Team:CCU Taiwan/Design - 2021.igem.org

Team:CCU iGEM/Parts

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Biobrick

Purpose: specialized to kill extracellular bacteria

T7
Promoter

RBS

6x His

EK site

CBD

Linker

Thrombin releasable DPK

T7
terminator

T7
Promoter

RBS

6x His

EK site

CBD

Linker

Thrombin releasable D2A

T7
terminator

Purpose: specialized to kill intracellular bacteria

T7
Promoter

RBS

6x His

EK site

CBD

Linker

Thrombin-TAT

CTSS-DPK

T7
terminator

Purpose: To examine the release rate of modified thrombin cleavage sites.

T7
Promoter

RBS

6x His

EK site

CBD

Linker

Thrombin/DPK mimic

eGFP

T7
terminator

T7
Promoter

RBS

6x His

EK site

CBD

Linker

Thrombin/D2A mimic

eGFP

T7
terminator

T7
Promoter

RBS

6x His

EK site

CBD

Linker

Thrombin/TAT mimic

eGFP

T7
terminator

Purpose: To verify our design, removing the first amino acid of TAT, Tyrosine, can increase the probability of Thrombin Cleavage.

T7
promoter

RBS

6x His

CBD

Linker

Thrombin releasable TAT_47-57

eGFP

T7
terminator

T7
promoter

RBS

6x His

CBD

Linker

Thrombin releasable TAT_48-57

eGFP

T7
terminator

Purpose: These biobricks are specialized to examine the cell penetration ability of
TAT_48-57.

T7
promoter

RBS

Thrombin

eGFP

T7
terminator

T7
promoter

RBS

Thrombin

TAT _48-57

eGFP

T7
terminator

T7 promoter

T7 DNA polymerase would recognize DNA sequence of this region and start to transcript.

RBS

Ribosome binding site

Ribosome would recognize the RNA sequence of this region and start to translate.

Thrombin cleavage site

We design LVPR links TAT_47-57 and _48-57 to test the release ability.

6xHis

6xHistidine

The hexa hisitidine-tag for protein purification or recognization.

CBD

Collagen binding domain

CBD is coloned from FN1 genes and is able to bind to collagen.

AMP

Antimicrobial peptides

Short cationic peptides that exhibit antimicrobial activity.

T7 terminator

T7 DNA polymerase would recognize the DNA sequence of this region and stop the transcript.

LVPRGK/LVPRFA/
LVPRGR

The modified thrombin cleavage site. The amino acids at P1 and P2' positions of thrombin cleavage site are modified to fit the first two amino acids at N-terminal of selected AMPs or TAT_48-57

eGFP

enhanced green fluorescent protein

The enhanced green fluorescent protein

Linker

Linker provides the space to allow thrombin docking and cleavage.

EK site

Enterokinase
cleavage site

Enterokinase can recognize the amino acid sequence of this region to purify our target protein.

Thrombin cleavage site

Thrombin cleavage site can be recognized and cut by thrombins in the wounds in order to release AMP. Moreover, in a normal Thrombin site, the sequence is LVPRGS. After the enzyme reaction, Glycine and Serine will be remained, which have the possibility of affecting AMP’s function. To prevent it from happening, we altered the sequence to LVPR. After a few experiments, it was proof that the alteration won’t affect the function of thrombin.

Thrombin releasable DPK-060

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by G and K to fit the first two amino acids at the N-terminal of DPK-060.

Thrombin releasable D2A21

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by F and A to fit the first two amino acids at the N-terminal of D2A21.

Thrombin releasable TAT_48-57

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by G and R to fit the first two amino acids at the N-terminal of TAT_48-57. TAT_48-57 is small peptides that can carry cargoes into macrophages.

Thrombin DPKmimic

The modified thrombin cleavage site LVPRGK was linked at the N-terminal of eGFP.

Thrombin D2Amimic

The modified thrombin cleavage site LVPRFA was linked at the N-terminal of eGFP.

Thrombin TATmimic

The modified thrombin cleavage site LVPRGR was linked at the N-terminal of eGFP.

Thrombin releasable TAT_47-57

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by Y and G to fit the first two amino acids at the N-terminal of TAT_47-57.

Thrombin releasable TAT_48-57

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by G and R to fit the first two amino acids at the N-terminal of TAT_48-57.

Cathepsin S site

Cathepsin S cleavage site can be recognized and cleaved by the lysosomal endopeptidase CTSS.

CTSS releasable DPK060

CTSS cleavage site can be recognized and cleaved by Cathepsin S in phagolysosomes. The amino acids at the P1’ and P2’ sites of CTSS stie cleavage site were replaced by G and K to fit the two amino acids at the N-terminal of DPK060.

TAT_48-57

TAT_48-57 is small peptides that can carry cargoes into macrophages.

Basic part

Part name	Description	Length(bp)
BBa_K3778012	T7 promoter	19
BBa_K3778013	RBS (Ribosome-binding site)	23
BBa_K3778014	6xHis	18
BBa_K3778015	Enterokinase cleavage site	24
BBa_K3778016	CBD (Collagen Binding Domain)	618
BBa_K3778017	Linker	24
BBa_K3778020	eGFP	717
BBa_K3778018	T7 terminator	48
BBa_K3778006	DPK-060	60
BBa_K3778022	Thrombin releasable DPK-060	72
BBa_K3778007	D2A21	69
BBa_K3778023	Thrombin releasable D2A21	81
BBa_K3778010	TAT_48-57	30
BBa_K3778026	Thrombin releasable TAT_48-57	42
BBa_K1202006	TAT(Trans-Activator of Transcription) protein	33
BBa_K3778027	Thrombin releasable TAT	45
BBa_K3778019	Cathepsin S cleavage site	12
BBa_K3778028	Cathepsin S-releasable DPK-060	72
BBa_K3778001	Thrombin cleavage site for DPK-060 mimic release (LVPRGK)	18
BBa_K3778000	Thrombin cleavage site (LVPRGS)	18
BBa_K3778002	Thrombin cleavage site for D2A21 mimic release (LVPRFA)	18
BBa_K3778005	Thrombin cleavage site for TAT_48-57 mimic release (LVPRGR)	18

Composite part

Part name	Description	Length(bp)
BBa_K3778021	T7P-RBS-6xHis-CBD-linker-LVPRGK-eGFP-T7T	1539
BBa_K3778029	T7P-RBS-6xHis-CBD-linker-LVPRFA-eGFP-T7T	1539
BBa_K3778032	T7P-RBS-6xHis-CBD-linker-LVPRGR-eGFP-T7T	1539
BBa_K3778033	T7P-RBS-6xHis-Enterokinase cleavage site-CBD-linker-Thrombin releasable TAT-T7T	875
BBa_K3778034	T7P-RBS-6xHis-Enterokinase cleavage site-CBD-linker-Thrombin releasable TAT_48-57-T7T	872
BBa_K3778035	T7P-RBS-6xHis-Enterokinase cleavage site-CBD-linker-Thrombin releasable DPK-060-T7T	902
BBa_K3778036	T7P-RBS-6xHis-Enterokinase cleavage site-CBD-linker-Thrombin releasable D2A21-T7T	911
BBa_K3778039	Thrombin releasable TAT_48-57-6xHis-Cathepsin S releasable DPK-060	978
BBa_K3778040	T7P-RBS-6xHis-LVPRGS-TAT_48-57-eGFP-T7T	919

Team:CCU Taiwan/Parts

Team:CCU iGEM/Parts

Biobrick

Purpose: specialized to kill extracellular bacteria

Purpose: specialized to kill intracellular bacteria

Purpose: To examine the release rate of modified thrombin cleavage sites.

Purpose: To verify our design, removing the first amino acid of TAT, Tyrosine, can increase the probability of Thrombin Cleavage.

Purpose: These biobricks are specialized to examine the cell penetration ability of TAT48-57.

T7 promoter

T7 DNA polymerase would recognize DNA sequence of this region and start to transcript.

RBS

Ribosome binding site

Ribosome would recognize the RNA sequence of this region and start to translate.

Thrombin cleavage site

We design LVPR links TAT47-57 and 48-57 to test the release ability.

6xHis

6xHistidine

The hexa hisitidine-tag for protein purification or recognization.

CBD

Collagen binding domain

CBD is coloned from FN1 genes and is able to bind to collagen.

AMP

Antimicrobial peptides

Short cationic peptides that exhibit antimicrobial activity.

T7 terminator

T7 DNA polymerase would recognize the DNA sequence of this region and stop the transcript.

LVPRGK/LVPRFA/LVPRGR

The modified thrombin cleavage site. The amino acids at P1 and P2' positions of thrombin cleavage site are modified to fit the first two amino acids at N-terminal of selected AMPs or TAT48-57

eGFP

enhanced green fluorescent protein

The enhanced green fluorescent protein

Linker

Linker provides the space to allow thrombin docking and cleavage.

EK site

Enterokinasecleavage site

Enterokinase can recognize the amino acid sequence of this region to purify our target protein.

Thrombin cleavage site

Thrombin releasable DPK-060

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by G and K to fit the first two amino acids at the N-terminal of DPK-060.

Thrombin releasable D2A21

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by F and A to fit the first two amino acids at the N-terminal of D2A21.

Thrombin releasable TAT48-57

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by G and R to fit the first two amino acids at the N-terminal of TAT48-57. TAT48-57 is small peptides that can carry cargoes into macrophages.

Thrombin DPKmimic

The modified thrombin cleavage site LVPRGK was linked at the N-terminal of eGFP.

Thrombin D2Amimic

The modified thrombin cleavage site LVPRFA was linked at the N-terminal of eGFP.

Thrombin TATmimic

The modified thrombin cleavage site LVPRGR was linked at the N-terminal of eGFP.

Thrombin releasable TAT47-57

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by Y and G to fit the first two amino acids at the N-terminal of TAT47-57.

Thrombin releasable TAT48-57

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by G and R to fit the first two amino acids at the N-terminal of TAT48-57.

Cathepsin S site

Cathepsin S cleavage site can be recognized and cleaved by the lysosomal endopeptidase CTSS.

CTSS releasable DPK060

CTSS cleavage site can be recognized and cleaved by Cathepsin S in phagolysosomes. The amino acids at the P1’ and P2’ sites of CTSS stie cleavage site were replaced by G and K to fit the two amino acids at the N-terminal of DPK060.

TAT48-57

TAT48-57 is small peptides that can carry cargoes into macrophages.

Basic part

Composite part

Purpose: These biobricks are specialized to examine the cell penetration ability of
TAT_48-57.

We design LVPR links TAT_47-57 and _48-57 to test the release ability.

LVPRGK/LVPRFA/
LVPRGR

The modified thrombin cleavage site. The amino acids at P1 and P2' positions of thrombin cleavage site are modified to fit the first two amino acids at N-terminal of selected AMPs or TAT_48-57

Enterokinase
cleavage site

Thrombin releasable TAT_48-57

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by G and R to fit the first two amino acids at the N-terminal of TAT_48-57. TAT_48-57 is small peptides that can carry cargoes into macrophages.

Thrombin releasable TAT_47-57

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by Y and G to fit the first two amino acids at the N-terminal of TAT_47-57.

Thrombin releasable TAT_48-57

The amino acids at the P1’ and P2’ sites of Thrombin cleavage sites were replaced by G and R to fit the first two amino acids at the N-terminal of TAT_48-57.

TAT_48-57

TAT_48-57 is small peptides that can carry cargoes into macrophages.