Team-iGEM SJTU BioX 201

This year we have submitted 25 parts, with 13 basic parts, and 12 composite parts. Don't think we win at quantity, we indeed provided a series of data with high-quality. This page provides an overview of all BioBricks that we have created and submitted to the iGEM Registry of Standard Biological Parts. For more detailed information and characterization of these parts, use the links below to reach the Registry page of each BioBrick.

【favorite basic part】

The cassette consists of 69 bp of the respective leader, including the sequence elements essential for adaptation, and one spacer flanked by two repeats. Wildtype E.coli has two types of CRISPRER array, with their repeat sequence differs in one base pair. Its biological function is explained and characterized in BBa_K2996011.

【favorite composite part】

RSRL-eGFP downstream of pTac

The ribosome-binding site and the translation initiation codon of this reading device are indicated. Translation of transcripts generated from the tac promoter will stop at the leader, for those two stop codons. The wrong reading frame and existing stop codon together will insure no expression of the florescent protein in the original situation.

Upon information input, that is addition of corresponding inducer, spacer adaptation from cas1/2 will result in an addition of 61 base pairs, 33bp new spacer and 28bp replicated repeat, into the integration cassette. Stop codon will be moved out of frame, while EGFP in the open reading frame. Thus, successful translation of EGFP is our desired information output signal, observed by eyes and microplate reader.

Figure 1. Schematic representation of pRead before induction.

Figure 2. Schematic representation of pRead after induction.

Basic part

part number	name	type
BBa_K2996500	zinc finger with flag tag	coding
BBa_K2996501	Terminator put downstream of zinc finger	coding
BBa_K2996502	C-luciferase put downstream of J23119	coding
BBa_K2996504	binding sites for dCas9 and zinc finger	other
BBa_K2996505	Lure sequence and DBD binding site spaced by 15 base pairs	other
BBa_K2996506	combination of sgRNA production module and lure-DBD binding site	other
BBa_K2996507	combination of sgRNA production module and target-DBD binding site	other
BBa_K2996700	Flexible Linker 4 that separates RpoA and dCas9	other
BBa_K2996703	RpoA, DNA-directed RNA polymerase subunit alpha	coding
BBa_K2996000	repL, replication initiator protein	coding
BBa_K2996001	Cas1-Cas2 complex	coding
BBa_K2996002	minimal CRISPR array-A	Signalling
BBa_K2996003	Lac promoter and operator plus RBS	Intermediate

Composite part

part number	name	type
BBa_K2996007	pTet upstream of Cas1/2	Composite
BBa_K2996008	pLuxR upstream of Cas1/2	Composite
BBa_K2996009	EGFP gene downstream of the synthetic RSRL sequence	Composite
BBa_K2996011	RSRL-EGFP downstream of pTac	Composite
BBa_K2996012	pLac upstream of repL	Composite
BBa_K2996701	RpoA fused with dCas9 through FL4	Composite
BBa_K2996702	tetracycline inducible transcription activator	Composite
BBa_K2996706	mRFP downstream of activation unit containing target	Composite
BBa_K2996707	mRFP downstream of activation unit containing lure	Composite
BBa_K2996708	LuxI downstream of activation unit containing target	Composite
BBa_K2996709	LuxI downstream of activation unit containing lure	Composite
BBa_K2996503	a fusion protein of C-luciferase and zinc finger	coding

Part collection

off-target detection

part number	name	type
BBa_K2996503	a fusion protein of C-luciferase and zinc finger	coding
BBa_K2996504	binding sites for dCas9 and zinc finger	other
BBa_K2996505	Lure sequence and DBD binding site spaced by 15 base pairs	other
BBa_K2996506	combination of sgRNA production module and lure-DBD binding site	other
BBa_K2996507	combination of sgRNA production module and target-DBD binding site	other

Part collection [transcription activation]

part number	name	type
BBa_K2996700	Flexible Linker 4 that separates RpoA and dCas9	Conjugate
BBa_K2996701	RpoA fused with dCas9 through FL4	Coding
BBa_K2996702	tetracycline inducible transcription activator	Composite
BBa_K2996703	RpoZ, DNA-directed RNA polymerase subunit alpha	Coding
BBa_K2996704	target sequence upstream of promotor	Signaling
BBa_K2996705	lure sequence upstream of promotor	Signaling
BBa_K2996706	mRFP downstream of activation unit containing target	Composite
BBa_K2996707	mRFP downstream of activation unit containing lure	Composite
BBa_K2996708	LuxI downstream of activation unit containing target	Composite
BBa_K2996709	LuxI downstream of activation unit containing lure	Composite

Part collection [information storage]

part number	name	type
BBa_K2996000	repL, replication initiator protein	coding
BBa_K2996001	Cas1-Cas2 complex	coding
BBa_K2996002	minimal CRISPR array-A	Generator
BBa_K2996012	pLac upstream of repL	Composite
BBa_K2996006	pLuxR upstream of repL	Composite
BBa_K2996007	pTet upstream of Cas1/2	Composite
BBa_K2996008	pLuxR upstream of Cas1/2	Composite
BBa_K2996009	EGFP gene downstream of the synthetic RSRL sequence	Composite
BBa_K2996011	RSRL-EGFP under tac promoter	Composite

Part improvement

We inactivated a restriction digestion site within the Coding sequence of Cas1/2(BBa_K2761009) created by iGEM18_Tec-Monterrey and created the new part BBa_K2996800.

CRISPR-Cas is a prokaryotic immune system in bacteria. The function of Cas1/2 complex is to recognize foreign DNA and insert a 33bp fragment into CRISPR locus of the bacteria’s genome as a new spacer.

In the previous part(BBa_K2761009), there was a restrict digestion site of HindIII(AAGCTT) from 692-697 of Cas1 coding region. We used overlapping PCR to bring in a point mutation, changing it into AAGCAT so that HindIII could be used to assemble this part into other vectors.

We designed primers ‘F’, ‘R’ on both end of Cas1/2 sequence and ‘Fm’ ‘Rm’ on restriction site Hind, and then ‘F’ and ‘Rm’ , ‘R’ and ‘Fm’ as two pairs of PCR primers respectively to produce two halves of the Cas1/2 mutant.

Then overlap PCR is conducted with amplified products of the two PCR reactions above being template and ‘F’ and ‘R’ being primers.

The mutant and original Cas1/2 was digested by HindIII. It is shown that the mutant was not cut while the original one was, which means our work has been successful.

Part characterization

We characterized an AHL responding promoter lux pR-HS(BBa_K2558001) created by iGEM18_Tsinghua.

title with style

AHL is a key molecule of quorum sensing system which can induce gene expression by activating lux pR promotor when combined with LuxR. We tried to use AHL as the signal transmitter for off-target recording in our project, so we put this lux pR-HS, a lux pR mutant with high intensity and small leakage from Neon Coli by iGEM18_Tsinghua, upstream of repL.

repL (BBa_K2996000) is a P1 lytic replication element that can act on mini-F origin and promote plasmid replication. Thus, we quantified the effect of lux pR-HS indirectly by measuring plasmid copy number through qPCR in E.coli harbouring pLux-repL BBa_K2996006 and pLux-cas BBa_K2996008.

a title with style

We tried different AHL concentration from 0 to 10-6 M and measured copy number in the time course. Both absolute copy number and relevant amplification of copy number compared to non-induction were calculated. The result confirmed the small leakage of lux pR-HS and it also showed obvious responding to AHL, with the maximum effect reached at a concentration of 10-7 M at 5h of induction. We also tried to induce with 10^-6M AHL, but the effect began to decrease. We also observed that the bacteria growed slower in a higher AHL concentration.