Team:OUC-China/Basic Part

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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” )




Part
Characteristic
Description
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”.
Part
Characteristic
Description
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.
Part
Characteristic
Description
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.
Part
Characteristic
Description
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.