Our team decided to use cinnamaldehyde as an antimicrobial compound, and Glucan-binding protein C(GbpC) in order for our recombinant Lactococcus lactis to be combined with biofilm. We designed the parts producing cinnamaldehyde from phenylalanine and expressed GbpC on L.lactis surface are indicated below.

To biosynthesize cinnamaldehyde from L-phenylalanine takes three steps, and it required three enzymes, phenylalanine-ammonia lyase, 4-coumarate-CoA ligase, Cinnamoyl-CoA reductase. The two parts, phenylalanine-ammonia lyase, 4-coumarate-CoA ligase, were optimized for L.lactis subsp. cremoris by IDT, Inc. Codon Optimization Tool. Cinnamoyl-CoA reductase derived from Arabidopsis thaliana, AtCCR, was used in the existing part (BBa_K1129005). These three genes were synthesized by IDT Inc. and assembled by HiFi Assembly (New England Biolabs, Inc.). The antimicrobial activity of Cinnamaldehyde against Streptococcus mutans was confirmed in our project. An explanation more in detail is an experiment page.

Glucan-binding protein C was obtained from Streptococcus mutans gene. Using the protein that expressed S.mutans surface enables to bind the biofilm formed by S.mutans and creep in the biofilm and exhibit effects. This part was obtained from S.mutans gene, cloned to pMG36e vector plasmid, and transformed to L.lactis. An explanation more in detail is an experiment page.

Bacteriocin biosynthesis gene cluster was used as antimicrobial activity against S.mutans. This part is for the biosynthesis of bacteriocin which is originally produced by Lactiplantibacillus pentosus H99. L.pentosus H99 is known to live in the intestines of cattle and produce bacteriocin which has antimicrobial activity. The gene cluster contains 4 sets of genes: Bacteriocin precursor, Bacteriocin immunity protein, PedC/BrcD family Bacteriocin maturation disulfide and ABC transporter ATP-binding protein. The antimicrobial activity of This Bacteriocin against Streptococcus mutans was confirmed in our project. An explanation more in detail is an experiment page.

We also used a shuttle vector plasmid, pMG36e, that can replicate E.coli and B.subtilis. The vector is based on pWVO1, originally obtained from L. lactis subsp. cremoris Wg2. The part of the vector containing the pWVO1 origin of replication was obtained from pGK11. This plasmid also has the Erythromycin resistance gene as an antibiotic resistance marker. By using this plasmid, the recombinant plasmid is transformed from E.coli to L.lactis directly without any additional plasmid cloning.

Parts table
Name Type Description Length
BBa_K4019001 Coding Optimized SmPAL (phenylalanine-ammonia lyase from Streptomyces maritimus) 1575
BBa_K4019002 Coding Optimized ScCCR (4-coumarate-CoA ligase from Streptomyces coelicolor) 1572
BBa_K4019004 Coding gbpC (Glucan-Binding Protein C of Streptococcus mutans) 1752
BBa_K4019005 Plasmid pMG36e 3610
BBa_K4019007 RBS RBS (upstream of 4-coumarate-CoA ligase) 39
BBa_K4019008 Composite RBS-Optimized ScCCL 1611
BBa_K4019009 Regulatory Putative promoter and RBS (upstream of gbpC) 240
BBa_K4019010 Composite Putative promoter-RBS-gbpC 1992
BBa_K4019011 Composite Bacteriocin biosynthesis gene cluster 3386