B12 SYNPORTERANOTHER BRICK IN THE WALL
iGEM TeamGöttingen 2016DepartmentforGenomicandAppliedMicrobiology,UniversityofGöttingen
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OUR TEAM
GregorHoffmann,TobiasSchilling,TobiasKrammer,AlexanderBräuerMiriamSchüler,InesFriedrich,MariekeEnders,NoraEulig,LarissaKrüger
Supervisors:Prof.Dr.RolfDaniel,Dr.AnjaPoehlein,Dr.AndreaThürmer,Genis AndresCastilloVillamizar
BACKGROUND
Since the chemical production of vitamin B12 isvery elaborate, production is facilitated bygenetically engineered microorganisms.However, the produced Vitamin B12 is notexported out of the organism and must beharvested by cell lysis. This prevents a costefficient production. To date, a natural cellularVitamin B12 exporter is unknown.
Vitamin B12 (Fig. 1) is anessential nutrient foralmost all organisms.However, its biosynthesisis confined to micro-organisms. Humans canmainly take it up viaanimal products or foodsupplements.
Figure 1:Structure ofVitaminB12(Hydroxycobalamin)
AIM
We intend to design, construct and introduce asynthetic Vitamin B12 exporter (“Synporter”,Fig. 2) into a production organism. Thereby, weaim for higher yields in the industrial VitaminB12 production without a required cell lysis.
ABOUT iGEM
TheannualiGEM (internationalGeneticallyEngineeredMachines)competitionisthelargestinthefieldofsyntheticbiology.ItisoperatedbytheiGEM FoundationthatwasestablishedbytheMassachusettsInstituteofTechnology.Theparticipantsareinterdisciplinarystudentteamsfromallovertheworldwhohavetoprovethemselvesinorganizingandexecutingtheentireprojectaswellasfindingfinancialsupport.
COURSE OF OUR PROJECTTheB12 Synporter consists of asignal for atwin arginine translocation (Tat)-mediatedexport that is linked to aB12-bindingprotein.Three differentconstructs are being tested(Tab.1).Table 1:Buildup of the B12 Synporter constructs.
Sequence OriginalPeptide(Species): Function Amino acids
Export signal TorA (E.coli) 39Linker 5aminoacids post TorA export signal 5
B12 binding proteinα-subunit MutB (P.shermanii): Methylmalonyl-CoA-Mutase;B12 co-factor 727BtuF (E.coliK12):B12import 265small subunit GlmS (C.cochlearium): Glutamatemutase; B12 co-factor 136
What has been done already…
After checking their identity, theproduction strains S. typhimurium TA100,S. blattae and R. planticola were cultivatedand their natural antibiotic resistanceswere determined. The vector pBAD202was chosen for expression and the strainswere made electrocompetent. The threedifferent constructs for the B12 Synporterwere designed and synthesized. (Fig. 3)
Figure 2: Model of the „Synporter“ bound to Vitamin B12 inside aGram-negative production organism.
Figure 3: Course of our project. Checkmarks ( ) indicate completed tasks.
How we will proceed…
The constructs will be cloned into thevector (Fig. 4) and introduced into E. colivia electroporation to check forexpression. This will be followed by thetransformation of the production strains.The success of the Vitamin B12 export willbe tested using a microbiological assay aswell as spectrophotometry. Finally, we willpresent our results at the Giant Jamboréein Boston.
Figure 4: Model for a B12 Synporter constructinserted into the pBAD202 plasmid vector.The vector includes the origin of replication (ori), akanamycin resistance gene (kanR), the promoter ofan L-arabinose operon (araBAD promotor) and theL-arabinose regulatory gene (araC).
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