Difference between revisions of "Part:BBa K4274035"

Revision as of 11:43, 12 October 2022

PvanP*-sfp_target-sfp_HA_US-p43-K4274013-sfp-K4274014-degQ-sfp_HA_DS
This part starts with the promoter PvanP* (Part: BBa_K4274006), a semi-synthetic promoter that promotes the constitutive expression of the single guide RNAs used in this composite part. Then, we have encoded sfp_target (Part: BBa_K4274008), the guide RNA for sfp (Part: BBa_K4274009) and degQ (Part: BBa_K4274010) knock-in. sfp_HA_US (part: BBa_K4274011) is the following sequence in this composite part. It performs the role of the donor DNA. We then encoded p43 (Part: BBa_K143013), a constitutive promoter for the following sequences. The promoter is followed by synthetic ribosome binding site in pJOE8999 (Part: BBa_K4274013). P43 is followed by degQ (Part: BBa_K4274010), a regulator gene that assists in the production of lipopeptide. The last coding sequence in this composite part is sfp_HA_DS (Part: BBa_K4274012), the donor DNA that provides the knock-in gene sequence. The composite part ends with the terminator B0015 (Part: BBa_B0015).
This part contains the parts that conducts the knock-out of the the mutant sfp gene in Bacillus subtilis, which comprises patB_target (Part: BBa_K4274007), PvanP* (Part: BBa_K4274006), and PvanP*-patB_target (Part: BBa_K4274036). On the other hand, the series of parts participating in sfp and degQ knock-in comprises Sfp_target (BBa_K4274008), sfp (Part: BBa_K4274009), degQ (Part: BBa_K4274010), sfp_HA_US (part: BBa_K4274011), sfp_HA_DS (Part: BBa_K4274012), synthetic RBS1 in pJOE8999 (Part: BBa_K4274013), synthetic RBS2 in pJOE8999 (Part: BBa_K4274014), and this composite part (Part: BBa_K4274035).

Usage and Biology

We applied the CRISPR_Cas9 mechanism to achieve sfp and degQ knocked-in. The coding sequence sfp¬¬¬_target (Part: BBa_K4274008) is the single guide RNA that assists the Cas9 protein to locate the targeted sequence. Both sfp_HA_US (part: BBa_K4274011) and sfp_HA_DS (Part: BBa_K4274012) are donor DNA sequences that inserts the desired modified gene sequence in between the broken DNA fragments. Our sfp (Part: BBa_K4274009) gene sequence is a part derived from B. amyloliquefaciens FZB42, performing the role of a primer for nonribosomal peptide synthesis. K4274013 (Part: BBa_K4274013) and K4274014 (Part: BBa_ K4274014) are respectively ribosome binding sites cloned into pJOE8999 to express sfp and degQ. After the modification of knocking-in sfp and degQ genes in the region of mutant sfp, it would enable Bacillus subtilis 168 to produce fengycins.

Source

sfp from B. amyloliquefaciens FZB42 and degQ from Bacillus subtilis 168.

Sequence and Features