Team:OUC-China/Parts
This year, OUC-China constructed many well-characterized parts based on our standardized design principle. We hope our parts will make it easier and more efficient for synthetic biologists and future iGEM teams to get the predictable results during using riboswitch. Our parts include six different Tuners so that they can be easily incorporated into a user circuit of interest when constructing modular riboswitch. We also include many modular riboswitches by utilizing four kinds of riboswitches. Additionally, many composite parts was constructed to verify our design principle of modular riboswitch.(named “RiboLegoes” )
| K2904000 | Regulatory | Tuner A |
| K2904001 | Regulatory | Tuner B |
| K2904002 | Regulatory | Tuner C |
| K2904003 | Regulatory | Tuner D |
| K2904004 | Regulatory | Tuner E |
| K2904005 | Regulatory | Tuner S |
Tuners
Based on the previous attenuator design, we designed six Tuners. Each Tuner confers a distinct level of riboswitch regulation; Tuners are lettered (A-E) in order of their capability of regulating riboswitch response curve, from strongest to weakest. Besides, a special Tuner was also designed to degrade Stabilizer, reducing the metabolic burden of cells. These basic parts should allow future teams to efficiently construct more “RiboLegoes”.
Based on the previous attenuator design, we designed six Tuners. Each Tuner confers a distinct level of riboswitch regulation; Tuners are lettered (A-E) in order of their capability of regulating riboswitch response curve, from strongest to weakest. Besides, a special Tuner was also designed to degrade Stabilizer, reducing the metabolic burden of cells. These basic parts should allow future teams to efficiently construct more “RiboLegoes”.
| K2904010 | Regulatory | Modular Adda riboswitch containing Tuner A |
| K2904011 | Regulatory | Modular Adda riboswitch containing Tuner B |
| K2904012 | Regulatory | Modular Adda riboswitch containing Tuner C |
| K2904013 | Regulatory | Modular Adda riboswitch containing Tuner D |
| K2904014 | Regulatory | Modular Adda riboswitch containing Tuner E |
| K2904015 | Regulatory | Modular Adda riboswitch containing a different source of Stabilizer and Tuner B |
| K2904020 | Regulatory | Modular Btub riboswitch containing Tuner A |
| K2904021 | Regulatory | Modular Btub riboswitch containing Tuner E |
| K2904022 | Regulatory | Modular Btub riboswitch containing Tuner S |
| K2904030 | Regulatory | Modular Four U riboswitch |
| K2904040 | Regulatory | Modular cobalamin riboswitch |
Modular riboswitches
Our next collection of parts consist of many modular riboswitches. First, we employing Adda riboswitch to construct modular Adda riboswitches by utilizing different Tuners respectively. Then we selected three typical Tuners to design modular Btub riboswitch based on the original Btub riboswitch. An improved cobalamin riboswitch was constructed to verify our design principle. Finally, one thermodynamic switch was also chosen to engineer modular riboswitch.
Our next collection of parts consist of many modular riboswitches. First, we employing Adda riboswitch to construct modular Adda riboswitches by utilizing different Tuners respectively. Then we selected three typical Tuners to design modular Btub riboswitch based on the original Btub riboswitch. An improved cobalamin riboswitch was constructed to verify our design principle. Finally, one thermodynamic switch was also chosen to engineer modular riboswitch.
| K2904050 | Device | aTc inducible sfGFP regulated by modular Adda riboswitch containing Tuner A |
| K2904051 | Device | aTc inducible sfGFP regulated by modular Adda riboswitch containing Tuner B |
| K2904052 | Device | aTc inducible sfGFP regulated by modular Adda riboswitch containing Tuner C |
| K2904053 | Device | aTc inducible sfGFP regulated by modular Adda riboswitch containing Tuner D |
| K2904054 | Device | aTc inducible sfGFP regulated by modular Adda riboswitch containing Tuner E |
| K2904055 | Device | aTc inducible sfGFP regulated by modular Adda riboswitch containing a different source Stabilizer |
| K2904022 | Device | aTc inducible sfGFP regulated by modular Btub riboswitch containing Tuner A |
| K2904030 | Device | aTc inducible sfGFP regulated by modular Btub riboswitch containing Tuner E |
| K2904040 | Device | aTc inducible sfGFP regulated by modular Btub riboswitch containing Tuner S |
Reporter gene regulated by modular riboswitch
To demonstrate that our modular riboswitch system can work with different proteins, we constructed this collection. Different riboswitches was utilized, including three kinetic switches: Adda riboswitch, Btub riboswitch, cobalamin biosensor, and one thermodynamic switch: Four U riboswitch. What’s more, we used three different kinds of GOI: sfGFP, mRFP1 and EYFP.
To demonstrate that our modular riboswitch system can work with different proteins, we constructed this collection. Different riboswitches was utilized, including three kinetic switches: Adda riboswitch, Btub riboswitch, cobalamin biosensor, and one thermodynamic switch: Four U riboswitch. What’s more, we used three different kinds of GOI: sfGFP, mRFP1 and EYFP.
| K2904020 | Composite | sfGFP regulated by modular Adda riboswitch containing STA81 and Tuner A |
| K2904021 | Composite | sfGFP regulated by modular Adda riboswitch containing STA129 and Tuner A |
| K2904092 | Composite | sfGFP regulated by modular Four U riboswitch |
| K2904100 | Measurement | Improved vitamin B12 biosensor, composed by modular cobalamin riboswitch in front of an mRFP1 |
| K2904120 | Composite | sfGFP regulated by modular Adda riboswitch containing Tuner A |
| K2904121 | Composite | sfGFP regulated by modular Adda riboswitch containing Tuner B |
| K2904122 | Composite | sfGFP regulated by modular Adda riboswitch containing Tuner C |
| K2904123 | Composite | sfGFP regulated by modular Adda riboswitch containing Tuner D |
| K2904124 | Composite | sfGFP regulated by modular Adda riboswitch containing Tuner E |
| K2904125 | Composite | sfGFP regulated by modular Adda riboswitch containing a different source of Stabilizer and Tuner B |
| K2904126 | Composite | EYFP regulated by modular Adda riboswitch containing Tuner A |
| K2904130 | Composite | sfGFP regulated by modular Btub riboswitch containing Tuner A |
| K2904131 | Composite | sfGFP regulated by modular Btub riboswitch containing Tuner E |
| K2904132 | Composite | sfGFP regulated by modular Btub riboswitch containing Tuner S |
aTc inducible sfGFP regulated by modular riboswitch.
We further include a collection of aTc-inducible sfGFP reporter constructs regulated by modular riboswitch, all under the control of the Ptet promoter. These parts allowed us to characterize the response curve of modular riboswitch. We successfully demonstrated distinct expression levels of sfGFP by changing different Tuners or riboswitches.
We further include a collection of aTc-inducible sfGFP reporter constructs regulated by modular riboswitch, all under the control of the Ptet promoter. These parts allowed us to characterize the response curve of modular riboswitch. We successfully demonstrated distinct expression levels of sfGFP by changing different Tuners or riboswitches.
| K2904140 | RNA | asRNA helping modular Adda riboswitch turn off gene expression |
| K2904141 | RNA | asRNA helping modular Adda riboswitch turn off gene expression |
| K2904142 | RNA | asRNA helping modular Btub riboswitch turn on gene expression |
asRNA
To control the riboswitches without medium replacement for removing the inducer, we used the model to design different asRNAs which target different regions to activate or deactivate the riboswitch. These parts can achieve these functionality that expand another method to regulate on-off state of riboswitch besides the ligand.
To control the riboswitches without medium replacement for removing the inducer, we used the model to design different asRNAs which target different regions to activate or deactivate the riboswitch. These parts can achieve these functionality that expand another method to regulate on-off state of riboswitch besides the ligand.
