Team:OUC-China/Parts
1. Overview
This year, OUC-China constructed numerous parts that can perform logical functions. Once the basic components of our design are put together, they can well form logic gates. We hope our parts will make it easier and more efficient for synthetic biologists and future iGEM teams to assemble the logic gates they need. Our parts include the switches and triggers of toehold (ON-switch), 3WJ repressors (OFF-switch) and six types of logic gates (OR, AND, NOT, NIMPLY, IMPLY, XOR). In addition, we added new data collected from laboratory experiments to the existing parts and used new reporter.
1.1 Toehold Switch
The toehold switches we tested have the characteristics of low leakage and high ON/OFF ratio, we choose it as ON switch. And we used it in the construction of logic gates and the design of some more complex circuits.
At the same time, we hope to optimize the ON/OFF ratio of toehold switch by improving the stability of trigger RNA. Therefore, we tried to add the hairpin structure at the 5' end of triggers (BBa_K3328010, BBa_K3328011, BBa_K3328012).
| Part | Type | Description |
|---|---|---|
| BBa_K3328008 | RNA | an ON-switch to regulate the expression of the downstream gene |
| BBa_K3328009 | RNA | binding to Toehold switch(K3328008) to express the downstream gene (no hairpin) |
| BBa_K3328010 | RNA | binding to Toehold switch(K3328008) to express the downstream gene |
| BBa_K3328011 | RNA | binding to Toehold switch(K3328008) to express the downstream gene |
| BBa_K3328012 | RNA | binding to Toehold switch(K3328008) to express the downstream gene |
1.2 3WJ Repressors
3WJ repressors showed remarkable ON/OFF ratio and had good orthogonality, we choose it as OFF switch. And we used it in the construction of logic gates and the design of some more complex circuits.
| Part | Type | Description |
|---|---|---|
| BBa_K3328000 | RNA | an OFF-switch to regulate the expression of the downstream gene |
| BBa_K3328001 | RNA | an OFF-switch to regulate the expression of the downstream gene |
| BBa_K3328002 | RNA | an OFF-switch to regulate the expression of the downstream gene |
| BBa_K3328003 | RNA | an OFF-switch to regulate the expression of the downstream gene |
| BBa_K3328004 | RNA | binding to 3WJ switch(K3328000) to inhibit the expression of the downstream gene |
| BBa_K3328005 | RNA | binding to 3WJ switch(K3328001) to inhibit the expression of the downstream gene |
| BBa_K3328006 | RNA | binding to 3WJ switch(K3328002) to inhibit the expression of the downstream gene |
| BBa_K3328007 | RNA | binding to 3WJ switch(K3328003) to inhibit the expression of the downstream gene4 |
1.3 Logic Gate
In the verification of the logic circuit, the inductors corresponding to the promoter before the switch structure were taken as the inputs, and the expression quantity of fluorescent protein was taken as the output, so as to test the existing logic operations and the logic operations designed by us (NIMPLY2, IMPLY1, IMPLY2, XOR1 and XOR2).
| Part | Type | Description |
|---|---|---|
| BBa_K3328053 | RNA | switch of OR |
| BBa_K3328054 | RNA | triggerA of OR |
| BBa_K3328055 | RNA | triggerB of OR |
| BBa_K3328027 | Composite | realize the boolean calculation of OR |
| BBa_K3328028 | Composite | two input trigger of OR logic gate to perform the truth table |
OR Gate
| Part | Type | Description |
|---|---|---|
| BBa_K3328044 | RNA | switch of AND |
| BBa_K3328045 | RNA | triggerA of AND |
| BBa_K3328046 | RNA | triggerB of AND |
| BBa_K3328021 | Composite | realize the boolean calculation of AND |
| BBa_K3328022 | Composite | two input trigger of AND logic gate to perform the truth table |
AND Gate
| Part | Type | Description |
|---|---|---|
| BBa_K3328000 | RNA | an OFF-switch to regulate the expression of the downstream gene |
| BBa_K3328001 | RNA | an OFF-switch to regulate the expression of the downstream gene |
| BBa_K3328002 | RNA | an OFF-switch to regulate the expression of the downstream gene |
| BBa_K3328003 | RNA | an OFF-switch to regulate the expression of the downstream gene |
| BBa_K3328004 | RNA | binding to 3WJ switch(K3328000) to inhibit the expression of the downstream gene (no hairpin) |
| BBa_K3328005 | RNA | binding to 3WJ switch(K3328001) to inhibit the expression of the downstream gene (no hairpin) |
| BBa_K3328006 | RNA | binding to 3WJ switch(K3328002) to inhibit the expression of the downstream gene (no hairpin) |
| BBa_K3328007 | RNA | binding to 3WJ switch(K3328003) to inhibit the expression of the downstream gene (no hairpin) |
NOT Gate
| Part | Type | Description |
|---|---|---|
| BBa_K3328047 | RNA | switch of NIMPLY1 |
| BBa_K3328048 | RNA | triggerA of NIMPLY1 |
| BBa_K3328049 | RNA | triggerB of NIMPLY1 |
| BBa_K3328050 | RNA | switch of NIMPLY2 |
| BBa_K3328051 | RNA | triggerA of NIMPLY2 |
| BBa_K3328052 | RNA | triggerB of NIMPLY2 |
| BBa_K3328023 | Composite | realize the boolean calculation of NIMPLY1 |
| BBa_K3328024 | Composite | two input trigger of NIMPLY1 logic gate to perform the truth table |
| BBa_K3328025 | Composite | realize the boolean calculation of NIMPLY2 |
| BBa_K3328026 | Composite | two input trigger of NIMPLY2 logic gate to perform the truth table |
NIMPLY Gate
| Part | Type | Description |
|---|---|---|
| BBa_K3328038 | RNA | switch of IMPLY1 |
| BBa_K3328039 | RNA | triggerA of IMPLY1 |
| BBa_K3328004 | RNA | triggerB of IMPLY1 |
| BBa_K3328041 | RNA | switch of IMPLY2 |
| BBa_K3328042 | RNA | triggerA of IMPLY2 |
| BBa_K3328004 | RNA | triggerB of IMPLY2 |
| BBa_K3328017 | Composite | realize the boolean calculation of IMPLY1 |
| BBa_K3328018 | Composite | two input trigger of IMPLY1 logic gate to perform the truth table |
| BBa_K3328019 | Composite | realize the boolean calculation of IMPLY2 |
| BBa_K3328020 | Composite | two input trigger of IMPLY2 logic gate to perform the truth table |
IMPLY Gate
| Part | Type | Description |
|---|---|---|
| BBa_K3328032 | RNA | switch of XOR1 |
| BBa_K3328033 | RNA | triggerA of XOR1 |
| BBa_K3328034 | RNA | triggerB of XOR1 |
| BBa_K3328035 | RNA | switch of XOR2 |
| BBa_K3328036 | RNA | triggerA of XOR2 |
| BBa_K3328037 | RNA | triggerB of XOR2 |
| BBa_K3328013 | Composite | realize the boolean calculation of XOR1 |
| BBa_K3328014 | Composite | two input trigger of XOR1 logic gate to perform the truth table |
| BBa_K3328015 | Composite | realize the boolean calculation of XOR2 |
| BBa_K3328016 | Composite | two input trigger of XOR2 logic gate to perform the truth table |
XOR Gate
1.4 New Date For Existing Parts
In many situations，the inducible promoters with low transcriptional leakage and high dynamic range are very important. Since the project needs, we tested a lot of inducible promoters. After a series of experimental tests, we selected Tet promoter and Lux promoter with good performance for our project. And this data is also useful for future teams.
|
|
Figure 1. The fluorescence data of promoters
(A) The Tet promoter fluorescence data in E. coli BL21 (DE3) at different aTc concentrations (0mg/mL, 0.01mg/mL, 0.1mg/mL, 0.25mg/mL, 0.5mg/mL and 1mg/mL). The Tet promoter showed the highest fluorescence intensity when the aTc concentration was 0.25mg/mL. (B) The Lux promoter fluorescence data in E. coli BL21 (DE3) at different HSL (N-(Ketocaproyl)-L-homoserine Lactone) concentrations (0mg/mL, 0.001mg/mL, 0.002mg/mL, 0.01mg/mL, 0.1mg/mL and 1mg/mL). The Lux promoter showed the highest fluorescence intensity when the HSL concentration was 0.01mg/mL. Error bar: SD (n=9).
| Part | Type | Description |
|---|---|---|
| BBa_K2904111 | Regulatory | Tet promoter |
| BBa_R0062 | Regulatory | Lux promoter |
1.5 Reporter
GFPmut3b protein with ASV-tagged whose half-life is about 110 minutes.
| Part | Type | Description |
|---|---|---|
| BBa_K3328031 | Reporter | GFPmut3b-ASV |
2. Improvement
We hope that the logic gates and some of the basics we designed will be widely used. Therefore, we add these structures after the promoter to assemble them into the entire circuit.
For example, we added 3WJ to the T7 promoter (BBa_K2150031) committed by the 2016 UCAS team. Our new part (BBa_K3328000) is an OFF-switch to regulate the expression of the downstream gene. This design changes the original functionality of the T7 promoter and gives it new features. In addition, it is an integral part of NOT and IMPLY boolean calculation.
2.1 Lower Leakage And Higher ON/OFF Ratio
Our experiments were done with both the improved part (BBa_K3328000) and the original part (BBa_K2150031) as a control. Our part has lower leakage and a higher ON/OFF ratio than the T7 promoter.
Figure 2. The comparison between T7 promoter and 3WJ switch1
In the blank control (IPTG=0 M), the fluorescence of T7 promoter (BBa_K2150031) is higher than 3WJ Switch1 (BBa_K3328000). This indicates that our part (BBa_K3328000) has the advantage of low leakage. When the inducer is added (IPTG=0.1 M), 3WJ Switch1 showed a high ON/OFF ratio of up to 15-fold, and the T7 promoter has the ON/OFF ratio of only 3-fold.
2.2 New Functions
Our part can be turned off by the trigger. In addition, it is an integral part of NOT and IMPLY boolean calculation.
2.2.1 The OFF-switch
We chose three-way Junction (3WJ) repressor as the OFF-switch. Three-way junction (3WJ) repressors switch RNA employs an unstable hairpin secondary structure that contains an RBS in the loop region and a start codon in the stem region.
Figure 3. The secondary structure of 3WJ repressor
Despite its high secondary structure, this unstable hairpin was previously demonstrated to be translationally active in toehold switch mRNA sensors. However, When the trigger RNA is expressed, the trigger will bind to the switch RNA. The resulting trigger–switch complex has a stable 3WJ structure that effectively sequesters the RBS and start codon within the loop and stem of the switch RNA, respectively, and strongly represses translation.
Figure 4. Demonstration of 3WJ repressor
Figure 5. The inhibitory effect of 3WJ repressors
In the validation of 3WJ, as with toehold, we also set blank control (IPTG=0 M, aTc=0 mg/mL). Compared with the group without trigger expression (IPTG=0.1 M, aTc=0 mg/mL), the group with trigger expression (IPTG=0.1 M, aTc=0.25 mg/mL) showed inhibitory effect. Error bar: SD (n=9).
Figure 6. Orthogonality of 3WJ repressors
Crosstalk was determined by dividing the arithmetic mean of the GFP fluorescence from a given trigger switch pair by the arithmetic mean of the GFP fluorescence for the cognate trigger switch interaction. GFP fluorescence was measured from n=9 biologically independent samples.
2.2.2 NOT Gate
We used 3WJ repressor to build NOT gate. Its unstable hairpin was previously demonstrated to be translationally active. When a complementary trigger RNA is expressed, the trigger will bind to the switch RNA, making the originally unstable 3WJ structure stable, and represses translation.
Figure 7. Demonstration of NOT gate
Figure 8. Two-input 3WJ repressor NOT gate
The left figure shows that when INPUT=0, the fluorescence of GFP is high. And the fluorescence intensity of GFP was low when INPUT=1. This corresponds to the situation described in the truth table on the right. INPUT=1 means that aTc (0.25 mg/mL) is added. Error bar: SD (n=9).
2.2.3 IMPLY Gate
We combined the 3WJ switch and toehold switch to realize the IMPLY Boolean calculation.
Figure 9. The secondary structure of the switch
When no trigger is expressed, this logic gate just likes a 3WJ switch. When trigger A expressed, the trigger will bind to the switch RNA. The binding allows for a branch migration process, exposing AUG and RBS for translation initiation. When trigger B expressed, the trigger will bind to 3WJ switch RNA. The resulting trigger–switch complex has a stable 3WJ structure that effectively sequesters the RBS and start codon within the loop and stem of the switch RNA, respectively, and strongly represses translation. When trigger A and B both expressed, the binding of trigger RNA to the toehold sequence allows the ribosome to bind to the former RBS and break open the 3WJ stable hairpin.
Figure 10. Demonstration of IMPLY gate
Figure 11. Two-input toehold and 3WJ repressor IMPLY gate
The left figure shows that when INPUT A=0, INPUT B=1, the fluorescence of GFP is low. And the fluorescence intensity of GFP was high in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/mL) is added, INPUT B=1 means that HSL (0.1 mg/mL) is added. Error bar: SD (n=9).