PART COLLECTION

OVERVIEW

Our project is based on an excellent light control system that can convert light and sound signals into chemical signals, and finally produce scents and colors. The system consists of 4 modules, “sensor array”, circuit, “resource allocator” and actuators.

Firstly, the “sensor array” module combines 3 light sensors, Cph8* (asterisk), YF1 and CcaSR, which could be activated predominantly by red, blue and green light. These optogenetic tools enables our E. coli to response to light of different wavelengths respectively. Cph8 is a chimeric histidine kinase that is switched on by infrared (705 nm) light and off by red (650 nm) light. So it can be used as a red light sensor. CcaSR is a green-light sensor based on the membrane-associated histidine kinase CcaS and its response regulator CcaR, which can be switched on by green (535 nm) light, inducing the promoter PcpcG2-172 (BBa_K592003) and off by far-red (672 nm) light. YF1 is a fusion protein of YtvA (B subtilis) and FixL (B japonicum) that can sense blue light. The fixJ is the wild-type response regulator to YF1. They can form a blue light sensor and transfer the signal to next module.

Secondly, the circuit module has two NOT gates to invert signals from red and blue light sensor, in which two repressors CI and PhIF can be expressed respectively activated by red and blue input to switch off corresponding red and blue output promoters.

Next, the “resource allocator” module, connects the input with the output. The resource-allocation system is a split-phage RNA polymerase system, enabling signals easily connected to the circuit outputs. The resource-allocation system also allocate the resource and stabilize the transcription level in the cell. In the system, three well orthogonal sigma fragments which has specific DNA binding domain are expressed under the control of 3 sensors. And a core fragment is expressed by a constitutive promoter. Only when a core fragment conjugate with specific sigma fragment to form a full-functional RNA polymerase, then the transcription of corresponding output gene is initiated. In this way, we not only keep the proportion of the response product precisely through the compete among 3 sigma fragments but reduce the metabolic burden of E. coli and make the whole system work more orderly. Here we sincerely appreciate Dr. Christopher Voigt’s laboratory that created this system, and sent us plasmids to support our work this year.

Last, the “actuator” module implement the biological functions that are the outputs of the RGB sysytem. There are three output genes responding to three light sensors activated by different wavelength of light. In order to implement customized outcome, theoretically, you can change them into any genes you want.

To make it work more well, ribozyme-based insulators and 18 strong synthetic terminators are also added to the whole system. We collected all these parts together.

PARTS OF SENSOR

Type	Parts Name	Basic/Composite	Number	Description
CDS	Cph8*	basic	BBa_K2598006	Cph8 is a chimeric histidine kinase that is switched on by infrared (705 nm) light and off by red (650 nm) light. So it can be used as a red light sensor. Here is promoter+Cph8+terminator
CDS	CcaSR	basic	BBa_K2598005	This part is a green-light sensor based on the membrane-associated histidine kinase CcaS and its response regulator CcaR, which can be switched on by green (535 nm) light, inducing the promoter PcpcG2-172 (BBa_K592003) and off by far-red (672 nm) light. So it can be used as a green light sensor
CDS	CcaSR	composite	BBa_K2598003	promoter+CcaSR+terminator
CDS	YF1+fixJ	composite	BBa_K2598009	YF1 is a fusion protein of YtvA (B subtilis) and FixL (B japonicum) that can sense blue light. fixJ is the wild-type response regulator to YF1. They can form a blue light sensor and transfer the signal to next module
CDS	ho1+pcyA	composite	BBa_K2598018	ho1 oxidizes the heme group using a ferredoxin cofactor, generating biliverdin Ixalpha and pcyA converts biliverdin IXalpha (BV) to phycocyanobilin (PCB), the immediate precursor of cyanobacterial phytochromes, which achieve phycocyanobilin biosynthetic process. Here is promoter+ho1+pcyA+terminator
insulator	BydvJ	basic	BBa_K2598014	ribozyme-based insulator
insulator	RiboJ	basic	BBa_K2598010	ribozyme-based insulator
promoter	laclq35	basic	BBa_K2598007	a promoter promotes expression of YF1 and fixJ
whole plasmid	pJFR1 (KX011464)	composite	BBa_K2598049	This plasmid codes three light sensors, Cph8*, CcaSR and YF1, under the promoters of J23106, J23108 and laclq35 to sense red, green and blue light respectively

PARTS OF CIRCUIT

Type	Parts Name	Basic/Composite	Number	Description
repressor	PhIF	composite	BBa_K2598016	PhIF is a repressor that can repress promoter, PPhIF(BBa_K1725000), of blue light output under promoter PfixK2 which can be activated by fixJ when blue light sensor is activated. Here is promoter+PhIF+terminator
repressor	CI	composite	BBa_K2598021	CI is a repressor that can repress promoter, Pλ(BBa_K1145005), of red light output under promoter Pomp which can be activated by activated red light sensor. Here is promoter+CI+terminator
sensor+repressor	YF1+fixJ+PhlF	composite	BBa_K2598039	blue light sensor YF1+fixJ(regulator to YF1)+repressor PhIF
sensor+repressor	Cph8*+CI	composite	BBa_K2598038	red light sensor composite Cph8*+repressor CI

PARTS OF RNAP

Type	Parts Name	Basic/Composite	Number	Description
sigma fragment	CGG	basic	BBa_K2598011	a T7 RNAP sigma fragment with DNA-binding domain that can be expressed when green light sensors are activated and be able to combine to non-active T7 RNAP core fragment (BBa_K2598001) to form a full-functional RNA polymerase and direct it to specific promoter PCGG(BBa_K2598023)
sigma fragment	CGG	composite	BBa_K2598013	promoter+CGG+terminator
sigma fragment	T3	basic	BBa_K2598015	a T7 RNAP sigma fragment with DNA-binding domain that can be expressed when blue light sensors are activated and be able to combine to non-active T7 RNAP core fragment (BBa_K2598001) to form a full-functional RNA polymerase and direct it to specific promoter PT3(BBa_K2598026)
sigma fragment	T3	composite	BBa_K2598033	promoter+T3+terminator
sigma fragment	K1F	composite	BBa_K2598022	K1F is a T7 RNAP sigma fragment with DNA-binding domain that can be expressed when red light sensors are activated and be able to combine to non-active T7 RNAP core fragment (BBa_K2598001) to form a full-functional RNA polymerase and direct it to specific promoter PK1F(BBa_K2598030). Here is promoter+K1F+terminator
sigma fragment+output	T3+BFP	composite	BBa_K2598040	sigma fragment T3+a blue fluorescent protein BFP
sigma fragment+output	CGG+GFP	composite	BBa_K2598041	sigma fragmentCGG+a green fluorescent protein GFP
sigma fragment+output	K1F+mRFP	composite	BBa_K2598042	sigma fragment K1F+a red fluorescent protein mRFP
sensor+sigma fragment	CcaSR+CGG	composite	BBa_K2598034	green light sensor CcaSR+sigma fragment CGG
core fragment	T7	basic	BBa_K2598000	Non-active T7 RNAP core fragment being able to combine to being able to combine to sigma fragment to form a full-functional RNA polymerase
core fragment	T7	composite	BBa_K2598001	promoter+T7+terminator
promoter	PCGG	basic	BBa_K2598023	a promoter that can be regulated by the full-functional RNA polymerase consisting of T7 RNAP sigma fragment T3 and T7 RNAP core fragment and promotes blue light output
promoter	PT3	basic	BBa_K2598026	a promoter that can be regulated by the full-functional RNA polymerase consisting of T7 RNAP sigma fragment CGG and T7 RNAP core fragment and promotes green light output
promoter	PK1F	basic	BBa_K2598030	a promoter that can be regulated by the full-functional RNA polymerase consisting of T7 RNAP sigma fragment K1F and T7 RNAP core fragment and promotes red light output
teminator	ECK120033737	basic	BBa_K2598002	a strong teminator
teminator	L3S2P11	basic	BBa_K2598004	a strong teminator
teminator	L3S2P55	basic	BBa_K2598008	a strong teminator
teminator	DT25	basic	BBa_K2598012	a strong teminator
teminator	L3S1P22	basic	BBa_K2598017	a strong teminator
teminator	DT11	basic	BBa_K2598020	a strong teminator
teminator	T7	basic	BBa_K2598024	a strong teminator
teminator	DT5	basic	BBa_K2598028	a strong teminator
teminator	DT16	basic	BBa_K2598031	a strong teminator
teminator	L3S3P11	basic	BBa_K2598054	a strong teminator
whole plasmid	pJFR2 (KX011465)	composite	BBa_K2598050	a strong teminator
whole plasmid	pJFR3 (KX011466)	composite	BBa_K2598051	a strong teminator

PARTS OF ACTUATOR