41.webp)

Figure 41: 5TS-3 performance

### 3TS Family (3′-T7 Hairpin with Mismatches)

We also designed the 3TS family, whose principle is based on the SSS-n family. At the 3′ end of the sequence, a reverse complementary sequence of the T7 promoter followed by the T7 promoter itself was appended, forming a hairpin structure that blocks the T7 promoter. When vancomycin is present, the aptamer undergoes a conformational change, disrupting the hairpin and releasing the T7 promoter to initiate transcription. We used the NUPACK software to predict the secondary structures of the aptamers, ensuring that the secondary structure of the vancomycin recognition domain remained intact. To explore the effect of T7 promoter closure, we introduced 0–2 groups of base mismatches (3 bp each) at different positions within the complementary strand. In addition, we altered bases at the 5′ end that participate in vancomycin-induced conformational changes, thereby changing the number of base pairs formed in the LBO after the induced hairpin structure is generated, in order to investigate how intrinsic aptamer properties influence performance. These two variables were designed independently, resulting in eight sequences, designated 3TS-1 through 3TS-8. Among them, 3TS-1 to 3TS-4 and 3TS-5 to 3TS-8 form two subgroups, differing in the number of 5′-end bases used to form the antibiotic-induced hairpin structures, while the rest of their sequence designs correspond one-to-one.

![5TS-4 test result](https://static.igem.wiki/teams/5772/eng2/figure42.webp)

Figure 42: 5TS-4 performance

Table 27: 3TS Family Sequences

| Sensor | Sequence (5' to 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 |

![3TS-1 test result](https://static.igem.wiki/teams/5772/eng2/figure43.webp)

Figure 43: 3TS-1 performance

![3TS-2 test result](https://static.igem.wiki/teams/5772/eng2/figure44.webp)

Figure 44: 3TS-2 performance

![3TS-3 test result](https://static.igem.wiki/teams/5772/eng2/figure45.webp)

Figure 45: 3TS-3 performance

![3TS-4 test result](https://static.igem.wiki/teams/5772/eng2/figure46.webp)

Figure 46: 3TS-4 performance

![3TS-6 test result](https://static.igem.wiki/teams/5772/eng2/figure48.webp)

Figure 48: 3TS-6 performance

![3TS-5 test result](https://static.igem.wiki/teams/5772/eng2/figure47.webp)

Figure 47: 3TS-5 performance

![3TS-7 test result](https://static.igem.wiki/teams/5772/eng2/figure49.webp)

Figure 49: 3TS-7 performance

![3TS-8 test result](https://static.igem.wiki/teams/5772/eng2/figure50.webp)

Figure 50: 3TS-8 performance

### Build

Ordered DNA sequences from company.

### Test

EWe performed transcription tests on the 3TS-n and 5TS-n sensors in sequence. Each aptamer was first annealed individually in hybridization buffer and then added to the transcription system. The annealing buffer, referred to as the Binding Buffer, was PBS supplemented with 50 mM MgCl₂.ach sensor was tested for vancomycin responsiveness.

Table 28: Annealing System

| Component | Concentration |
| --- | --- |
| 3TS-n or 5TS-n | 5 μM |
| Binding Buffer | 1x |
| Grade I water | / |

Table 29: Transcription System

| Component | Concentration |
| --- | --- |
| T7 Template strand | 0.25 μM |
| 3TS-n or 5TS-n | 0.25 μM |
| VAN | 0, 100 μM |
| T7 polymerase | 2.5 μM |

The test results for different groups are as follows

![](https://static.igem.wiki/teams/5772/eng2/figure51.webp)

Figure 51

### Learn

**5TS family:** Bulge number affected overall fluorescence intensity, but none showed vancomycin responsiveness.
**3TS family:** Mismatch position and count influenced performance. Sensors with longer 5′ sequences and inner mismatches (e.g., 3TS-6, 3TS-8) showed up to ~13% increase with VAN, indicating partial success. However, background fluorescence was high.
**Conclusion:** Future designs must balance promoter sealing (low background) with efficient conformational switching (high dynamic range).

### References

1. Tan, J.H., Fraser, A.G. Quantifying metabolites using structure-switching aptamers coupled to DNA sequencing. *Nat Biotechnol* (2025).
2. Xiaona Fang, Tian Gao, Zhaofeng Luo, Renjun Pei. Efficient selection of vancomycin-specific aptamers via particle display and development of a high-sensitivity fluorescent apta-sensor for vancomycin detection. *Sensors and Actuators B: Chemical*, Volume 436, 2025, 137681.

## 2.5 LBO modification for reduced signal leakage

## 2.5.1 Design and Initial Test

To prevent leakage from the T7 promoter, we extended the 5′ end of the LBO strand. The extended segment was designed to be complementary to the T7 promoter on the SRO, so that in the absence of vancomycin, the template strand could not hybridize with the T7 promoter sequence and transcription would not occur. In the presence of vancomycin, however, the LBO undergoes a conformational change to form a hairpin structure, promoting the displacement and release of the SRO strand. The T7 promoter of the freed SRO can then hybridize with the template strand, be recognized by T7 polymerase, and initiate transcription.

Another possible mechanism is that the conformational change of the LBO exposes a T7 sequence toehold originally bound to LBO, which facilitates the displacement of SRO by the template strand and triggers transcription.

![LBO extension design diagram](https://static.igem.wiki/teams/5772/eng2/figure52.webp)

Figure 52: LBO extension strategy for promoter sealing

### Build

In our initial design, the SRO's T7 promoter contained 19 bases with no complementarity to the LBO strand. Here, we extended the 5′ end of the LBO strand by 0, 4, 8, 12, 16, or 19 complementary bases, designated L1, L2, L3, L4, L5, and L6, respectively. The SRO carrying the T7 promoter was designated S1.

Table 30: Modified LBO and SRO Sequences

| Component | Sequence (5' to 3') |
| --- | --- |
| S1 | AGCCGAACTGAGTAATACGACTCACTATAGG |
| L1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| L2 | ATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| L3 | TCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| L4 | TGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| L5 | ATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| L6 | CCTATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |

### Test

Our objective was to identify combinations of L-n and S1 that could respond to the antibiotic. Negative group: 0 μM VAN; Positive group: 100 μM VAN.

Annealing conditions:

Table 31: Annealing Conditions

| Component | Concentration |
| --- | --- |
| L-n | 2 μM |
| S1 | 1 μM |
| PBS | 1x |

(Heated at 95 °C for 5 min, slowly cooled.)

Transcription system:

Table 32: Transcription System Setup

| Component | Concentration |
| --- | --- |
| Transcription buffer | 1x |
| T7 Template strand | 100 nM |
| L-n | 200 nM |
| S1 | 100 nM |
| NTP mix | 2 mM |
| DFHBI | 1 μM |
| Pyrophosphatase | 0.00001 U |
| VAN | 0, 100 μM |
| T7 polymerase | 2.5 μM |

![Initial test results for LBO modifications](https://static.igem.wiki/teams/5772/eng2/figure53.webp)

Figure 53: Performance of different LBO extension variants

### Learn

The best response was S1+L2 (fluorescence ratio ~1.21:1). S1+L2 validated the strategy, but high background remained. Fluorescence decreased from L1 to L6, indicating stronger sealing, yet responsiveness dropped.

## 2.5.2 Optimized Design and Performance Evaluation

To improve S1+L4/L5, we extended their 3′ ends to destabilize the duplex and favor hairpin formation upon vancomycin binding.

### Build

Table 33: Optimized LBO Variants

| Variant | Sequence (5' to 3') |
| --- | --- |
| L4\_1 | TGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAG |
| L4\_2 | TGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTAAT |
| L4\_3 | TGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTAATACGA |
| L5\_1 | ATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAG |
| L5\_2 | ATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTAAT |
| L5\_3 | ATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTAATACGA |
| L5\_4 | ATAGTGAGTCGTATTACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACTGAGTAATACGACTCA |

### Test

![Test results for L4 variants](https://static.igem.wiki/teams/5772/eng2/figure54.webp)

Figure 54: Performance of L4 variants with 3′ extensions

Used DFHBI-1T dye for higher signal contrast.

![Test results for L4 and L5 variants](https://static.igem.wiki/teams/5772/eng2/figure55.webp)

Figure 55: Comparative performance of L4 and L5 variants with DFHBI-1T

![Final comparison of L5 variants](https://static.igem.wiki/teams/5772/eng2/figure56.webp)

Figure 56: Detailed analysis of L5 variant performance

### Learn

S1+L5\_3 showed improved response (1.35:1). Extending the 3′ end transitioned the system from over-sealed to functional. Optimal state lies near 13 added bases.

## 2.5.3 Template Concentration Optimization

We varied template:S1 ratios (0.5, 1, 5, 10) to optimize signal-to-background.

### Test

![Template concentration optimization results](https://static.igem.wiki/teams/5772/eng2/figure57.webp)

Figure 57: Effect of template concentration on system performance

### Learn

Best response at 1:1 ratio. Too low: weak signal; too high: template displaces S1 regardless of vancomycin. Narrow optimal window exists.

### References

1. Tan, J.H., Fraser, A.G. Quantifying metabolites using structure-switching aptamers coupled to DNA sequencing. Nat Biotechnol (2025).
2. Xiaona Fang, Tian Gao, Zhaofeng Luo, Renjun Pei. Efficient selection of vancomycin-specific aptamers via particle display and development of a high-sensitivity fluorescent apta-sensor for vancomycin detection. Sensors and Actuators B: Chemical, Volume 436, 2025, 137681.

# Cycle 3: Cas system amplification

## Design

From the substitution results, it can be seen that the vancomycin final concentration range detectable by the sensor system alone is roughly 50 μM – 100 μM, which does not match our expected concentration range. Therefore, we employed Cas12a as a tool for signal amplification.

## Build

Once Cas12a is activated, it non-specifically cleaves all single-stranded DNA (ssDNA) in the system. At this point, pre-introduced reporter probes (short ssDNA fragments labeled with a fluorophore and a quencher) are cleaved. When the probe is cut, the separation of the fluorophore from the quencher generates a fluorescence signal.

We designed the reporter as:

```
5’-CCCCCC-3’
with 6-FAM labeled at the 5’ end and BHQ at the 3’ end.
```

## Test

### Step 1. Test on Cas system

**Procedure**

#### 1. Strand hybridization

Table 1: Strand Hybridization Components

|  | 0 | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- | --- |
| LBO1-Cas (μL) | 2 | 2 | 2 | 2 | 2 | 2 |
| SRO2-Cas (μL) | 2 | 2 | 2 | 2 | 2 | 2 |
| VAN(μL) | 0 | 10 | 10 | 10 | 10 | 10 |
| PBS(μL) | 16 | 6 | 6 | 6 | 6 | 6 |
| Overall (μL) | 20 | 20 | 20 | 20 | 20 | 20 |

Final concentrations in the hybridization system:

Table 2: Final Concentrations After Hybridization

|  | 0 | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- | --- |
| LBO1-Cas (μM) | 1 | 1 | 1 | 1 | 1 | 1 |
| SRO2-Cas (μM) | 1 | 1 | 1 | 1 | 1 | 1 |
| VAN (μM) | 0 | 25 | 50 | 100 | 250 | 500 |

#### 2. Construct Cas system (20 μL)

Table 3: Cas Reaction Setup

| Component | Volume | Final concentration |
| --- | --- | --- |
| 10x Reaction buffer | 2 μL |  |
| 1μM reporter | 2 μL | 200 nM |
| 20x Cas12a | 2 μL | 100 nM |
| 1μM crRNA | 2 μL | 100 nM |
| DEPC water | 10 μL |  |
| Target (SRO2-Cas solution) | 2 μL |  |

#### 3. Prepare Master Mix

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

Table 4: Master Mix Preparation (648 μL total)

| Component | Volume |
| --- | --- |
| 10x Reaction buffer (μL) | 72 |
| 1μM reporter (μL) | 14.4 |
| 20x Cas12a (μL) | 36 |
| crRNA (μL) | 72 |
| DEPC water (μL) | 453.6 |
| Overall (μL) | 648 |

Add 18 μL of master mix + 2 μL of target to each tube. Perform experiment in triplicate per concentration. Measure fluorescence using a microplate reader.

![Results of Cas system test](https://static.igem.wiki/teams/5772/eng3/figure1.webp)

Figure 1

### Step 2. Test on Cas system upon increasing the ratio of LBO:SRO

We hypothesized that an excess of free SRO2-Cas in the system was interfering with the detection. To reduce background signals and minimize interference from SRO2-Cas, we increased the ratio of LBO1-Cas to SRO2-Cas to 1.3.

#### 1. Strand hybridization

Table 5: Hybridization with Increased LBO:SRO Ratio

|  | 0 | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- | --- |
| LBO1-Cas (μL) | 2.6 | 2.6 | 2.6 | 2.6 | 2.6 | 2.6 |
| SRO2-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 |

Final concentrations in the hybridization system:

Table 6: Final Concentrations with Modified Ratio

|  | 0 | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- | --- |
| LBO1-Cas (μM) | 1.3 | 1.3 | 1.3 | 1.3 | 1.3 | 1.3 |
| SRO2-Cas (μM) | 1 | 1 | 1 | 1 | 1 | 1 |
| VAN (μM) | 0 | 25 | 50 | 100 | 250 | 500 |

#### 2. Construct Cas system (same as step 1)

Same reaction setup as Table 3.

#### 3. Prepare Master Mix (same composition)

Same master mix as Table 4.

![Kinetic measurement of fluorescence changes](https://static.igem.wiki/teams/5772/eng3/en2.avif)

Figure 2

![Comparison of normalized fluorescence](https://static.igem.wiki/teams/5772/eng3/en3.avif)

Figure 3

**Figure 2:** Kinetic measurement of fluorescence changes within the Cas system over 30 minutes.

**Figure 3:** Comparison of normalized fluorescence between systems with and without the Cas component. (The fluorescence of the no-antibiotic group was set to 1.)

In the figure above, the left panel shows the fluorescence intensity from a strand displacement assay without the Cas amplification system, while the right panel displays the fluorescence intensity of the Cas system after coupling it with the strand displacement system. It was observed that the system without Cas achieved a minimum detection limit of 25 μM for vancomycin, whereas the Cas system could detect vancomycin at concentrations as low as 5 μM. This clearly demonstrates that the Cas amplification system can effectively enhance the overall detection resolution of the system.

## Learn

We introduced the Cas cleavage-based amplification system, which can effectively improve the sensor’s resolution for vancomycin concentrations.

### References

1. Janice S. Chen et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436-439 (2018).

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

* [Preparation of Buffer Solutions](#buffer)
  + [Buffer Formulations](#buffer-formulations)
  + [Procedure](#buffer-procedure)
* [Strand Displacement Module](#strand-displacement)
  + [Strand Hybridization Protocol](#strand-hybridization)
  + [Strand Displacement Protocol 1](#displacement-protocol1)
  + [Strand Displacement Protocol 2](#displacement-protocol2)
  + [Strand Proportional Hybridization Protocol](#proportional-hybridization)
  + [Fluorescence Quantification Protocols](#fluorescence-quantification)
* [T7 Transcription Module](#t7-transcription)
  + [Transcription Protocol for SSS-M Aptamer Sensor](#transcription-sss-m)
  + [Template Concentration Gradient](#template-gradient)
  + [Low-template System](#low-template)
  + [Modified Annealing Protocols](#modified-annealing)
  + [T7 Polymerase Concentration Series](#t7-polymerase-series)
  + [VAN Gradient at 1.25 µM T7](#van-gradient)
  + [SSS-S vs SSS-L](#sss-s-vs-sss-l)
  + [TS Families](#ts-families)
  + [Double-Strand Extension](#double-strand-extension)
  + [S1:L Variants](#s1-l-variants)
  + [Template Concentration Gradient for S1 + L5\_3](#template-ratio)
* [CRISPR-Cas12a Amplification Module](#crispr-cas12a)
  + [Cas12a System](#cas12a-system)
  + [Procedure](#cas12a-procedure)

# Preparation of Buffer Solutions

## Buffer Formulations

### (1) Binding Buffer: PBS pH 7.4 (used in hybridization)

| Reagent | Concentration |
| --- | --- |
| NaCl | 137 mM |
| KCl | 2.7 mM |
| Na₂HPO₄ | 10 mM |
| KH₂PO₄ | 1.8 mM |

### (2) 5x Transcription Buffer (used in T7 transcription)

| Reagent | Concentration |
| --- | --- |
| HEPES pH 7.5 | 400 mM |
| MgCl₂ | 120 mM |
| Spermidine | 10 mM |
| DTT (Dithiothreitol) | 200 mM |

### (3) 10x Reaction in Cas system (used in Cas reaction)

| Reagent | Concentration |
| --- | --- |
| Tris-HCl (pH 7.9) | 10 mM |
| NaCl | 50 mM |
| MgCl₂ | 10 mM |
| BSA | 100 µg/mL |

## Procedure

1. Calculate the required mass of solid solutes using the formula: Mass (mg) = Concentration (mM) × Volume (mL) × Molecular Weight (g/mol)
2. Rinse reagent bottles with Type I water. Weigh the solids using a balance, transfer them to the bottles, and add Type I water up to the target volume mark. Cap the bottles and label them with the solution names.
3. Place the prepared solutions in an ultrasonic cleaner and sonicate for approximately 3 minutes to accelerate solute dissolution.
4. Adjust the pH of the buffer solutions:
   * Remove the electrode from the KCl solution and rinse the pH meter electrode with Type I water and dry it
   * Calibrate the pH meter using standard buffer solutions (pH 4.00, 6.98, and 8.18)
   * First measure the buffer's pH and adjust it by adding NaOH or HCl dropwise until pH reaches standard.
   * Rinse the electrode with Type I water between measurements
   * After completion, rinse the electrode and store it in KCl solution
5. Filter the solutions for homogeneity and to remove any undissolved solids:
   * Rinse the filtration apparatus with Type I water
   * Set up the vacuum filtration system using 45 μm filter paper
   * Secure the collection flask and funnel with clamps to prevent air leaks
   * Turn on the vacuum pump to begin filtration
   * After filtration, disconnect the air tube before turning off the pump
   * Dismantle the filtration setup and transfer the filtrate to reagent bottles
   * Rinse the filtration apparatus with Type I water between different solutions

# Strand Displacement Module

## Strand Hybridization Protocol for Polyacrylamide Gel Electrophoresis

**Scope of Application:** The experiments in the following table were conducted according to this protocol. The terms "LBO" and "SRO" mentioned hereafter are collective designations for these sequences.

DNA Sequences Used in Strand Displacement Experiments

| Name | Sequence |
| --- | --- |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| SRO1 | AGCCGAACTGAG |
| SRO1-T7 | AGCCGAACTGAGTAATACGACTCACTATAGG |
| LBO2 | TTTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG |
| SRO2-T7 | AGCCGGAGAGAGAAATAATACGACTCACTATAGG |
| SRO2 | AGCCGGAGAGAGAAA |
| LBO3 | TCTCTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG |
| SRO3 | AGCCGGAGAGAGAGAGA |
| LBO4 | CCTGAACCCCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGGTT |
| SRO4 | TCGGGGTTCAGG |
| LBO5 | GCCTGAACCCCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGGTT |
| SRO5 | TCGGGGTTCAGGC |
| LBO6 | TAAGATCTCTCGGGACGACCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGTCGTCCC |
| SRO6 | GTCGTCCCGAGAGTA |
| LBO7 | TAAGATCTCTCAGGGACGACCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGTCGTCCCT |
| SRO7 | GTCGTCCCTGAGAGAA |
| LBO8 | TAAGATCTCTCAGGGGACGACCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGTCGTCCCCT |
| LBO1-Cas | GACGTATCGACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACT |
| SRO1-Cas | GGAGACGCCGAACTGAGTCGATACGTCTAGATTG |
| SRO2-Cas | GGAGACCACTGAGCCGAACTGAGTCGCTAGATTG |

### Objective

For both the T7 and Cas systems, we initially hybridized the LBO and SRO strands at a 1:1 ratio and verified the hybridization efficiency via PAGE electrophoresis.

### Procedure

1. Prepare the hybridization strands according to the table below.

   | Group Reactants | LBO1 | SRO1 | LBO:SRO 1:1 |
   | --- | --- | --- | --- |
   | 10μM LBO1(μL) | 1 | 0 | 1 |
   | 10μM SRO1(μL) | 0 | 1 | 1 |
   | PBS(μL) | 9 | 9 | 8 |
   | Overall system | 10 μL | | |
2. Place the prepared hybridization strands in boiling water and allow them to cool naturally to room temperature to facilitate hybridization. The annealed solution can then be used as a sample for polyacrylamide gel electrophoresis.

## Strand Displacement Protocol 1 for Polyacrylamide Gel Electrophoresis

**Scope of Application:** The experiments in the following table were conducted according to this protocol.

DNA Sequences Used in Strand Displacement Protocol 1

| Name | Sequence |
| --- | --- |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| SRO1 | AGCCGAACTGAG |
| SRO1-T7 | AGCCGAACTGAGTAATACGACTCACTATAGG |
| LBO2 | TTTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG |
| SRO2-T7 | AGCCGGAGAGAGAAATAATACGACTCACTATAGG |
| SRO2 | AGCCGGAGAGAGAAA |
| LBO3 | TCTCTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG |
| SRO3 | AGCCGGAGAGAGAGAGA |

### Objective

Using polyacrylamide gel electrophoresis for preliminary qualitative study on whether strand displacement reaction can occur after adding antibiotics.

### Procedure

1. Prepare the hybridization strands according to the table below.

   | Group Reactants | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
   | --- | --- | --- | --- | --- | --- | --- | --- | --- |
   | 10μM LBO(μL) | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
   | 10μM SRO(μL) | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
   | PBS(μL) | 9 | 9 | 8 | 7 | 7 | 7 | 7 | 7 |
2. After preparation, vortex the mixture thoroughly and centrifuge for 10 seconds. Then place the prepared hybridization stands in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization.
3. After annealing and hybridization, add antibiotic solution to each tube according to the table below to initiate the strand displacement reaction.

   | Reactants | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
   | --- | --- | --- | --- | --- | --- | --- | --- | --- |
   | VAN(μL) | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 |
   | Overall(μL) | 10 | | | | | | | |

   **Note:** The concentrations of added vancomycin were 50 μM, 100 μM, 200 μM, 500 μM, and 1 mM respectively for group 4-8, and are diluted ten times in the reaction system, and the final concentrations are respectively 5μM, 10μM, 20μM, 50μM, 100μM.
4. The annealed solution can then be used as a sample for polyacrylamide gel electrophoresis.

## Strand Displacement Protocol 2 for Polyacrylamide Gel Electrophoresis

**Scope of Application:** The experiments in the following table were conducted according to this protocol.

DNA Sequences Used in Strand Displacement Protocol 2

| Name | Sequence |
| --- | --- |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| SRO1-T7 | AGCCGAACTGAGTAATACGACTCACTATAGG |
| LBO2 | TTTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG |
| SRO2-T7 | AGCCGGAGAGAGAAATAATACGACTCACTATAGG |
| LBO1-Cas | GACGTATCGACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACT |
| SRO1-Cas | GGAGACGCCGAACTGAGTCGATACGTCTAGATTG |
| SRO2-Cas | GGAGACCACTGAGCCGAACTGAGTCGCTAGATTG |

### Improvements

We added the antibiotic solution during the preparation of the hybridization chains, rather than adding it after annealing as previously done, to ensure thorough occurrence of the strand displacement reaction.

### Procedure

1. Prepare the hybridization chains according to the table below.

   | Group Reactants | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
   | --- | --- | --- | --- | --- | --- | --- | --- | --- |
   | 10μM LBO (μL) | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
   | 10μM SRO (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 | 10 μL | | | | | | | |

   **Note:** The concentrations of added vancomycin 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.
2. Place the prepared hybridization chains in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used as a sample for polyacrylamide gel electrophoresis.

## Strand Proportional Hybridization Protocol for Polyacrylamide Gel Electrophoresis

**Scope of Application:** The experiments in the following table were conducted according to this protocol.

DNA Sequences Used in Proportional Hybridization Protocol

| Name | Sequence |
| --- | --- |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| SRO1-T7 | AGCCGAACTGAGTAATACGACTCACTATAGG |

### Objective

We found that for the LBO and SRO strands of the T7 system, the hybridization efficiency between LBO and SRO is not sufficiently high. When hybridized at a 1:1 ratio of LBO:SRO-T7, a significant amount of free SRO remains in the system, leading to signal leakage. Therefore, it is necessary to identify an appropriate SRO:LBO ratio that keeps the background signal within an acceptable range. (In contrast, the LBO and SRO of the Cas system hybridize at a ratio close to 1:1. So during the strand displacement experiments, the ratio of LBO to SRO was not optimized through screening at this stage.)

We first conducted a preliminary study on the hybridization of LBO and SRO strands using polyacrylamide gel electrophoresis to find a suitable ratio.

### Procedure

1. Take 8 centrifuge tubes (1.5ml each) and label them as LBO, SRO, LBO:SRO=1:1, LBO:SRO=1:1.5, LBO:SRO=1:2, LBO:SRO=1:2.5, LBO:SRO=1:3, LBO:SRO=1:3.5.
2. Prepare the hybridization strands according to the table below.

   | Group Reactants | LBO1 | SRO1 | LBO:SRO 1:1 | LBO:SRO 1:1.5 | LBO:SRO 1:2 | LBO:SRO 1:2.5 | LBO:SRO 1:3 | LBO:SRO 1:3.5 |
   | --- | --- | --- | --- | --- | --- | --- | --- | --- |
   | 10μM LBO(μL) | 1 | 0 | 1 | 1.5 | 2 | 2.5 | 3 | 3.5 |
   | 10μM SRO(μL) | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
   | PBS(μL) | 9 | 9 | 8 | 7.5 | 7 | 6.5 | 6 | 5.5 |
   | Overall system | 10 μL | | | | | | | |
3. Place the prepared hybridization chains in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used as a sample for polyacrylamide gel electrophoresis.

## Fluorescence Quantification Protocols

### Strand Proportional Hybridization Protocol for Fluorescence Quantification (used in T7 system)

**Scope of Application:** The experiments in the following table were conducted according to this protocol.

DNA Sequences Used in Fluorescence Quantification

| Name | Sequence |
| --- | --- |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| SRO1-T7 | AGCCGAACTGAGTAATACGACTCACTATAGG |
| LBO2 | TTTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG |
| SRO2-T7 | AGCCGGAGAGAGAAATAATACGACTCACTATAGG |

#### Objective

In the absence of antibiotics, hybridize SRO with LBO and measure the fluorescence of the system. The fluorescence value reflects the amount of free SRO in the system, which may activate downstream amplification pathways and generate background signals. Therefore, it is necessary to identify an appropriate SRO:LBO ratio that keeps the background signal within an acceptable range.

#### Procedure

1. Different SRO:LBO ratios were tested to evaluate the background fluorescence levels under each condition, enabling the selection of an optimal SRO:LBO ratio for sensor construction.

   | Reagent | 1:1 | 1:2 | 1:3 | 1:5 | 1:7 | 1:9 | Positive control |
   | --- | --- | --- | --- | --- | --- | --- | --- |
   | 10μM LBO(μL) | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
   | 10μM SRO(μL) | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
   | 10μM LBO-BHQ(μL) | 3 | 6 | 9 | 15 | 21 | 27 | 0 |
   | 10μM SRO-FAM(μL) | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
   | PBS(μL) | 24 | 21 | 18 | 12 | 6 | 0 | 27 |
   | Overall(μL) | 30 | 30 | 30 | 30 | 30 | 30 | 30 |
2. After preparation, vortex the mixture thoroughly and centrifuge for 10 seconds.
3. Place the prepared hybridization chains in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used for constructing the strand displacement reaction system.

### Strand Displacement Protocol for Fluorescence Quantification (used in T7 system)

DNA Sequences Used in T7 System Fluorescence Quantification

| Name | Sequence |
| --- | --- |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| SRO1-T7 | AGCCGAACTGAGTAATACGACTCACTATAGG |
| LBO2 | TTTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG |
| SRO2-T7 | AGCCGGAGAGAGAAATAATACGACTCACTATAGG |

#### Objective

Based on the ratio hybridization experiments, we determined that a 5:1 ratio of LBO:SRO is optimal in the T7 system. Therefore, this ratio was selected for quantitative fluorescence analysis to assess whether the system could produce antibiotic concentration-dependent fluorescence changes after the strand displacement reaction was initiated by adding antibiotics.

#### Procedure

1. Prior to the strand displacement reaction, we first prepare the required hybridization chains in a unified manner to ensure that all experimental groups utilize the same hybridization chains when constructing the reaction system. Prepare the hybridization chains according to the table below.

   | Reagent | Experimental group | Positive control | Negative control |
   | --- | --- | --- | --- |
   | 10μM LBO(μL) | 0 | 0 | 15 |
   | 10μM SRO(μL) | 0 | 0 | 3 |
   | 10μM LBO-BHQ(μL) | 100 | 0 | 0 |
   | 10μM SRO-FAM(μL) | 20 | 3 | 0 |
   | PBS(μL) | 80 | 27 | 12 |
   | Overall(μL) | 200 | 30 | 30 |
2. After preparation, vortex the mixture thoroughly and centrifuge for 10 seconds.
3. Place the prepared hybridization chains in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used for construct the strand displacement reaction system.
4. Set up the reaction system as follows:

   | Groups | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
   | --- | --- | --- | --- | --- | --- | --- | --- | --- |
   | VAN (μL) | 0 | 0 | 0 | 5 | 5 | 5 | 5 | 5 |
   | Hybridized strands (μL) | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
   | PBS(μL) | 45 | 45 | 45 | 40 | 40 | 40 | 40 | 40 |
   | Types of hybridized strands(μL) | LBO+SRO-FAM | LBO+SRO | LBO-BHQ + SRO-FAM | | | | | |

   **Microplate reader parameters:**

   * Temperature: 37°C
   * Excitation wavelength: 490 nm
   * Emission wavelength: 520 nm
   * Time: 30 min

### Strand Displacement Protocol for Fluorescence Quantification (used in Cas system)

**Scope of Application:** The experiments in the following table were conducted according to this protocol.

DNA Sequences Used in Cas System Fluorescence Quantification

| Name | Sequence |
| --- | --- |
| LBO1-Cas | GACGTATCGACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACT |
| SRO1-Cas | GGAGACGCCGAACTGAGTCGATACGTCTAGATTG |
| SRO2-Cas | GGAGACCACTGAGCCGAACTGAGTCGCTAGATTG |
| crRNA-1 | UAAUUUCUACUAAGUGUAGAUACGUAUCGACUCAGUUCGGC |
| crRNA-2 | UAAUUUCUACUAAGUGUAGAUCGACUCAGUUCGGCUCAGUG |

#### Objective

In the Cas system, the LBO and SRO strands can hybridize at a ratio close to 1:1. Consequently, this ratio was selected for quantitative fluorescence analysis to determine whether the system exhibits antibiotic concentration-dependent fluorescence changes following the strand displacement reaction triggered by antibiotic addition.

#### Procedure

1. Prior to the strand displacement reaction, we first prepare the required hybridization chains in a unified manner to ensure that all experimental groups utilize the same hybridization chains when constructing the reaction system. Prepare the hybridization chains according to the table below.

   | Reagent | Experimental group | Positive control |
   | --- | --- | --- |
   | 10mM LBO-BHQ(μL) | 200 | 0 |
   | 10mM SRO-FAM(μL) | 40 | 3 |
   | PBS(μL) | 160 | 27 |
   | Overall(μL) | 400 | 30 |
2. After preparation, vortex the mixture thoroughly and centrifuge for 10 seconds.
3. Place the prepared hybridization chains in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used for construct the strand displacement reaction system.
4. Construct strand displacement reaction system (Each group repeated 5 times)

   | Reagents | 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 |
   | Strands (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 |
   | Types of hybridized strands | LBO-BHQ + SRO-FAM | | | | | | | | | | | | SRO-FAM |

   **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.
5. **Microplate reader parameters:**
   * Temperature: 37°C
   * Excitation wavelength: 490 nm
   * Emission wavelength: 520 nm
   * Time: 30 min

#### Improvement

In subsequent experiments, to further reduce signal leakage, we slightly increased the LBO:SRO ratio to 1.3:1 and conducted the experiments using the same method as described in the protocol.

## Polyacrylamide Gel Electrophoresis (used in T7 and Cas system)

### Procedure

#### 1. Gel preparation

| Reagent | Volume |
| --- | --- |
| H2O | 3.93 mL |
| 30% ACS | 4 mL |
| 5x TBE | 2 mL |
| 10% AP | 70 μL |
| TEMED | 7 μL |

1. Assemble the gel cassette and check for leaks.
2. Prepare the gel according to the formulation in the table below.
3. After preparation, vortex to mix thoroughly. Add the gel into the electrophoresis tank using a pipette, insert a comb, and avoid generating bubbles during the process.
4. Wait 30-45 minutes for the gel to solidify. Add 1x TBE as the electrophoresis buffer until the outer liquid level is half the height of the inner liquid level. Then, remove the comb.
5. Add 10x Loading Buffer to the annealed sample at a 1/10 volume ratio, mix well, and centrifuge.
6. Add 10 μL of sample to each well of the solidified gel, and load an equal amount of marker into the wells at both ends.

#### 2. Running the gel

1. Load the DNA samples into the wells of the gel.
2. Apply electricity at 120V with a maximum current of 600mA, and run the electrophoresis for about 30 minutes.

#### 3. Gel Staining and Observation

1. Prepare the developing solution.
2. Cut the gel and place it into a gel staining box. Add approximately 25 ml of developing solution, and incubate protected from light on a shaker for 15 minutes.
3. Develop and observe the gel bands using a gel imaging system.

# T7 Transcription Module

## Transcription Protocol for SSS-M Aptamer Sensor (Before Annealing-Condition Change)

### Objective

To evaluate the transcriptional response of the SSS-S single-stranded aptamer sensor under different vancomycin (VAN) concentrations.

### Materials & Reagents

* 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9)
* T7 Template DNA strand (10 µM stock)
* SSS-M aptamer DNA (10 µM stock; annealed to 1 µM before use)
* NTP mix (25 mM)
* DFHBSI (1 mM stock)
* Pyrophosphatase (PPase) 0.001 U/µL
* T7 RNA polymerase (50 µM)
* Vancomycin (VAN) (1 mM stock; also prepare 50 µM and 200 µM working solutions)
* DEPC-treated RNase-free water

### Procedure

1. Annealing SSS-M DNA
   * Objective: Generate a 1 µM annealed stock of SSS-S aptamer DNA for direct use in transcription reactions.
   * Mix 10.0 µL of 10 µM SSS-M DNA with 90.0 µL DEPC water to obtain 1 µM SSS-S DNA (100 µL total).
   * Annealing program:
     + Heat at 95 °C for 5 min
     + Slowly cool to room temperature (e.g., 0.1 °C/s)
     + Hold on ice until use
   * For this experiment, 30 µL of 1 µM SSS-M is required for the transcription system. Preparing 100 µL provides extra volume to account for pipetting losses.
2. Transcription Master Mix (510 µL)

   Mix in a 1.5 mL centrifuge tube (without VAN and T7 polymerase):

   | Component | Final conc. | Volume (µL) |
   | --- | --- | --- |
   | 5× Transcription buffer | 1× | 120.0 |
   | T7 Template strand (10 µM) | 1 µM | 60.0 |
   | Annealed SSS-M (1 µM) | 50 nM | 30.0 |
   | NTP mix (25 mM) | 2 mM | 48.0 |
   | DFHBSI (1 mM) | 1 µM | 0.6 |
   | Pyrophosphatase (0.001 U/µL) | 0.00001 U | 6.0 |
   | DEPC water | --- | 245.4 |
   | Total | --- | 510.0 |

   This master mix volume is sufficient for 12 wells at 42.5 µL/well.
3. Plate Setup

   Distribute 42.5 µL of transcription master mix per well. Then add VAN and T7 polymerase to reach a final volume of 50 µL/well.

   | Group | Transcription Mix (µL) | VAN (µL) | VAN conc. (final) | T7 polymerase (µL, 50 µM) | DFHBSI only (µL) |
   | --- | --- | --- | --- | --- | --- |
   | Group 0 | 42.5 | 5.0 (PBS) | 0 µM | 2.5 | 0 |
   | Group 1 | 42.5 | 5.0 (50 µM) | 50 µM | 2.5 | 0 |
   | Group 2 | 42.5 | 5.0 (200 µM) | 200 µM | 2.5 | 0 |
   | Group 3 | 42.5 | 5.0 (1 mM) | 1 mM | 2.5 | 0 |
   | Negative Ctrl | 0 | 0 | --- | 0 | 50 (1 µM DFHBSI) |
4. Anneal SSS-M DNA as described in Step 1.
5. Prepare transcription master mix (510 µL) in a 1.5 mL centrifuge tube (Step 2).
6. Distribute 42.5 µL of the master mix into each designated well of a 384-well plate.
7. Quickly add VAN (5 µL/well):
   * Group 0: PBS 5 µL
   * Group 1: 50 µM VAN 5 µL
   * Group 2: 200 µM VAN 5 µL
   * Group 3: 1 mM VAN 5 µL
   * Negative control: none
8. Immediately add T7 RNA polymerase (2.5 µL/well, 50 µM) to all wells except negative control. Seal the plate with film, gently tap or briefly centrifuge to collect liquid at the bottom. Incubate at 37 °C for 10 hours. Record fluorescence every 1 minutes at Ex/Em = 506/560 nm.

### Controls

* Group 0 (PBS) serves as negative VAN control.
* Negative control wells (50 µL DFHBSI without transcription components or T7) measure background fluorescence.

## Transcription Protocol for SSS-M Aptamer Sensor: Template Concentration Gradient

### Purpose

Evaluate how template DNA concentration (≈1 µM / 100 nM / 10 nM final) affects the transcriptional response of the SSS-M sensor under 0 µM vs 1 mM vancomycin (VAN) conditions.

### Materials & Reagents

* 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9)
* SSS-M aptamer DNA (10 µM stock → anneal to 1 µM before use)
* T7 Template DNA stocks for per-well addition: 10 µM, 1 µM, 100 nM
* NTP mix (25 mM)
* DFHBSI (1 mM stock)
* Pyrophosphatase (PPase) 0.001 U/µL
* T7 RNA polymerase (50 µM)
* Vancomycin (VAN) (1 mM stock; PBS for 0 µM)
* DEPC-treated RNase-free water

### Procedure

1. Annealing SSS-M DNA (to 1 µM)
   * Mix 10.0 µL of 10 µM SSS-M with 90.0 µL DEPC water → 1 µM SSS-S (100 µL total).
   * Anneal: 95 °C for 5 min → slow cool to RT (e.g., 0.1 °C/s) → hold on ice.
   * This run needs 50 µL of the 1 µM SSS-M for the master mix; preparing 100 µL provides headroom.
2. Transcription Master Mix (Total 750 µL)

   This master mix does not contain VAN, T7 polymerase, or template DNA. Template will be added per well to set the gradient.

   | Component | Final conc. (in reaction) | Volume |
   | --- | --- | --- |
   | 5× Transcription buffer | 1× | 200 µL |
   | SSS-M (1 µM, annealed) | 50 nM | 50 µL |
   | NTP mix (25 mM) | 2 mM | 80 µL |
   | DFHBSI (1 mM) | 1 µM | 1 µL |
   | DEPC water | --- | 409 µL |
   | Pyrophosphatase (0.001 U/µL) | 0.00001 U | 10 µL |
   | Total | --- | 750 µL |

   Mix gently, vortex briefly, spin down. This volume supports 20 wells at 37.5 µL/well of master mix.
3. Plate Setup (Per-well totals = 50 µL)

   Distribute 37.5 µL master mix per well, then add VAN, T7 polymerase, and 5 µL template stock as indicated:

   | Group | Master Mix (µL) | VAN (µL) | T7 (µL, 50 µM) | Template per well | Notes (Final template in 50 µL) |
   | --- | --- | --- | --- | --- | --- |
   | 1 | 37.5 | 5.0 (0 µM, PBS) | 2.5 | 5 µL of 10 µM | ~1 µM |
   | 2 | 37.5 | 5.0 (1 mM) | 2.5 | 5 µL of 10 µM | ~1 µM |
   | 3 | 37.5 | 5.0 (0 µM, PBS) | 2.5 | 5 µL of 1 µM | ~100 nM |
   | 4 | 37.5 | 5.0 (1 mM) | 2.5 | 5 µL of 1 µM | ~100 nM |
   | 5 | 37.5 | 5.0 (0 µM, PBS) | 2.5 | 5 µL of 100 nM | ~10 nM |
   | 6 | 37.5 | 5.0 (1 mM) | 2.5 | 5 µL of 100 nM | ~10 nM |
   | Negative | 0 | 0 | 0 | --- | 50 µL of 1 µM DFHBSI only |

   Template working stocks (from 10 µM):

   * 1 µM: 10 µL of 10 µM + 90 µL water.
   * 100 nM: 10 µL of 1 µM + 90 µL water (or 1 µL of 10 µM + 99 µL water).
4. Anneal SSS-M to 1 µM (Step 1).
5. Prepare the 750 µL master mix (Step 2).
6. Dispense 37.5 µL/well master mix to the 384-well plate.
7. Immediately add VAN (5 µL/well) per group (PBS for 0 µM; 1 mM VAN for +VAN groups).
8. Immediately add T7 polymerase (2.5 µL/well, 50 µM).
9. Add template DNA (5 µL/well) using the specified stock for each group (10 µM / 1 µM / 100 nM).
10. Seal the plate. Incubate at 37 °C for 10 h. Record fluorescence every 1 min at Ex/Em =506 / 560nm.

### Controls

* 0 µM VAN (PBS) serves as the no-VAN control at each template level.
* Negative wells: 50 µL of 1 µM DFHBSI only for background fluorescence.

## Transcription Protocol for SSS-M (Low-template System; T7 in Master)

This experiment followed the same procedure as "Experiments --- SSS-M Aptamer Sensor (Before Annealing-Condition Change)", except that the template strand concentration was diluted to 10 nM to evaluate how lower template input affects transcription efficiency and signal-to-background ratio.

## Transcription Protocol for SSS-M (Modified Annealing: Co-anneal with VAN vs PBS; T7 in Master)

### Purpose

Assess whether co-annealing SSS-M with the T7 reverse strand in the presence of VAN (vs PBS) changes transcriptional output.

### Materials & Reagents

* 10 µM SSS-M DNA
* 10 µM T7 reverse strand DNA
* Vancomycin (VAN) 1 mM (PBS as 0 µM control)
* 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9)
* Template DNA working solution (2.5 µM) --- added later
* NTP mix 25 mM
* DFHBSI 1 mM
* T7 RNA polymerase 50 µM
* Pyrophosphatase (PPase) 0.001 U/µL
* DEPC-treated water

### Procedure

1. DNA Annealing (SSS-M + T7 Reverse, ±VAN)

   Tube A (VAN-annealed "template")

   | Component | Volume |
   | --- | --- |
   | 10 µM SSS-M | 8 µL |
   | 10 µM T7 reverse strand | 8 µL |
   | 1 mM VAN | 16 µL |
   | Total | 32 µL |

   Tube B (PBS-annealed "template")

   | Component | Volume |
   | --- | --- |
   | 10 µM SSS-M | 8 µL |
   | 10 µM T7 reverse strand | 8 µL |
   | PBS | 16 µL |
   | Total | 32 µL |

   Annealing program (both tubes): Hot water bath >95 °C for 5 min, then cool to RT (bench) → keep on ice.
2. In-vitro Transcription System (Master, 300 µL total)

   Do NOT add the 2.5 µM template yet. Prepare the base mix first (300 µL recipe shown below), then split, then add annealed material.

   Base Mix (without template, 240 µL):

   | Component | Volume |
   | --- | --- |
   | 5× Transcription buffer | 60 µL |
   | 25 mM NTP mix | 15 µL |
   | 50 µM T7 polymerase | 15 µL |
   | DEPC water | 144 µL |
   | 1 mM DFHBSI | 3 µL |
   | 0.001 U/µL PPase | 3 µL |
   | Subtotal | 240 µL |

   Split the 240 µL base mix into two tubes, 120 µL each.

   Now add template (2.5 µM) + annealed materials:

   * To Tube 1 (VAN condition): add 30 µL of the annealed Tube A material.
   * To Tube 2 (PBS condition): add 30 µL of the annealed Tube B material likewise.

   At this point, each condition tube is ~150 µL (120 µL base + 30 µL template/annealed additions).
3. Plate Setup (per-well totals = 50 µL, 3 replicates/condition)

   For each condition (VAN-annealed vs PBS-annealed), dispense 50 µL/well into 3 wells (total 150 µL per condition). Include a background well if desired.

   | Condition | Per-well volume | Replicates |
   | --- | --- | --- |
   | VAN-annealed (Tube A) | 50 µL | 3 |
   | PBS-annealed (Tube B) | 50 µL | 3 |
   | Negative (optional) | 50 µL of 1 µM DFHBSI only | ≥1 |
4. Anneal SSS-M + T7 reverse with VAN (Tube A) or PBS (Tube B) as in Step 1.
5. Prepare the 240 µL base mix (Step 2) without template; split 120 µL + 120 µL.
6. Add 30 µL of 2.5 µM template to each tube and incorporate the respective annealed material (VAN or PBS) per your run plan → ~150 µL per condition.
7. Quickly dispense 50 µL/well into a 384-well plate (3 wells per condition).
8. Seal the plate. Incubate at 37 °C for 10 h. Read fluorescence every 1 min (Ex/Em506 /560 nm).

### Controls

* PBS-annealed condition serves as the 0-VAN control.
* Optional background wells: 1 µM DFHBSI only (50 µL) for instrument background.

## Transcription Protocol for SSS-M (Modified Annealing; T7 Polymerase Concentration Series)

### Purpose

Assess how T7 RNA polymerase level affects transcriptional output when SSS-M and the T7 Reverse strand are co-annealed either with 1 mM VAN or with PBS (0 µM).

### Materials & Reagents

* 10 µM SSS-M DNA
* 10 µM T7 Reverse strand DNA
* Vancomycin (VAN) 1 mM; PBS (0 µM)
* 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9)
* NTP mix 25 mM
* DFHBSI 100 µM
* Pyrophosphatase (PPase) 0.001 U/µL
* T7 RNA polymerase stocks: 50 µM, 40 µM, 25 µM (and optionally 50 µM, 5 µM, 0.5 µM for the second set)
* DEPC-treated RNase-free water

### Procedure

1. DNA Annealing (SSS-M + T7-Reverse, ±VAN)

   Tube A (VAN-annealed duplex)

   | Component | Volume |
   | --- | --- |
   | 10 µM SSS-M | 24 µL |
   | 10 µM T7 Reverse strand | 24 µL |
   | 1 mM VAN | 48 µL |
   | Total | 96 µL |

   Tube B (PBS-annealed duplex)

   | Component | Volume |
   | --- | --- |
   | 10 µM SSS-M | 24 µL |
   | 10 µM T7 Reverse strand | 24 µL |
   | PBS | 48 µL |
   | Total | 96 µL |

   Annealing program (both tubes): Hot water bath >95 °C for 5 min, then allow to cool to room temperature; keep on ice until use.
2. In-vitro Transcription System (Base Master, 900 µL total)

   Prepare in a 1.5 mL tube (no template yet):

   | Component | Volume |
   | --- | --- |
   | 5× Transcription buffer | 200 µL |
   | 25 mM NTP mix | 50 µL |
   | DEPC water | 480 µL |
   | DFHBSI 100 µM | 8 µL |
   | Pyrophosphatase 0.001 U/µL | 10 µL |
   | Subtotal (no T7, no template) | 748 µL |

   Now split this 748 µL base into three equal systems (≈ 249--250 µL each).

   Standardize each system to 225 µL (discard/retain excess so each is exactly 225 µL), then add T7 as below:

   | System | Base (pre-T7) | T7 polymerase | T7 stock | T7 volume |
   | --- | --- | --- | --- | --- |
   | Sys-1 (High) | 225 µL | + 15 µL | 50 µM | 15 µL |
   | Sys-2 (Mid) | 225 µL | + 15 µL | 40 µM | 15 µL |
   | Sys-3 (Low) | 225 µL | + 15 µL | 25 µM | 15 µL |

   Mix each system gently after adding T7.

   Second set: repeat the same workflow with T7 stocks at 50 µM, 5 µM, 0.5 µM (still 15 µL per 225 µL system) to create an additional high/medium/low enzyme series
3. Split per Condition (±VAN duplex "template")

   Do not add "template" earlier. From each T7 system (Sys-1/2/3), split into two tubes of 110 µL (for 0 µM and 1 mM conditions):

   * Sys-X / PBS condition: take 110 µL of the T7-containing system + 27.5 µL of the PBS-annealed duplex (from Step 1, Tube B).
   * Sys-X / VAN condition: take 110 µL of the T7-containing system + 27.5 µL of the VAN-annealed duplex (from Step 1, Tube A).

   This yields six tubes total (High-PBS, High-VAN, Mid-PBS, Mid-VAN, Low-PBS, Low-VAN), each ~137.5 µL.
4. Plate Setup (per-well totals)

   Dispense 50 µL/well to the 384-well plate from each tube.

   At ~137.5 µL/tube, you can fill 2 wells (100 µL) comfortably (with ~37.5 µL dead volume).

   If you need triplicates, scale each split to ≥150 µL (e.g., use 120 µL split + 30 µL annealed duplex) so you can plate 3 × 50 µL per tube.

   Negative control (optional): 50 µL of 1 µM DFHBSI only to measure background fluorescence.
5. Anneal SSS-M + T7-Reverse with 1 mM VAN or PBS (Step 1).
6. Prepare the 900 µL base transcription system (Step 2, no template).
7. Create three T7 systems by adding 15 µL of the specified T7 stock to 225 µL of base (Sys-1/2/3).
8. From each T7 system, split 110 µL + 110 µL; add 27.5 µL of PBS-annealed duplex to one, and 27.5 µL of VAN-annealed duplex to the other (Step 3).
9. Plate 50 µL/well for each tube into the 384-well plate (2 wells per tube as prepared; scale if need 3).
10. Seal the plate; incubate at 37 °C for 10 h. Record fluorescence every 1 min (Ex/Em 506/560 nm).

## Transcription Protocol for SSS Sensor: VAN Gradient at 1.25 µM T7

This experiment followed the same conditions as "Experiments --- SSS-M (Modified Annealing; T7 Polymerase Concentration Series)" under the 1.25 µM T7 polymerase setting, but extended the vancomycin gradient to include 0, 5, 20, and 100 µM concentrations to achieve a finer resolution of the dose--response relationship.

## Transcription Protocol for SSS-S vs SSS-L

This experiment was conducted under the same conditions as "Experiments --- SSS-M (Modified Annealing: Co-anneal with VAN vs PBS; T7 in Master)", except that the DNA strands were replaced with SSS-S and SSS-L sequences to compare their structural effects on transcription activation and vancomycin responsiveness.

## Transcription Protocol for TS Families (5TS-1~4; 3TS-1~8)

### Purpose

Evaluate redesigned TS aptamer sensors (bulge/mismatch families) for vancomycin responsiveness under a standardized in-vitro transcription assay. Each batch runs four variants in parallel with VAN = 0 or 1 mM.

### Materials & Reagents

* 5× Binding Buffer (PBS containing MgCl₂; your note: 5×, PBS with 10 mM MgCl₂)
* 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9 @ 25 °C)
* T7 Template DNA 10 µM (used in master; target 250 nM in system)
* TS variants: 5TS-1, -2, -3, -4 (and separately 3TS-1 ... -8) --- 10 µM stocks
* NTP mix 25 mM
* DFHBSI 100 µM stock (final 1 µM)
* Pyrophosphatase (PPase) 0.001 U/µL
* T7 RNA polymerase 50 µM
* Vancomycin (VAN): 1 mM stock; PBS for 0 µM
* DEPC-treated RNase-free water

### Procedure

1. DNA Annealing (per variant)

   Prepare one annealing tube per variant (works for either 5TS-n or 3TS-n):

   | Component | Volume |
   | --- | --- |
   | 10 µM TS variant (5TS-n or 3TS-n) | 8.0 µL |
   | 1st-grade water | 4.8 µL |
   | 5× Binding Buffer (PBS with Mg²⁺) | 3.2 µL |
   | Total per variant | 16.0 µL |

   Program: Hot water bath >95 °C for 5 min, then cool to room temperature; keep on ice. (This yields an annealed TS solution you'll add to each system in Step 3.)
2. Transcription Master (Total 1,020 µL)

   (no VAN, no T7 added here; T7 and VAN are added at the plating step)

   | Component | Final conc. (system) | Volume |
   | --- | --- | --- |
   | 5× Transcription buffer | --- | 240 µL |
   | 10 µM T7 Template strand | 250 nM | 30 µL |
   | 25 mM NTP mix | 2 mM | 96 µL |
   | DFHBSI 100 µM | 1 µM | 12 µL |
   | DEPC water | --- | 570 µL |
   | PPase 0.001 U/µL | 0.001 U | 12 µL |
   | Total | --- | 1,020 µL |
3. Split for Four Variants & Spike in Annealed TS

   Split the 1,020 µL master into four tubes: 240 µL × 4 (960 µL used; ~60 µL spare is dead volume)

   To each 240 µL tube, add 15 µL of the corresponding annealed TS from Step 1 → 255 µL per variant system.

   This gives four variant systems (e.g., 5TS-1, -2, -3, -4) each at ~255 µL prior to plating.

   Repeat the same for 3TS in two batches of four variants each (3TS-1~4, then 3TS-5~8).
4. Plate Setup (per-well totals = 40 µL)

   Per your grouping:

   | Group | Transcription system (µL) | VAN (µL) | T7 polymerase (µL; 50 µM) | DFHBSI only (µL) |
   | --- | --- | --- | --- | --- |
   | 0 (0 µM VAN) | 34 | 4 (PBS) | 2 | 0 |
   | 3 (1 mM VAN) | 34 | 4 (1 mM) | 2 | 0 |
   | Negative | 0 | 0 | 0 | 50 (1 µM DFHBSI) |

   Quick check: 34 + 4 + 2 = 40 µL / well.
5. Procedure (per batch of four variants)
   * Anneal each TS variant (Step 1).
   * Prepare the 1,020 µL transcription master (Step 2).
   * Split master into 4 × 240 µL and add 15 µL annealed TS to each (Step 3).
   * Plate per well:
     + Add 34 µL of the corresponding variant system to each designated well.
     + Add 4 µL VAN: PBS for Group 0 (0 µM) or 1 mM VAN for Group 3 (100 µM final).
     + Add 2 µL T7 polymerase (50 µM) per well.
   * Seal; incubate at 37 °C for 10 h. Record fluorescence every 1 min at Ex/Em =506 /560 nm.

### Controls

* Group 0 (PBS) is the no-VAN control for each variant.
* Negative wells: 50 µL of 1 µM DFHBSI only for instrument/background reference.

## Transcription Protocol for Double-Strand Extension (S1:L1--L6)

### Purpose

Evaluate the vancomycin responsiveness of the extended hybrid duplex sensors built from S1 + Ln (n = 1...6) under a standardized in-vitro transcription assay. The workflow mirrors the TS runs: prepare a 1,020 µL transcription master, split for variants, spike annealed S1:L duplex, and plate with VAN = 0 or 1 mM.

### Materials & Reagents

* S1 strand (10 µM)
* L strands (10 µM): L1, L2, L3, L4, L5, L6
* 5× PBS (so that the annealing mix is 1× PBS in the 32 µL anneal)
* 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9 @ 25 °C)
* T7 Template DNA 10 µM (target 250 nM in the transcription system)
* NTP mix 25 mM
* DFHBSI 100 µM (final 1 µM)
* Pyrophosphatase (PPase) 0.001 U/µL
* T7 RNA polymerase 50 µM
* Vancomycin (VAN) 1 mM stock; PBS for 0 µM
* DEPC-treated RNase-free water

### Procedure

1. Annealing (per variant, S1:L with 5× PBS)

   Prepare one annealing tube per L variant (S1:L1, S1:L2, ... S1:L6):

   | Component (10 µM stocks) | Volume |
   | --- | --- |
   | S1 | 3.2 µL |
   | L (L1...L6) | 6.4 µL |
   | 5× PBS | 6.4 µL |
   | Water (1st-grade) | 16.0 µL |
   | Total | 32.0 µL |

   Program: Hot water bath >95 °C for 5 min, then cool to room temperature; hold on ice. (6.4 µL of 5× PBS in 32 µL → 1× PBS during annealing.)
2. Transcription Master (Total 1,020 µL; no VAN, no T7)

   Same system as above:

   | Component | Final conc. (system) | Volume |
   | --- | --- | --- |
   | 5× Transcription buffer | --- | 240 µL |
   | T7 Template (10 µM) | 100 nM | 12 µL |
   | NTP mix (25 mM) | 2 mM | 96 µL |
   | DFHBSI (100 µM) | 1 µM | 12 µL |
   | DEPC water | --- | 648 µL |
   | PPase (0.001 U/µL) | 0.001 U | 12 µL |
   | Total | --- | 1020 µL |

   Mix gently.
3. Split for Variants & Spike Annealed Duplex

   Split the master into four equal aliquots for a 4-variant batch (repeat the batch for remaining Ls): 225 µL × 4 (uses 900 µL; ~120 µL remains as dead volume).

   To each 225µL tube, add 30µL of the corresponding annealed S1:L duplex (from Step 1) → 255 µL per variant system.

   Batching:

   * Batch A: L1, L2, L3, L4 (4 variants).
   * Batch B: L5, L6 (use the same workflow; you may replicate some variants or allocate more replicates to fill the four-way split if desired).
4. Plate Setup (per-well totals = 40 µL)

   Use the same group volumes as the TS runs:

   | Group | Transcription system (µL) | VAN (µL) | T7 polymerase (µL; 50 µM) | DFHBSI only (µL) |
   | --- | --- | --- | --- | --- |
   | 0 (0 µM VAN) | 34 | 4 (PBS) | 2 | 0 |
   | 3 (1 mM VAN) | 34 | 4 (1 mM) | 2 | 0 |
   | Negative | 0 | 0 | 0 | 50 (1 µM DFHBSI) |

   Quick check: 34 + 4 + 2 = 40 µL/well.
5. Procedure (per 4-variant batch)
   * Anneal S1 with each L variant using Step 1 (5× PBS → 1× PBS in 32 µL).
   * Prepare the 1,020 µL transcription master (Step 2).
   * Split into 4 × 225µL; add 30µL annealed S1:L to each to make variant systems (Step 3).
   * Plate per well:
     + Add 34 µL of the variant system to each designated well.
     + Add 4 µL VAN: PBS for Group 0 (0 µM) or 1 mM VAN for Group 3 (100 µM final).
     + Add 2 µL T7 polymerase (50 µM) per well.
   * Seal; incubate at 37 °C for 10 h. Record fluorescence every 1 min (Ex/Em506 /560 nm).

### Controls

* Group 0 (PBS) is the no-VAN control for each S1:L variant.
* Negative wells: 50 µL of 1 µM DFHBSI only for background.

## Transcription Protocol for S1 : L4, L4-1, L4-2, L4-3, L5, L5-1, L5-2, L5-3, L5-4

This experiment followed the same procedure as "Experiments --- Double-Strand Extension (S1:L1--L6)", except that the L strands were replaced with L4, L4-1, L4-2, L4-3, L5, L5-1, L5-2, L5-3, L5-4, and the fluorescent dye DFHBI-1T was used instead of DFHBSI for improved signal clarity.

## Transcription Protocol for S1 + L5\_3: Template Concentration Gradient (0.5:1, 1:1, 5:1, 10:1)

This experiment was conducted under the same conditions as "Experiments --- S1 : L4, L4-1, L4-2, L4-3, L5, L5-1, L5-2, L5-3, L5-4", using the S1 + L5\_3 duplex, but the template-to-S1 ratio was varied (0.5:1, 1:1, 5:1, 10:1) instead of the original 1:1 ratio to examine the effect of template concentration on transcriptional output.

# CRISPR-Cas12a Amplification Module

## Cas12a System

The Cas12a system can be used for nucleic acid detection. A typical Cas12a detection system consists of the following components:

* **Cas12a protein:** Cas12a is a nuclease belonging to the type II CRISPR--Cas system, subtype V.
* **crRNA:** CRISPR RNA (crRNA) is typically an RNA of 42--44 nucleotides. Unlike Cas9, which requires both crRNA and tracrRNA, the Cas12a system requires only a single crRNA.
* **Reporter:** A short single-stranded DNA (ssDNA) labeled with a fluorophore at one end and a quencher at the other.

Cas12a recognizes the target DNA through complementary base pairing with the crRNA. Once Cas12a is activated, it non-specifically cleaves all ssDNA in the system. At this point, the pre-introduced reporter probe (short ssDNA labeled with a fluorophore and a quencher) is cleaved. Upon cleavage, the fluorophore separates from the quencher, producing a fluorescence signal.

## Procedure

1. Strand hybridization

   | Reagents | 0 | 1 | 2 | 3 | 4 | 5 |
   | --- | --- | --- | --- | --- | --- | --- |
   | 10 μM LBO-Cas(μL) | 2.6 | 2.6 | 2.6 | 2.6 | 2.6 | 2.6 |
   | 10 μM SRO-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 |

   **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.

   6 groups of solution this step contains free SRO-Cas, serving as the target for the Cas system
2. Preparation of master mix: To ensure that each target group reacts in a uniform solution, a master mix is prepared first according to the table below, by pre-mixing all components except the target.

   | Component | Volume |
   | --- | --- |
   | 10x Reaction buffer(μL) | 72 |
   | 1μM reporter(μL) | 14.4 |
   | 20 x Cas12a(μL) | 36 |
   | 1μM crRNA(μL) | 72 |
   | DEPC water(μL) | 453.6 |
   | Overall(μL) | 648 |
3. 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. Each group is repeated 5 times. The composition of each 20 μL reaction system is shown in the table below.

   | Component | Volume | Final concentrations |
   | --- | --- | --- |
   | 10x Reaction buffer | 2 μL |  |
   | Target | 2 μL |  |
   | 1μM reporter | 2 μL | 200 nM |
   | 20 x Cas12a | 2 μL | 100 nM |
   | 1μM crRNA | 2 μL | 100 nM |
   | DEPC water | 10 μL |  |
4. Loading sample
   1. Place the centrifuge tubes on an ice tray.
   2. For each sample, add the substituted solution to the pre-mix solution to reach a final volume of 20 μL.
   3. Transfer the mixture into a 384-well plate.
5. Measurement of fluorescence intensity
   * Temperature: 37°C
   * Excitation wavelength: 490 nm
   * Emission wavelength: 520 nm
   * Time: 30 min

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

* [Strand Displacement Module](#strand-displacement)
  + [Coupled with T7 Transcription](#t7-coupled)
  + [Coupled with Cas System](#cas-coupled)
* [T7 Transcription Module](#t7-transcription)
  + [Problem](#problem)
  + [Single-Strand System](#single-strand)
  + [Extended Double-Strand System](#double-strand)
* [Cas Module](#cas-module)

# Experimental results of strand displacement module

## Strand displacement module coupled with T7 transcription

In the strand displacement module coupled with T7 transcription, we identified an optimal LBO1-SRO1-T7 pair through PAGE electrophoresis and quantitative fluorescence analysis. At an LBO:SRO hybridization ratio of 5:1, this pair demonstrated low background noise (below 20%) and produced concentration-dependent fluorescence changes at antibiotic concentrations exceeding 10 µM, thereby establishing a foundation for coupling with the T7 amplification system.

### Results

#### 1. Design LBO-Cas and SRO-Cas

Table 1: LBO and SRO Sequences

| Name | Sequence |
| --- | --- |
| LBO1 | CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA |
| SRO1 | AGCCGAACTGAG |
| SRO1-T7 | AGCCGAACTGAGTAATACGACTCACTATAGG |
| LBO2 | TTTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG |
| SRO2-T7 | AGCCGGAGAGAGAAATAATACGACTCACTATAGG |
| SRO2 | AGCCGGAGAGAGAAA |
| LBO3 | TCTCTCTCTCTCCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGGAG |
| SRO3 | AGCCGGAGAGAGAGAGA |
| LBO4 | CCTGAACCCCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGGTT |
| SRO4 | TCGGGGTTCAGG |
| LBO5 | GCCTGAACCCCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGGTT |
| SRO5 | TCGGGGTTCAGGC |
| LBO6 | TAAGATCTCTCGGGACGACCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGTCGTCCC |
| SRO6 | GTCGTCCCGAGAGTA |
| LBO7 | TAAGATCTCTCAGGGACGACCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGTCGTCCCT |
| SRO7 | GTCGTCCCTGAGAGAA |
| LBO8 | TAAGATCTCTCAGGGGACGACCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGGTCGTCCCCT |

1. PAGE electrophoresis verification showed that LBO1-SRO1 and LBO2-SRO2 exhibited high hybridization efficiency, and strand displacement could be initiated upon antibiotic addition.

![PAGE electrophoresis result 1](https://static.igem.wiki/teams/5772/re/re1.webp)

Figure 1: PAGE electrophoresis verification

![PAGE electrophoresis result 2](https://static.igem.wiki/teams/5772/re/re2.webp)

Figure 2: PAGE electrophoresis verification

2. After incorporating the T7 promoter sequence, LBO1-SRO1-T7 and LBO2-SRO2-T7 were hybridized at a 1:1 ratio for quantitative fluorescence analysis. It was found that LBO1-SRO1-T7 demonstrated relatively stable hybridization.

![Quantitative fluorescence analysis 1](https://static.igem.wiki/teams/5772/re/re3.webp)

Figure 3: Quantitative fluorescence analysis

![Quantitative fluorescence analysis 2](https://static.igem.wiki/teams/5772/re/re4.webp)

Figure 4: Quantitative fluorescence analysis

3. To decrease background signal, we performed the ratio hybridization experiment for LBO1-SRO1-T7. The background signal was observed to be lower at an LBO1:SRO1-T7 ratio of 5:1. Although further increasing the ratio could reduce background noise, it would compromise the sensor's sensitivity. Therefore, this ratio was selected for subsequent experiments.

![Ratio hybridization experiment](https://static.igem.wiki/teams/5772/re/re5.webp)

Figure 5: Ratio hybridization experiment

4. At an LBO1:SRO1-T7 ratio of 5:1, quantitative fluorescence analysis was performed after antibiotic addition to investigate the strand displacement effect. It was observed that antibiotic concentrations greater than 10 µM induced concentration-dependent changes in fluorescence intensity, thereby establishing a foundation for coupling with the T7 amplification system.

![Strand displacement effect](https://static.igem.wiki/teams/5772/re/re6.webp)

Figure 6: Strand displacement effect

## Strand displacement module coupled with Cas system

In the strand displacement module coupled with the Cas system, we identified an optimal LBO1-SRO2-Cas pair through PAGE electrophoresis and quantitative fluorescence analysis. When hybridized at an LBO:SRO ratio of 1:1, this pair exhibited low background noise (below 25%) and produced concentration-dependent fluorescence changes at antibiotic concentrations above 20 µM, thereby establishing a foundation for coupling with the Cas amplification system.

### Results

1. PAGE electrophoresis analysis confirmed that the LBO1-SRO2-Cas complex exhibits high hybridization efficiency at a 1:1 ratio and undergoes strand displacement upon antibiotic addition. However, the proportion of hybrid complexes participating in strand displacement remains relatively low.

![LBO1+SRO2 PAGE electrophoresis](https://static.igem.wiki/teams/5772/re/re7.webp)

Figure 7: LBO1+SRO2 PAGE electrophoresis

2. When using a 1:1 ratio of LBO1:SRO1-Cas, quantitative fluorescence measurements were performed after antibiotic introduction to evaluate the strand displacement effect. The results demonstrated antibiotic concentration-dependent fluorescence changes at concentrations above 20 µM, establishing the foundation for integration with the Cas amplification system.

![Normalized fluorescence intensity comparison](https://static.igem.wiki/teams/5772/re/re8.webp)

Figure 8: Normalized fluorescence intensity comparison

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.

3. Comparison of normalized fluorescence intensity results from quantitative fluorescence experiments conducted with and without crRNA confirmed that crRNA enhances the strand displacement reaction.

# Experimental results of T7 transcription module

## Problem

When the screened LBO1-SRO1-T7 strand displacement system was directly coupled with the transcription system, signal leakage occurred because the T7 promoter sequence in SRO1-T7 remained unblocked. Even when SRO1-T7 was hybridized with LBO1 and not released, the exposed T7 promoter could still initiate transcription. To address this, we explored various methods to block the T7 promoter and reduce signal leakage. The following section documents our some effective attempts and results.

## Experimental results of the single-strand system

### Overview

We aimed to construct a single-strand aptamer sensor to detect vancomycin through structural switching. Multiple design iterations were carried out, testing variations in sensor length, template concentrations, T7 polymerase levels, and promoter sealing strategies. Although several constructs showed partial improvements, consistent antibiotic-dependent fluorescence responses remain a challenge. Below, we summarize our results and the outcomes.

### Experimental Findings

#### Initial SSS Family Designs

![Schematic diagram of single-strand sensor (SSS) design](https://static.igem.wiki/teams/5772/re/re9.webp)

Figure 9: Schematic diagram of single-strand sensor (SSS) design

Table 2: Single-Strand Sensor Designs

| Construct | Description | Key Result |
| --- | --- | --- |
| SSS-S | Short 5′ blocking sequence | Showed ~16.2% higher fluorescence at 100 µM VAN vs. 0 µM after 10 h |
| SSS-M | Medium 5′ blocking sequence | No significant VAN response |
| SSS-L | Long 5′ blocking sequence | No significant VAN response |

#### Template and Polymerase Optimization

Table 3: Optimization Parameters

| Parameter | Range Tested | Outcome |
| --- | --- | --- |
| Template concentration | 10 nM -- 1 µM | At 10 nM, a difference between groups appeared, but high noise and poor reproducibility |
| T7 polymerase concentration | 0.025 -- 2.5 µM | At high concentrations, reactions were fast but non-specific; at low concentrations, no transcription occurred |

### Results

![Fluorescence intensity after 10 hours incubation](https://static.igem.wiki/teams/5772/re/re10.webp)

Figure 10: Fluorescence intensity after 10 hours incubation

The SSS family of designs tested whether a single-stranded promoter hairpin could prevent premature transcription while still responding to vancomycin. Among the three variants, SSS-S (short blocking sequence) performed best, showing reduced background fluorescence and a modest but measurable increase (~16.2%) in fluorescence at 100 µM vancomycin compared to the control. This suggests that a shorter blocking sequence provides tighter promoter sealing without completely disrupting the aptamer's ability to switch conformation. In contrast, SSS-M and SSS-L failed to demonstrate any concentration-dependent response, likely due to excessive complementarity that stabilized the hairpin too strongly or interfered with the aptamer binding domain. These results highlighted an important design principle: over-engineering the promoter lock can inadvertently abolish ligand sensitivity, while a carefully balanced short blocker may offer a better trade-off between background control and responsiveness.

#### Extended Families: 5TS and 3TS Designs

To overcome incomplete promoter sealing and disrupted aptamer binding, we developed two new families:

5TS family: T7 promoter at 5′ end with complementary bulges (3--0 bulges).

![5TS family design with complementary bulges](https://static.igem.wiki/teams/5772/re/re1.avif)

Figure 11: 5TS family design with complementary bulges

![Fluorescence analysis of 5TS variants](https://static.igem.wiki/teams/5772/re/re2.avif)

Figure 12: Fluorescence analysis of 5TS variants

3TS family: T7 promoter at 3′ end with mismatch designs (0--2 mismatches).

![3TS family design with mismatch patterns](https://static.igem.wiki/teams/5772/re/re3.avif)

Figure 13: 3TS family design with mismatch patterns

![Fluorescence analysis of 3TS variants](https://static.igem.wiki/teams/5772/re/re4.avif)

Figure 14: Fluorescence analysis of 3TS variants

#### Key Findings:

5TS variants: Bulge number influenced total fluorescence but not vancomycin response.

3TS variants: More mismatches and mismatches near the inner hairpin increased fluorescence intensity.

Some 3TS constructs showed ~11–13% fluorescence increase in vancomycin-treated samples, but background leakage was high

![Fluorescence intensity after 5 hours incubation](https://static.igem.wiki/teams/5772/re/re15.webp)

Figure 15: Fluorescence intensity after 5 hours incubation

![3TS family fluorescence comparison](https://static.igem.wiki/teams/5772/re/re16.webp)

Figure 16: 3TS family fluorescence comparison

![Extended 3TS variants fluorescence analysis](https://static.igem.wiki/teams/5772/re/re17.webp)

Figure 17: Extended 3TS variants fluorescence analysis

### Summary of Observations:

Incomplete promoter sealing was the main cause of background leakage.

SSS-S and some 3TS constructs showed relatively better vancomycin responsiveness, but the sensitivity was still insufficient.

Template and polymerase concentration adjustments alone did not solve the problem.

NUPACK simulations highlighted that maintaining the aptamer's recognition domain is critical, as design modifications risk disrupting ligand binding.

### Analysis & Interpretation:

Our cycles demonstrate that while single-strand promoter-locking is a promising strategy, balancing low background leakage with ligand-dependent switching remains difficult. The trade-off lies between strong promoter sealing (which prevents leakage but can also hinder switching) and weak sealing (which allows switching but suffers from high background).

### Lessons Learned

Experimental errors (e.g., in polymerase concentration setup) led to misleading results in early trials, highlighting the importance of replicates and strict operation standards.

Sequence design trade-offs are non-trivial: adding complementary regions risks distorting aptamer binding motifs.

Secondary structure prediction (NUPACK) is essential to pre-validate that recognition domains remain intact before synthesis.

### Future Plans

Refined design: Explore hybrid single-/double-strand systems that combine strong promoter sealing with flexible release mechanisms.

Reduce background leakage: Introduce rational bulge/mismatch engineering guided by systematic NUPACK and ViennaRNA simulations.

Application direction: If successful, the platform could be expanded to detect antibiotics beyond vancomycin (e.g., tetracycline, streptomycin), or adapted for food safety and clinical diagnostics.

Transparency: Although results so far are not ideal, we report them in full to remain scientifically honest and to help future teams avoid repeating our pitfalls.

In summary, our single-strand aptamer sensor development showed partial successes (especially in SSS-S and certain 3TS variants), but failed to achieve a stable concentration-dependent response. The lessons from these attempts will guide us toward improved promoter-sealing strategies and more reliable aptamer--antibiotic detection systems.

## Experimental results of the extended double-strand system

### Overview

To further optimize promoter sealing and antibiotic responsiveness, we explored a double-strand extension strategy. By progressively extending the 5′ or 3′ ends of the LBO strand, we aimed to adjust the degree of promoter--aptamer complementarity and achieve an optimal balance between background suppression and vancomycin-dependent release.

### Experimental Findings

#### 1. Initial LBO 5′-Extension

We tested six variants (L1–L6) with 0–19 complementary bases added to the 5′ end of the LBO strand, paired with a constant SRO carrying the T7 promoter (S1).

![ExtensionSchematic of LBO 5′-extension strategy](https://static.igem.wiki/teams/5772/re/re18.webp)

Figure 18:Schematic of LBO 5′-extension strategy

Table 4: LBO 5′-Extension Results

| Pair | Complementarity added | Key Result |
| --- | --- | --- |
| S1+L1 | 0 bases | High background, weak response |
| S1+L2 | 4 bases | Best response (~1.21-fold increase at 100 µM VAN) |
| S1+L3–L6 | 8–19 bases | Strong sealing, but unresponsive to VAN |

![Fluorescence analysis of LBO 5′-extension variants](https://static.igem.wiki/teams/5772/re/re19.webp)

Figure 19:Fluorescence analysis of LBO 5′-extension variants

Among the six designs, S1+L2 struck the best balance: the promoter was partially sealed by four complementary bases, enabling vancomycin to trigger displacement and transcription with ~21% higher fluorescence in the positive group. In contrast, L3–L6 sealed the promoter too tightly, abolishing responsiveness. These results demonstrated that moderate complementarity improves background control while preserving aptamer switching capacity. However, even in the best group, background fluorescence remained high and the overall signal-to-background ratio was unsatisfactory.

#### 2. LBO 3′-Extension

Building on the results above, we modified L4 and L5 by progressively extending their 3′ ends (L4\_1–L4\_3 and L5\_1–L5\_4). The aim was to destabilize the LBO–SRO duplex upon vancomycin binding, thereby improving responsiveness.

![ LBO 3′-extension fluorescence after 10 hours](https://static.igem.wiki/teams/5772/re/re20.webp)

Figure 20:LBO 3′-extension fluorescence after 10 hours

![LBO 3′-extension fluorescence after 45 minutes](https://static.igem.wiki/teams/5772/re/re21.webp)

Figure 21: LBO 3′-extension fluorescence after 45 minutes

**Key Findings:**

All S1+L4\_n variants remained insensitive to vancomycin, suggesting that the tested increments overshot the optimal range.

S1+L5\_3 (13-base extension) exhibited a clear response, with ~1.35-fold higher fluorescence at 100 µM vs. 0 µM VAN — stronger than S1+L2.

Extensions beyond this point (e.g., L5\_4) destabilized the system completely, producing high but non-specific fluorescence.

The L5 series followed a transition from over-sealed → intermediate → unsealed states as more bases were added. At the intermediate state (L5\_3), the promoter was sealed enough to suppress background yet still able to open in response to vancomycin. By contrast, L4 variants skipped over this "sweet spot," indicating that our step sizes were too large. These results suggest that fine-tuned base additions (smaller increments) are necessary to locate the optimal trade-off.

#### 3. Template Concentration Effects

To further refine the best-performing construct (S1+L5\_3), we varied template concentrations relative to S1 (ratios 0.5–10).

![Template concentration optimization for S1+L5_3](https://static.igem.wiki/teams/5772/re/re22.webp)

Figure 22: Template concentration optimization for S1+L5\_3

**Key Findings:**

At a 1:1 template:S1 ratio, vancomycin responsiveness was most pronounced.

At low template concentrations, overall signal was too weak, masking group differences.

At excessively high template concentrations, the template strand displaced S1 directly, overriding vancomycin dependence.

The transcription system displayed classic concentration trade-offs: too little template resulted in insufficient signal, while too much template led to non-specific displacement. A narrow intermediate range allowed antibiotic-dependent differences to be observed. This highlights the importance of carefully balanced stoichiometry in aptamer-based transcription systems.

### Summary of Observations

S1+L2 (4-base 5′ extension) and S1+L5\_3 (13-base 3′ extension) were the most responsive constructs, though both still suffered from high background.

Too few complementary bases → leakage and weak specificity.

Too many complementary bases → over-sealing and loss of sensitivity.

System performance is strongly influenced by template concentration, which must be optimized within a narrow window.

### Analysis & Interpretation

Our double-strand extension strategy confirmed that small, carefully tuned modifications to the aptamer–promoter interface can dramatically alter performance. The results demonstrate a U-shaped trade-off: both insufficient and excessive complementarity abolish responsiveness, while intermediate levels yield the best signal-to-background balance.

### Lessons Learned

Even minor base additions (4 vs. 13) can completely shift aptamer performance, underscoring the sensitivity of nucleic acid design.

Using different dyes (DFHBI-1T vs. DFHBSI) improved visualization of subtle changes, suggesting the choice of reporter is critical.

Step increments in base addition were too large in the L4 series, preventing us from capturing the optimal range.

### Future Plans

Fine-resolution tuning: Explore smaller step extensions (e.g., +6, +7, +8 bases) to pinpoint the optimal LBO–SRO balance.

Combine strategies: Integrate promoter-sealing principles from the single-strand system with double-strand extension to reduce leakage.

Practical applications: If optimized, this strategy could serve as a modular framework for designing aptamer sensors responsive to a wide range of antibiotics or other small molecules.

Scientific transparency: By reporting both successful and failed constructs, we ensure that future teams can learn from our design space exploration without duplicating ineffective attempts.

In summary, the double-strand extension approach confirmed that promoter sealing can be tuned to improve antibiotic responsiveness. S1+L2 and S1+L5\_3 emerged as promising leads, but further refinements are required to reduce background leakage and enhance reliability.

# Experimental results of Cas module

We applied the Cas12a system as an amplification module, which successfully enhanced the fluorescence signal.

### Results

#### 1. Design LBO-Cas and SRO-Cas

To implement signal amplification, Cas12a was introduced and used to construct LBO-Cas (BHQ-labeled) and SRO-Cas (6-FAM-labeled). Fluorescence was measured by microplate reader and displacement efficiencies were calculated by normalizing fluorescence against a positive control. Two SRO-Cas / crRNA combinations were designed and tested.

Table 5: Cas System Sequences

| Component | Sequence |
| --- | --- |
| LBO1-Cas | GACGTATCGACTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAACT |
| SRO1-Cas | GGAGACGCCGAACTGAGTCGATACGTCTAGATTG |
| crRNA-1 | UAAUUUCUACUAAGUGUAGAUACGUAUCGACUCAGUUCGGC |
| SRO2-Cas | GGAGACCACTGAGCCGAACTGAGTCGCTAGATTG |
| crRNA-2 | UAAUUUCUACUAAGUGUAGAUCGACUCAGUUCGGCUCAGUG |

#### 2. Measurement of Fluorescence Intensity After Cas12a Cleavage

In the Cas12a system, once activated, Cas12a non-specifically cleaves all single-stranded DNA in the system. At this point, the pre-incorporated reporter probe (a short ssDNA fragment bearing a fluorescent and quenching moiety) is cleaved. Following cleavage, the separation of the fluorescent group from the quencher group generates a fluorescent signal. The amount of SRO displaced at the front end correlates positively with the rate of fluorescence intensity increase. Therefore, vancomycin concentration can be determined by measuring the rate of fluorescence intensity increase.

1. The figure below shows the fluorescence changes of the system over 30 minutes after the addition of different antibiotic concentrations. It can be observed that antibiotic concentrations greater than 5 µM produce a concentration-dependent increase in fluorescence intensity.

![Real-time fluorescence kinetics with Cas12a amplification](https://static.igem.wiki/teams/5772/re/re23.webp)

Figure 23: Real-time fluorescence kinetics with Cas12a amplification

2. To verify whether the introduction of Cas12a can effectively amplify the signal, we designed two experiments. The only difference between the two experiments was the presence or absence of Cas12a protein. As can be seen, the addition of Cas12a protein significantly enhanced the resolution of the sensor.

![Signal amplification comparison with and without Cas12a](https://static.igem.wiki/teams/5772/re/rere1.avif)

Figure 24: Normalized fluorescence intensity without CRISPR-cas12a system（base line: positive control group）

![Normalized fluorescence without Cas coupling](https://static.igem.wiki/teams/5772/re/rere2.avif)

Figure 25: Normalized fluorescence intensity with CRISPR-cas12a system（base line: zero antibiotic group）

By comparing two figures, it can be observed that in the uncoupled Cas system, the strand displacement system exhibited concentration-dependent fluorescence changes at antibiotic concentrations greater than 20 µM. In contrast, after coupling with the Cas system, the strand displacement system produced concentration-dependent fluorescence changes at antibiotic concentrations above 5 µM. This demonstrates that the Cas system effectively lowered the minimum detectable concentration of the sensor and served as a signal amplification mechanism.

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