Basic parts

Role	Part type	BioBrick
T7 promoter	Regulatory	BBa_K4140000
TyrR	Coding	BBa_K4140001
TyrR RBS	RBS	BBa_K4140002
ParoF promoter	Regulatory	BBa_K4140003
P2A linker	coding	BBa_K4140004
L7AE	Coding	BBa_K4140005
TyrP promoter	Regulatory	BBa_K4140006
KP-SP	Tag	BBa_K4140007
lacZ alpha	Coding	BBa_K4140008
Phenylalanine hydroxylase (PAH)	Coding	BBa_K4140009
Permease	Coding	BBa_K4140010
Lac promoter	regulatory	BBa_K4140011
Human U6 promoter	regulatory	BBa_K4140012
VSVg-Fusogen	Coding	BBa_K4140013
glpD	Reporter	BBa_K4140014
Kink turn	RNA	BBa_K4140015
Cas12g	coding	BBa_K4140016
CMV promoter	Regulatory	BBa_K4140017
Phenylalanine aptamer 1	DNA	BBa_K4140018
Phenylalanine aptamer 2	DNA	BBa_K4140019
Phenylalanine aptamer 3	DNA	BBa_K4140020
AcrIIA5 v2 anti-CRISPR	Coding	BBa_K4140021
Peg10	coding	BBa_K4140022

Composite Parts

Role	Part type	BioBrick
BBa_K4140023	Device	Sensing device
BBa_K4140024	Reporter	Reporter device
BBa_K4140025	Composite	PAH releasing device
BBa_K4140026	Device	Regulatory device
BBa_K4140027	Composite	Permeability device
BBa_K4140028	Device	SEND delivery device

Characterization

Characterization

Our Part

The Characterized Part

Experimental characterization, Literature characterization, characterization of mutational landscape, and characterizatoin by mathematical modeling

BBa_K4140001

BBa_K2981004

Experimental characterization, characterization of mutational landscape, characterization by mathematical modeling, and Literature characterization

BBa_K4140009

BBa_K200008

Experimental characterization, characterization by mathematical modeling and literature characterization

BBa_K4140005

BBa_K3743006

Experimental characterization, characterization by mathematical modeling and literature characterization

BBa_K4140006

BBa_K2981003

Experimental characterization and characterization by mathematical modeling

BBa_K4140007

BBa_K2981006

Experimental characterization and Literature characterization

BBa_K4140017

BBa_K1861303

Experimental characterization and Literature characterization

BBa_K4140000

BBa_R0085

Experimental characterization and characterization by mathematical modeling

BBa_K4140002

BBa_K3585000

Experimental characterization and characterization by mathematical modeling

BBa_K4140003

BBa_K2981002

Improvements

The improvement

our part

The improved part

This year, we enhanced the LacZ alpha gene by incorporating a peptide signal that controls the secretion of B galactosidase extracellularly (KP-SP tagging) in an effort to improve the cleavage capacity of the X gal, heighten the intensity of the dark blue color emitted by the X gal product, and reduce the amount of time required to receive the results after performing the test on the chip.

BBa_K4140008

BBa_E0033

This year we employed this approach in our circuit as a regulatory domain, as we took advantage of Cas12g properties to control the expression of PAH in our therapeutic circuit as it differs from other Cas12 proteins by targeting a single-strand RNA substrate making it a potent platform for post-transcriptional modification, and reduces the chance of gene knockdown as it inhibits translation not the transcription by nucleotide deletion like other Cas protein due to inactivation of its NUC domain which mediates the nuclease activity of Cas protein. We carried out an improvement to its functionality limiting its off-targeting effect by adding a kink turn upstream to the 5� end of its mRNA (translation initiation site) as shown in figure 1, which acts as a translation switch for Cas12g by repressing its synthesis in the presence of L7Ae due to the formation of a crystal structure composed of L7Ae-boxC/D k-turn complex which inhibits ribosomal function on the mRNA of Cas12g but in the absence of L7Ae protein Cas12g is freely expressed. The presence or lack of L7Ae is correlated with the concentration of phenylalanine since TyrR dimer, ATP, and the paroF promoter are required for L7Ae's production.