Team:TokyoTech/Contribution
Contributions
This Contribution page explained how our project contributes to future iGEM. We did these two things below.
1. Parts addition related to Lactococcus
2. Create Biosafety “Sugoroku”
1. Parts addition related to Lactococcus
The parts related to heterologous gene expression in Lactococcus are registered. These parts can also be seen on the Parts page. The parts we used in our project to express our target proteins in lactococcus are table1. In addition to that, particularly new parts are explained in detail below.
| Name | Discription |
|---|---|
| BBa_K4019001 | Optimized SmPAL (phenylalanine-ammonia lyase from Streptomyces maritimus) |
| BBa_K4019002 | Optimized ScCCR (4-coumarate-CoA ligase from Streptomyces coelicolor) |
| BBa_K4019004 | gbpC (Glucan-Binding Protein C of Streptococcus mutans) |
| BBa_K4019005 | pMG36e |
| BBa_K4019007 | RBS (upstream of 4-coumarate-CoA ligase) |
| BBa_K4019008 | RBS-Optimized ScCCL |
| BBa_K4019009 | Putative promoter and RBS (upstream of gbpC) |
| BBa_K4019010 | Putative promoter-RBS-gbpC |
| BBa_K4019011 | Bacteriocin biosynthesis gene cluster |
Part:BBa_K4019004
gbpC (Glucan-Binding Protein C of Streptococcus mutans)
This part comes from our target strain, Streptococcus mutans,
and code Glucan-binding protein C. S.mutans express this protein and bind specifically
to glucan synthesized themselves. GbpC is a surface-anchored protein
and it contains LPXTG motif which is processed by sortases to anchor the peptide glycan layer[1].
We considered that this part can use L.lactis, the same gram-positive bacteria, on their surface.
Part:BBa_K4019005
pMG36e
The vector is based on pWVO1, originally obtained from L.
lactis subsp. cremoris Wg2, which also replicates in E. coli and B. subtilis.
The part of the vector containing the pWVO1 origin of replication was obtained from pGK11.
This plasmid was reported by Van De Guchte, M. et.al.[2] We want to express enzymes in E. coli first,
and then Lactococcus lactis finally.
So, we had to use the shuttle vector that can function in both E.coli and L.lactis.
Part:BBa_K4019008
RBS-Optimized ScCCL
To biosynthesize cinnamaldehyde from L-phenylalanine takes three steps,
and it required three enzymes, phenylalanine-ammonia lyase, 4-coumarate-CoA ligase,
cinnamoyl-CoA reductase. Optimized ScCCL is a 4-coumarate-CoA ligase derived from
Streptomyces coelicolor and catalyzed acid-thiol ligation of cinnamic acid to cinnamoyl- CoA.
This part is composed of RBS derived from upstream of Streptomyces coelicolor 4-coumarate-CoA
ligase and Optimized ScCCL coding part. Optimized ScCCL was optimized for L.lactis subsp.
cremoris by IDT, Inc. Codon Optimization Tool.
Part:BBa_K4019011
Bacteriocin biosynthesis gene cluster
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.
Its antimicrobial activity against Streptococcus mutans is confirmed in our project.
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.
2. Create Biosafety “Sugoroku”
Studying biosafety and biosecurity is indispensable for future iGEMers and beginners of synthetic biology. But it is sometimes bothering and boring because there are a lot of restrictions and laws. So, we made a sugoroku, one of the Japanese board games, to learn containment with fun. This Biosafety Sugoroku is explained in detail here. This can understand easily what is we should do and should not do regarding handling recombinant DNA and strains.
Reference
[1] Sato, Y., Yamamoto, Y. & Kizaki, H. Cloning and sequence analysis of the gbpC gene encoding a novel glucan- binding protein of Streptococcus mutans. Infect. Immun. 65, 668–675 (1997).
[2] Van De Guchte, M., Van Der Vossen, J. M. B. M., Kok, J. & Venema, G. Construction of a lactococcal expression vector: Expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl. Environ. Microbiol. 55, 224–228 (1989).