Home | HuBu-4-CHN - iGEM 2024

1. Overview

In order to construct an efficient OMVs gene editing target delivery platform, our team devised a series of fundamental and composite components to accomplish the corresponding functionalities. Leveraging the functions of these designed fundamental components, we progressively assembled and engineered them into composite parts. Our part designs were iteratively refined throughout the project's progression. (know more about our Contribution of parts)

Composite parts are included in the following design paths:

1. The design of path 1 for membrane surface display (refer to Engineering for details)
BBa_K5382120 InaK-linker-Im7
BBa_K5382130 CL7-linker-sfGFP

2. The design of path 2 for membrane surface display (refer to Engineering for details)
BBa_K5382110 InaK-linker-z domain
BBa_K5382160 InaK-linker-4z domain
3. Cas9 RNP self-assembly
BBa_K5382150 Cas9 ribonucleoproteins (gRNA PRDX4)

Our parts synergistically collaborate to accomplish the functionality of OMVs, which are designed for precise delivery of Cas9 RNP. All our registered components strictly adhere to the standardization criteria outlined in RFC [10] or RFC [1000]. Furthermore, we have examined these components and uploaded pertinent experimental data as reliable references, aiming to enhance accessibility and proficiency in utilizing these resources.

1. InaK-linker-z domain

Basic parts	Name	Description	Type	Length
BBa_K5382100	T7 promoter	The T7 promoter is a DNA sequence recognized and bound by T7 RNA polymerase, initiating transcription.	Regulatory	19 bp
BBa_K5382101	lac operator	*The lac operator is a DNA sequence in the E. coli* lactose operon that binds to the lac repressor protein.**	Coding	25 bp
BBa_K5382102	RBS	Efficient ribosome binding site	RBS	23 bp
BBa_K5382103	InaK-ice nucleoprotein	The InaK is an ice nucleoprotein found in Pseudomonas syringae KCTC1832, which promotes the formation of ice crystals in organisms. Its N-terminal domain acts as an anchor moda, tightly attaching to the surface of E. coli cells for surface display.	Coding	1353 bp
BBa_K5382105	GS linker-short peptide	The GS linker is made up of Gly and Ser.	Coding	6 bp
BBa_K5382106	z domain-protein domain	*The z domain is a synthetic protein that binds to the Fc region of IgG. In this study, it is displayed on E. coli* cells to bind antibodies.**	Coding	174 bp
BBa_K5382107	*6His-affinity tag**	*The 6His affinity tag is used as a label for nickel column elution purification in protein purification.**	Tag	18 bp
BBa_K5382108	T7 terminator	The T7 terminator is a DNA sequence that controls gene expression by instructing the T7 RNA polymerase to halt transcription.	Terminator	48 bp
Composite part	Name	Description	Type	Length
BBa_K5382110	InaK-linker-z domain_protein domain that have the ability to bind to lgG	Click the Parts link on the left for a detailed explanation of composite parts.	Composite	1722 bp

2. InaK-linker-4z domain

Basic parts	Name	Description	Type	Length
BBa_K5382100	T7 promoter	The T7 promoter is a DNA sequence recognized and bound by T7 RNA polymerase, initiating transcription.	Regulatory	19 bp
BBa_K5382101	lac operator	The lac operator is a DNA sequence in the E. coli lactose operon that binds to the lac repressor protein.	Coding	25 bp
BBa_K5382102	RBS	Efficient ribosome binding site	RBS	23 bp
BBa_K5382103	InaK-ice nucleoprotein	The InaK is an ice nucleoprotein found in Pseudomonas syringae KCTC1832, which promotes the formation of ice crystals in organisms. Its N-terminal domain acts as an anchor moda, tightly attaching to the surface of E. coli cells for surface display.	Coding	1353 bp
BBa_K5382105	GS linker-short peptide	The GS linker is made up of Gly and Ser.	Coding	6 bp
BBa_K5382151	4z domain-protein domain	The 4z domain, a tetramer of the z domain, can be surface displayed on the surface of E. coli cell to bind antibodies.	Coding	801 bp
BBa_K5382107	6*His-affinity tag	The 6*His affinity tag is used as a label for nickel column elution purification in protein purification.	Tag	18 bp
BBa_K5382108	T7 terminator	The T7 terminator is a DNA sequence that controls gene expression by instructing the T7 RNA polymerase to halt transcription.	Terminator	48 bp
Composite part	Name	Description	Type	Length
BBa_K5382160	InaK-linker-4z domain_protein domain that have the ability to bind to lgG	Click the Parts link on the left for a detailed explanation of composite parts.	Composite	2347 bp

3. InaK-linker-Im7

Basic parts	Name	Description	Type	Length
BBa_K5382100	T7 promoter	The T7 promoter is a DNA sequence recognized and bound by T7 RNA polymerase, initiating transcription.	Regulatory	19 bp
BBa_K5382101	lac operator	The lac operator is a DNA sequence in the E. coli lactose operon that binds to the lac repressor protein.	Coding	25 bp
BBa_K5382102	RBS	Efficient ribosome binding site	RBS	23 bp
BBa_K5382103	InaK-ice nucleoprotein	The InaK is an ice nucleoprotein found in Pseudomonas syringae KCTC1832, which promotes the formation of ice crystals in organisms. Its N-terminal domain acts as an anchor moda, tightly attaching to the surface of E. coli cells for surface display.	Coding	1353 bp
BBa_K5382112	GS linker-short peptide	The GS linker, composed of Gly and Ser, connects InaK and Im7 herein.	Coding	12 bp
BBa_K5382113	Im7-an immune protein with high affinity to CL7	The Im7 is an immune protein with a high affinity for CL7 (an engineered variant of colicin CE7).	Coding	261 bp
BBa_K5382114	HA tag-a tag for protein label	The HA (Hemagglutinin) tag is commonly used to label proteins.	Tag	27 bp
BBa_K5382107	6*His-affinity tag	The 6*His affinity tag is used as a label for nickel column elution purification in protein purification.	Tag	18 bp
BBa_K5382108	T7 terminator	The T7 terminator is a DNA sequence that controls gene expression by instructing the T7 RNA polymerase to halt transcription.	Terminator	48 bp
Composite part	Name	Description	Type	Length
BBa_K5382120	InaK-linker-Im7_Cell membrane anchoring protein and immune protein complex	Click the Parts link on the left for a detailed explanation of composite parts.	Composite	1849 bp

4. CL7-linker-sfGFP

Basic parts	Name	Description	Type	Length
BBa_K5382100	T7 promoter	The T7 promoter is a DNA sequence recognized and bound by T7 RNA polymerase, initiating transcription.	Regulatory	19 bp
BBa_K5382124	CL7-protein tag	The CL7 protein tag is derived from E. coli CE7 and has been engineered to remove DNA binding and catalytic activity, while still retaining its strong affinity for the inhibitory protein Im7. This makes CL7 a valuable tool for protein purification and molecular biology applications.	Coding	393 bp
BBa_K5382121	GS linker-short peptide	The GS linker is a short peptide sequence used to link CL7 and sfGFP.	Coding	30 bp
BBa_K5382122	HRV 3C site-enzyme cleavage site	The HRV 3C protease is commonly used in biotechnology to specifically recognize and cut the amino acid sequence Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro, removing the affinity label on fusion proteins.	Coding	24 bp
BBa_K5382123	sfGFP-superfolder green fluorescent protein	The sfGFP, also known as Superfolder GFP, is a genetically modified version of the green fluorescent protein (GFP).	Coding	717 bp
BBa_K5382107	6*His-affinity tag	The 6*His affinity tag is used as a label for nickel column elution purification in protein purification.	Tag	18 bp
BBa_K5382108	T7 terminator	The T7 terminator is a DNA sequence that controls gene expression by instructing the T7 RNA polymerase to halt transcription.	Terminator	48 bp
Composite part	Name	Description	Type	Length
BBa_K5382130	CL7-linker-sfGFP_Green fluorescent protein and CL7 complex	Click the Parts link on the left for a detailed explanation of composite parts.	Composite	1295 bp

5. Cas9 ribonucleoproteins (gRNA PRDX4)

Basic parts	Name	Description	Type	Length
BBa_K5382131	Tac promoter	The Tac promoter, a robust promoter in E. coli, combines sequences from a trp promoter and a lacUV5 promoter to enhance transcriptional efficiency and allow regulation by lac repressors.	Regulatory	29 bp
BBa_K5382101	lac operator	The lac operator is a DNA sequence in the E. coli lactose operon that binds to the lac repressor protein.	Coding	25 bp
BBa_K5382133	CRISPR-associated protein 9(Cas9)-an enzyme used for gene editing	The CRISPR-associated protein 9 (Cas9) is an enzyme used for precise gene editing.	Coding	4106 bp
BBa_K5382107	6*His-affinity tag	The 6*His affinity tag is used as a label for nickel column elution purification in protein purification.	Tag	18 bp
BBa_K5382134	3'UTR-3' untranslated region	In eukaryotes, the 3'UTR contains a signal for polyadenylation of mRNA's 3' end, which is crucial for mRNA transport, stability, and translation regulation.	Signalling	145 bp
BBa_K5382100	T7 promoter	The T7 promoter is a DNA sequence recognized and bound by T7 RNA polymerase, initiating transcription.	Regulatory	19 bp
BBa_K5382141	PRDX4 gRNA(crRNA)-CRISPR RNA	The CRISPR RNA (crRNA) in the CRISPR-Cas system specifies the genomic target for Cas9 nuclease cleavage.	Coding	20 bp
BBa_K5382136	Cas9 handle-reverse repeating sequence in gRNA	The Cas9 handle, specifically the stem-loop sequence of the guide RNA (gRNA), refers to a palindromic sequence that can fold back upon itself to form a stem-loop configuration due to hydrogen bonding between complementary nucleotide bases.	Coding	42 bp
BBa_K5382137	S.pyogenes terminator-terminator sequence	The S.pyogenes terminator, derived from Streptococcus pyogenes, ensures stable expression and prevents degradation of the sgRNA within the cell when added to its 3' end during construction.	Terminator	34 bp
BBa_K5382108	T7 terminator	The T7 terminator is a DNA sequence that controls gene expression by instructing the T7 RNA polymerase to halt transcription.	Terminator	48 bp
Composite part	Name	Description	Type	Length
BBa_K5382150	Cas9 ribonucleoproteins (gRNA PRDX4)-Co-expression and self-assembly of RNPs	Click the Parts link on the left for a detailed explanation of composite parts.	Composite	4562 bp

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