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− | <h2>1.Increase the production of B12 in B. megaterium</h2> | + | <h2>1.Increase the production of B12 in <i>B. megaterium</i></h2> |
<h3>1.1 Increase flux of glutamyl-tRNA to uroporphyrinogen Ⅲ by regulating feedback inhibition of glutamyl-tRNA reductase (HemA) </h3> | <h3>1.1 Increase flux of glutamyl-tRNA to uroporphyrinogen Ⅲ by regulating feedback inhibition of glutamyl-tRNA reductase (HemA) </h3> | ||
<h3>1.2 Reduce flux of uroporphyrinogen Ⅲ to heme by an antisense RNA(as) strategy </h3> | <h3>1.2 Reduce flux of uroporphyrinogen Ⅲ to heme by an antisense RNA(as) strategy </h3> | ||
− | <h3>1.3 Overexpress RdhANP to soak up excess B12 production </h3> | + | <h3>1.3 Overexpress <i>RdhANP</i> to soak up excess B12 production </h3> |
− | <h2>2.Express and verify RdhANP</h2> | + | <h2>2.Express and verify <i>RdhANP</i></h2> |
− | <h3>2.1 Clone | + | <h3>2.1 Clone <i>RdhANP</i> to suitable vectors </h3> |
− | <h3>2.2 Induce to express RdhANP in B. megaterium</h3> | + | <h3>2.2 Induce to express <i>RdhANP</i> in <i>B. megaterium</i></h3> |
− | <h3>2.3 Purify and concentrate RdhANP </h3> | + | <h3>2.3 Purify and concentrate <i>RdhANP</i> </h3> |
<h3>2.4 Construct dehalogenation-testing system in vitro and in vivo</h3> | <h3>2.4 Construct dehalogenation-testing system in vitro and in vivo</h3> | ||
<h3>2.5 Detect dehalogenation reaction by HPLC-MS</h3> | <h3>2.5 Detect dehalogenation reaction by HPLC-MS</h3> | ||
− | <h3>2.6 Overexpress RdhANP by testing different vectors </h3> | + | <h3>2.6 Overexpress <i>RdhANP</i> by testing different vectors </h3> |
<h3>2.7 Design synthetic RBS by 'Ribosome binding site calculator' and RBSDesigner and test their production level</h3> | <h3>2.7 Design synthetic RBS by 'Ribosome binding site calculator' and RBSDesigner and test their production level</h3> | ||
<h3>2.8 Perform Directed Enzyme& Protein Evolution by certain companies. </h3> | <h3>2.8 Perform Directed Enzyme& Protein Evolution by certain companies. </h3> | ||
<h3>2.9 Test the dehalogenation of these mutants our constructed wastewater-imitating system </h3> | <h3>2.9 Test the dehalogenation of these mutants our constructed wastewater-imitating system </h3> | ||
− | <h3>2.10 Coordinate B12 production and RdhANP expression</h3> | + | <h3>2.10 Coordinate B12 production and <i>RdhANP</i> expression</h3> |
− | <h2>3.Bind B. megaterium to cellulose membrane</h2> | + | <h2>3.Bind <i>B. megaterium</i> to cellulose membrane</h2> |
− | <h3>3.1 Coexpress Cellulose binding domain(CBD) and Membrane anchoring domain(MAD) in B. megaterium</h3> | + | <h3>3.1 Coexpress Cellulose binding domain(CBD) and Membrane anchoring domain(MAD) in <i>B. megaterium</i></h3> |
− | <h3>3.2 Test the binding of B. megaterium to cellulose membrane</h3> | + | <h3>3.2 Test the binding of <i>B. megaterium</i> to cellulose membrane</h3> |
− | <h2>4.Coexpress CBD, MAD and RdhANP in B. megaterium and test its binding to cellulose membrane and dehalogenation</h2> | + | <h2>4.Coexpress CBD, MAD and <i>RdhANP</i> in <i>B. megaterium</i> and test its binding to cellulose membrane and dehalogenation</h2> |
− | <h2>5.Construct biofilm containing RdhANP-expressing B. megaterium</h2> | + | <h2>5.Construct biofilm containing <i>RdhANP</i>-expressing <i>B. megaterium</i></h2> |
<h2>6.Introduce our engineered biofilm and cellulose membrane to HMBR</h2> | <h2>6.Introduce our engineered biofilm and cellulose membrane to HMBR</h2> | ||
<h2>7.Cooperate with JLU-iGEM to complete the halogenated phenol's degradation pathway and optimize organohalide-contained wastewater treatment</h2> | <h2>7.Cooperate with JLU-iGEM to complete the halogenated phenol's degradation pathway and optimize organohalide-contained wastewater treatment</h2> |
Revision as of 07:46, 30 October 2017