Team:UPF Barcelona/Part Collection

Team:UPF Barcelona - 2021.igem.org

Parts

In this page we expose a summary of our used Parts, classified in: ARIA basic parts, ARIA composite parts and existing parts.

Here you can find the parts that have made it possible to develop the ARIA project. Most of the basic parts used were found on the iGEM parts registry, with the exception of a total of 5 basic parts that were created and catalogued in the aforementioned registry by ourselves. Also, quite a few composite parts were designed by joining basic parts (existing and ARIA basic parts), all of this with the aim of building the biosensors library.

Part Collection

As already mentioned in the Project section, ARIA makes use of the CRISPR-Cas technology for diagnostic purposes. This way, most of our parts are related to it and created with the aim of constructing this system, and that is the reason why we decided to nest them in a Part collection called CRISPR-Cas for Diagnosis. With the emergence of fast diagnostic procedures based on Cas12 and Cas13, a collection like this one will be useful for future iGEM teams also making use of this system.

Our part collection aims to be easy-to-use and user-friendly, as it is structured and organized, being all the part pages linked between them in a way that an external user could comfortably replicate our design. As for the collection categories, the specifications can be consulted in the Collection page.

Composite part

To center our efforts, we specially focused on modelling and testing one of our composite parts: the complete Efficient gRNA Chloramphenicol construct. All the documentation is available on its parts registry page (BBa_K3791021).

Part improvement

Moreover, as we have used lots of already existing parts, we wanted to make our contribution in one of them. For this, we thought, designed and tested a new perspective for the usage of Protein E, which provokes cell autolysis. For this, we aimed to reduce its leakiness expression and accurately control it, so we used a repressible and inducible promoter. More details are available in both the original (BBa_K2500006) and the new part registry pages (BBa_K3791025).

All this information is better indicated in the tables below:

ARIA BASIC PARTS

Part Code	Name	Function	Part Collection Category
BBa_K3791000	Spacer gRNA Ampicillin	Target a specific DNA fragment of a certain ampicillin-resistance gene.	gRNA Spacer
BBa_K3791001	Spacer gRNA Chloramphenicol	Target a specific DNA fragment of a certain chloramphenicol-resistance gene.	gRNA Spacer
BBa_K3791002	Spacer gRNA Erythromycin	Target a specific DNA fragment of a certain erythromycin-resistance gene.	gRNA Spacer
BBa_K3791003	Spacer gRNA Kanamycin	Target a specific DNA fragment of a certain kanamycin-resistance gene.	gRNA Spacer
BBa_K3791004	Spacer gRNA Spectinomycin	Target a specific DNA fragment of a certain spectinomycin-resistance gene.	gRNA Spacer

Table 1: ARIA basic parts.

ARIA COMPOSITE PARTS

Part Code	Name	Structure	Function	Part Collection Category
BBa_K3791005	gRNA Ampicillin	Repeat + Spacer gRNA Ampicillin	Assembly with functional LbCas12a for DNase activity performance. Specific recognition of target ampicillin-resistance gene.	gRNA
BBa_K3791006	gRNA Chloramphenicol	Repeat + Spacer gRNA Chloramphenicol	Assembly with functional LbCas12a for DNase activity performance. Specific recognition of target chloramphenicol-resistance gene.	gRNA
BBa_K3791007	gRNA Erythromycin	Repeat + Spacer gRNA Erythromycin	Assembly with functional LbCas12a for DNase activity performance. Specific recognition of target erythromycin-resistance gene.	gRNA
BBa_K3791008	gRNA Kanamycin	Repeat + Spacer gRNA Kanamycin	Assembly with functional LbCas12a for DNase activity performance. Specific recognition of target kanamycin-resistance gene.	gRNA
BBa_K3791009	gRNA Spectinomycin	Repeat + Spacer gRNA Spectinomycin	Assembly with functional LbCas12a for DNase activity performance. Specific recognition of target spectinomycin-resistance gene.	gRNA
BBa_K3791010	gRNA Ampicillin construct	T7 promoter + Repeat + Spacer gRNA Ampicillin	gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target ampicillin-resistance gene.	gRNA construct
BBa_K3791011	gRNA Chloramphenicol construct	T7 promoter + Repeat + Spacer gRNA Chloramphenicol	gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target chloramphenicol-resistance gene.	gRNA construct
BBa_K3791012	gRNA Erythromycin construct	T7 promoter + Repeat + Spacer gRNA Erythromycin	gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target erythromycin-resistance gene.	gRNA construct
BBa_K3791013	gRNA Kanamycin construct	T7 promoter + Repeat + Spacer gRNA Kanamycin	gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target kanamycin-resistance gene.	gRNA construct
BBa_K3791014	gRNA Spectinomycin construct	T7 promoter + Repeat + Spacer gRNA Spectinomycin	gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target spectinomycin-resistance gene.	gRNA construct
BBa_K3791015	Efficient gRNA Ampicillin	Repeat + Spacer gRNA Ampicillin + 4 NTs + Repeat	Efficient assembly with functional LbCas12a for DNase activity performance. Specific recognition of target ampicillin-resistance gene.	Efficient gRNA
BBa_K3791016	Efficient gRNA Chloramphenicol	Repeat + Spacer gRNA Chloramphenicol + 4 NTs + Repeat	Efficient assembly with functional LbCas12a for DNase activity performance. Specific recognition of target chloramphenicol-resistance gene.	Efficient gRNA
BBa_K3791017	Efficient gRNA Erythromycin	Repeat + Spacer gRNA Erythromycin + 4 NTs + Repeat	Efficient assembly with functional LbCas12a for DNase activity performance. Specific recognition of target erythromycin-resistance gene.	Efficient gRNA
BBa_K3791018	Efficient gRNA Kanamycin	Repeat + Spacer gRNA Kanamycin + 4 NTs + Repeat	Efficient assembly with functional LbCas12a for DNase activity performance. Specific recognition of target kanamycin-resistance gene.	Efficient gRNA
BBa_K3791019	Efficient gRNA Spectinomycin	Repeat + Spacer gRNA Spectinomycin + 4 NTs + Repeat	Efficient assembly with functional LbCas12a for DNase activity performance. Specific recognition of target spectinomycin-resistance gene.	Efficient gRNA
BBa_K3791020	Efficient gRNA Ampicillin construct	T7 promoter + Repeat + Spacer gRNA Ampicillin + 4 NTs + Repeat + L3S2P21 terminator	Efficient gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target ampicillin-resistance gene.	Efficient gRNA construct
BBa_K3791021	Efficient gRNA Chloramphenicol construct	T7 promoter + Repeat + Spacer gRNA Chloramphenicol + 4 NTs + Repeat + L3S2P21 terminator	Efficient gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target chloramphenicol-resistance gene.	Efficient gRNA construct
BBa_K3791022	Efficient gRNA Erythromycin construct	T7 promoter + Repeat + Spacer gRNA Erythromycin + 4 NTs + Repeat + L3S2P21 terminator	Efficient gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target erythromycin-resistance gene.	Efficient gRNA construct
BBa_K3791023	Efficient gRNA Kanamycin construct	T7 promoter + Repeat + Spacer gRNA Kanamycin + 4 NTs + Repeat + L3S2P21 terminator	Efficient gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target kanamycin-resistance gene.	Efficient gRNA construct
BBa_K3791024	Efficient gRNA Spectinomycin construct	T7 promoter + Repeat + Spacer gRNA Spectinomycin + 4 NTs + Repeat + L3S2P21 terminator	Efficient gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target spectinomycin-resistance gene.	Efficient gRNA construct
BBa_K3791025	Autolysis protein E construct	pBAD-AraC promoter + efficient RBS + autolysis protE + E. coli his operon terminator	Transform it in bacteria to be able to accurately autolysate them avoiding leakiness protein expression.

Table 2: ARIA composite parts.

EXISTING PARTS

Part Code	Name
BBa_K2927005	LbCas12a
BBa_K2927006	guide RNA of LbCas12a
BBa_K1614000	T7 promoter for expression of functional RNA
BBa_K2500006	Protein E: Bacteria-Lysing Protein Encoded by Phage Phi X 17
BBa_K2306014	Efficient RBS from bacteriophage T7 gene 10 and Shine Dalgarno
BBa_I0500	Inducible pBad/araC promoter
BBa_B0053	E. coli his operon terminator
BBa_K731721	T7 terminator
BBa_K2675031	L3S2P21 Terminator

Table 3: Existing parts used.