Plasmid Design
Strain:
ClearColi BL21(DE3)
- Plasmid 1: pET-28a(+)
T7 promoter, ColE1/pMB1/pBR322/pUC origin of replication, KanR, IPTG inducible. - Plasmid 2: pBAD30 (available from suppliers such as Qi Yun Biology, etc.)
araBAD promoter, p15A origin of replication, AmpR, L-arabinose inducible.
The following are the original backbones of the plasmids:
Basis:
ClearColi BL21(DE3)
Contains the DE3 fragment, enabling the synthesis of T7 RNA polymerase, allowing high-efficiency recombinant protein expression using the T7 promoter; Lacks Lon protease and OmpT protease synthesis genes, preventing degradation of exogenous proteins.
Dual Plasmid System
pET-28a(+) is used for high-efficiency recombinant protein expression, induced by IPTG; the recombinant protein is a short peptide, non-toxic, thus no additional measures are currently taken to address T7 promoter leakage. pBAD30 carries a suicide switch, induced by L-arabinose; the pBAD promoter has low leakage, making it suitable for hosting a suicide switch.
The ColE1/pMB1/pBR322/pUC origin of replication and the p15A origin of replication are derived from different sources, indicating that the plasmids belong to different compatibility groups, thus the two plasmids can be used simultaneously; they possess Kan and Amp resistance, respectively, facilitating selection.
Recombinant Protein Sequence:
MVPKFWRFPMGGGGSGGGGSGGGGSYGRKKRRQRRRPQHHHHHH
MKDIGGGGSGGGGSGGGGSHHHHHH
N-terminal - MVPKFWRFPM - (GGGGS)×3 - YGRKKRRQRRRPQ - HHHHHH - C-terminal
N-terminal - MKDI - (GGGGS)×3 - HHHHHH - C-terminal
The short peptide sequence MPFRWFKPV-NH₂ will be used directly for now; the His-tag is added at the C-terminal.
Restriction Enzyme Sites to Be Avoided:
Codon Bias:
Optimized using the preferred codons for E. coli B strains (for this short sequence, the most preferred codons were used instead of a probabilistic distribution).
Suicide Switch Components
With reference to the principle of the suicide switch plasmid constructed by CAFA_China2022, SZ-SHD, which consists of two components: T4 holin and T4 endolysin.
Design from CAFA_China2022
Modification Strategy:
The pBAD30 plasmid is used to express T4 holin and T4 endolysin.
