Designs
1.Increase the production of B12 in B. megaterium
1.1 Increase flux of glutamyl-tRNA to uroporphyrinogen Ⅲ by regulating feedback inhibition of glutamyl-tRNA reductase (HemA)
1.2 Reduce flux of uroporphyrinogen Ⅲ to heme by an antisense RNA(as) strategy
1.3 Overexpress RdhANP to soak up excess B12 production
2.Express and verify RdhANP
2.1 Clone rdhANP to suitable vectors
2.2 Induce to express RdhANP in B. megaterium
2.3 Purify and concentrate RdhANP
2.4 Construct dehalogenation-testing system in vitro and in vivo
2.5 Detect dehalogenation reaction by HPLC-MS
2.6 Overexpress RdhANP by testing different vectors
2.7 Design synthetic RBS by 'Ribosome binding site calculator' and RBSDesigner and test their production level
2.8 Perform Directed Enzyme& Protein Evolution by certain companies.
2.9 Test the dehalogenation of these mutants our constructed wastewater-imitating system
2.10 Coordinate B12 production and RdhANP expression
3.Bind B. megaterium to cellulose membrane
3.1 Coexpress Cellulose binding domain(CBD) and Membrane anchoring domain(MAD) in B. megaterium
3.2 Test the binding of B. megaterium to cellulose membrane
4.Coexpress CBD, MAD and RdhANP in B. megaterium and test its binding to cellulose membrane and dehalogenation
5.Construct biofilm containing RdhANP-expressing B. megaterium
6.Introduce our engineered biofilm and cellulose membrane to HMBR
7.Cooperate with JLU-iGEM to complete the halogenated phenol's degradation pathway and optimize organohalide-contained wastewater treatment
8.Introduce downstream devices to eliminate microorganisms leaked into the effluent
