The core of the iGEM competition lies in the standardization of biological parts. Our project is committed to enhancing the lipopeptide production capacity of Bacillus velezensis through a systematic synthetic biology approach. Based on the actual needs of four rounds of DBTL cycles, we have developed the following core parts:

Composite Part

Dual gRNA expression cassette(BBa_25VUB2AP): Specifically designed for the knockout of long fragment gene cluster.

Gene Editing and Analysis Parts

Specific editing primers for key regulatory genes (relA, cdaA, cheB).

Primers for Gene editing of the cyclic lipopeptide synthesis gene cluster (NRPS cluster).

qPCR detection primer sets for assessing transcriptional level of NRPS genes.

All these parts have been specifically optimized for the genetic engineering of Bacillus velezensis. The dual gRNA editing system effectively addresses the bottlenecks of low efficiency and off-target effects in long fragment editing that are present in traditional single gRNA systems. All parts have been experimentally validated for their reliability and have been submitted to the iGEM Registry, providing a toolkit for long-fragment gene editing.

Biobricks	Short Description	Type	Length (bp)	Existing or newly added
BBa_25NLM78P	The forward primer of the upstream homologous arm of the srfB gene cluster	Primer	51	New
BBa_25AR44U5	The reverse primer of the upstream homologous arm of the srfB gene cluster	Primer	28	New
BBa_25JSL6PC	The forward primer of the downstream homologous arm of the srfB gene cluster	Primer	42	New
BBa_25RSLDDK	The reverse primer of the downstream homologous arm of the srfB gene cluster	Primer	49	New
BBa_25UFLOBB	The forward gRNA gene for CRISPR-mediated targeting of the srfB gene	gRNA_gene	30	New
BBa_25Y4IKIR	The reverse gRNA gene for CRISPR-mediated targeting of the srfB gene	gRNA_gene	30	New
BBa_25CUHMSM	The forward primer of the upstream homologous arm of the ituA-D gene cluster	Primer	47	New
BBa_25XSE72Q	The reverse primer of the upstream homologous arm of the ituA-D gene cluster	Primer	34	New
BBa_25EIP405	The forward primer of the downstream homologous arm of the ituA-D gene cluster	Primer	33	New
BBa_251XUSQW	The reverse primer of the downstream homologous arm of the ituA-D gene cluster	Primer	47	New
BBa_25301ES0	The forward primer of the upstream homologous arm of the srfA-D gene cluster	Primer	47	New
BBa_25FDDAGO	The reverse primer of the upstream homologous arm of the srfA-D gene cluster	Primer	28	New
BBa_254UX0WU	The forward primer of the downstream homologous arm of the srfA-D gene cluster	Primer	35	New
BBa_256HTVM7	The reverse primer of the downstream homologous arm of the srfA-D gene cluster	Primer	54	New
BBa_2541FU73	The forward primer of the upstream homologous arm of the ppsCD gene cluster	Primer	47	New
BBa_25047MFO	The reverse primer of the upstream homologous arm of the ppsCD gene cluster	Primer	36	New
BBa_25V572KZ	The forward primer of the downstream homologous arm of the ppsCD gene cluster	Primer	27	New
BBa_25I7EDST	The reverse primer of the downstream homologous arm of the ppsCD gene cluster	Primer	41	New
BBa_25HLNIAW	The forward primer for amplifying the dual gRNA expression cassette incorporating dual gRNAs targeting the ituA-D gene cluster	Primer	79	New
BBa_25YRODYA	The reverse primer for amplifying the dual gRNA expression cassette incorporating dual gRNAs targeting the ituA-D gene cluster	Primer	81	New
BBa_25C8ZT36	The forward primer for amplifying the dual gRNA expression cassette incorporating dual gRNAs targeting the srfA-D gene cluster	Primer	79	New
BBa_2542FZNQ	The reverse primer for amplifying the dual gRNA expression cassette incorporating dual gRNAs targeting the srfA-D gene cluster	Primer	81	New
BBa_25IWREBZ	The forward primer for amplifying the dual gRNA expression cassette incorporating dual gRNAs targeting the ppsCD gene cluster	Primer	79	New
BBa_25847M0C	The reverse primer for amplifying the dual gRNA expression cassette incorporating dual gRNAs targeting the ppsCD gene cluster	Primer	81	New
BBa_25TJJWIS	The forward primer of the ituD gene for qPCR expression analysis	Primer	20	New
BBa_25HOPIWA	The reverse primer of the ituD gene for qPCR expression analysis	Primer	20	New
BBa_25W039PL	The forward primer of the ituA gene for qPCR expression analysis	Primer	20	New
BBa_25OZ55ZB	The reverse primer of the ituA gene for qPCR expression analysis	Primer	21	New
BBa_259S25AQ	The forward primer of the ituB gene for qPCR expression analysis	Primer	18	New
BBa_25VNYAJ7	The reverse primer of the ituB gene for qPCR expression analysis	Primer	20	New
BBa_25XODHAI	The forward primer of the ituC gene for qPCR expression analysis	Primer	21	New
BBa_253JZH5W	The reverse primer of the ituC gene for qPCR expression analysis	Primer	21	New
BBa_25Z17GLL	The forward primer of the srfA gene for qPCR expression analysis	Primer	18	New
BBa_25HCH1P8	The reverse primer of the srfA gene for qPCR expression analysis	Primer	22	New
BBa_256E72MO	The forward primer of the srfB gene for qPCR expression analysis	Primer	20	New
BBa_25GB5WHZ	The reverse primer of the srfB gene for qPCR expression analysis	Primer	20	New
BBa_25J1513M	The forward primer of the srfC gene for qPCR expression analysis	Primer	20	New
BBa_25VUBDEF	The reverse primer of the srfC gene for qPCR expression analysis	Primer	20	New
BBa_257CIRO1	The forward primer of the srfD gene for qPCR expression analysis	Primer	20	New
BBa_25M25G2K	The reverse primer of the srfD gene for qPCR expression analysis	Primer	22	New
BBa_2597Z94M	The forward primer of the fenA gene for qPCR expression analysis	Primer	24	New
BBa_25MFKNRP	The reverse primer of the fenA gene for qPCR expression analysis	Primer	25	New
BBa_25FOYJ2Q	The forward primer of the fenB gene for qPCR expression analysis	Primer	25	New
BBa_256J1KRS	The reverse primer of the fenB gene for qPCR expression analysis	Primer	24	New
BBa_25BAV1VP	The forward primer of the fenC gene for qPCR expression analysis	Primer	22	New
BBa_25L5QXSZ	The reverse primer of the fenC gene for qPCR expression analysis	Primer	22	New
BBa_2537IFSN	The forward primer of the fenD gene for qPCR expression analysis	Primer	24	New
BBa_25GLS3YZ	The reverse primer of the fenD gene for qPCR expression analysis	Primer	22	New
BBa_25QWTS4G	The forward primer of the fenE gene for qPCR expression analysis	Primer	20	New
BBa_25UUYMGN	The reverse primer of the fenE gene for qPCR expression analysis	Primer	20	New
BBa_25D3CF9A	The forward primer that specifically binds to the highly conserved regions at the start of the bacterial 16S rDNA gene	Primer	20	Exisiting
BBa_258FRPPG	The reverse primer that specifically binds to the highly conserved regions at the end of the bacterial 16S rDNA gene	Primer	21	Exisiting
BBa_254KV7SG	The forward primer for qPCR normalization and reference gene analysis	Primer	20	Exisiting
BBa_25PE46QC	The reverse primer for qPCR normalization and reference gene analysis	Primer	22	Exisiting
BBa_25AWFQTE	The forward gRNA gene for CRISPR-mediated targeting of the cdaA gene	gRNA_gene	27	New
BBa_25PQ88PW	The reverse gRNA gene for CRISPR-mediated targeting of the cdaA gene	gRNA_gene	27	New
BBa_25HF4ENJ	The forward primer for HDR-mediated cdaA gene replacement	Primer	36	New
BBa_25MVW4H2	The reverse primer for HDR-mediated cdaA gene replacement	Primer	35	New
BBa_25B7RZUS	The forward gRNA gene for CRISPR-mediated targeting of the relA gene	gRNA_gene	27	New
BBa_252QTD1M	The reverse gRNA gene for CRISPR-mediated targeting of the relA gene	gRNA_gene	27	New
BBa_25HN3HWG	The forward primer for HDR-mediated relA gene replacement	Primer	35	New
BBa_25HGOLJ6	The reverse primer for HDR-mediated relA gene replacement	Primer	38	New
BBa_25SBC1TM	The forward gRNA gene for CRISPR-mediated targeting of the cheB gene	gRNA_gene	24	New
BBa_25WJ02RJ	The reverse gRNA gene for CRISPR-mediated targeting of the cheB gene	gRNA_gene	24	New
BBa_251D0MRN	The forward primer for HDR-mediated cheB gene replacement	Primer	35	New
BBa_253AY6GY	The reverse primer for HDR-mediated cheB gene replacement	Primer	35	New
BBa_25N11ZED	The forward primer for sequencing verification of CRISPR-induced mutations	Primer	20	New
BBa_25C4XXHA	The reverse primer for sequencing verification of CRISPR-induced mutations	Primer	25	New
BBa_25I2HXYV	The forward primer for amplifying inserted fragment 1 for constructing the dual gRNA expression cassette	Primer	48	New
BBa_25UDI69E	The reverse primer for amplifying inserted fragment 1 for constructing the dual gRNA expression cassette	Primer	32	New
BBa_25LD1EH3	The forward primer for amplifying inserted fragment 2 for constructing the dual gRNA expression cassette	Primer	38	New
BBa_25LD1EH3	The reverse primer for amplifying inserted fragment 2 for constructing the dual gRNA expression cassette	Primer	31	New
BBa_25USNDMG	The forward primer for amplifying inserted fragment 3 for constructing the dual gRNA expression cassette	Primer	29	New
BBa_25NNG7BM	The reverse primer for amplifying inserted fragment 3 for constructing the dual gRNA expression cassette	Primer	50	New
BBa_25HMDYQA	The forward primer for reverse PCR amplification of the pJOE8999 plasmid backbone	Primer	59	New
BBa_2597DDHF	The reverse primer for reverse PCR amplification of the pJOE8999 plasmid backbone	Primer	59	New
BBa_25IFT7BH	PvanP* promoter	Promoter	10	Exisiting
BBa_254J635F	oop Terminator	Terminator	30	Exisiting
BBa_25M8JTYD	The template plasmid pJOE8999	Plasmid	7794	Exisiting

Biobricks	Short Description（Title)	Type	Length (bp)	Existing or newly added
BBa_25VUB2AP	Dual gRNA expression cassette	Device	395	New
BBa_25UTM7Z8	Dual gRNA vector pWLS	Plasmid	7597	New