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Notes about Mgfp-5

9.11 Transformation of Mgfp-5

• 1.

  Take 50ul competent cell into EP tube on the ice to melt.

• 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.

• 3.

  After that, heat shock them in 42℃ water for 90 seconds.

• 4.

  Put them into ice for 5 minutes.

• 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.

• 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

9.12

• 1.

  Select monoclonal colonies of Mgfp-5 according to the protocol and add into 5ml LB liquid medium.

• 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

9.13 Induction of Mgfp-5

• 1.

  Add 1ml bacteria solution into 99ml K+ Lb.

• 2.

  Table concentrator in 37℃ for 1-1.5 hr.

• 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 4 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.

• 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

9.14 Natural purification of Mgfp-5 and SDS-page

• 1.

  Purify the protein by natural protocol.

• 2.

  Produce protein sample and load into the gel.

• 3.

  Gel electrophoresis.

• 4.

  Dye and wash the gel 1hr and scan using gel imager.

9.20 Transformation of Mgfp-5

• 1.

  Take 50ul competent cell into EP tube on the ice to melt.

• 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.

• 3.

  After that, heat shock them in 42℃ water for 90 seconds.

• 4.

  Put them into ice for 5 minutes.

• 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.

• 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

9.21

• 1.

  Select monoclonal colonies of Mgfp-5 according to the protocol and add into 5ml LB liquid medium.

• 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

9.22 Induction of Mgfp-5

• 1.

  Add 1ml bacteria solution into 99ml K+ Lb.

• 2.

  Table concentrator in 37℃ for 1-1.5 hr.

• 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 6 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.

• 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

9.23 Natural purification of Mgfp-5 and SDS-page

• 1.

  Purify the protein by Natural protocol.

• 2.

  Produce protein sample and load into the gel.

• 3.

  Gel electrophoresis.

• 4.

  Dye and wash the gel 1hr and scan using gel imager.

10.3 Transformation of Mgfp-5

• 1.

  Take 50ul competent cell into EP tube on the ice to melt.

• 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.

• 3.

  After that, heat shock them in 42℃ water for 90 seconds.

• 4.

  Put them into ice for 5 minutes.

• 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.

• 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.4

• 1.

  Select monoclonal colonies of Mgfp-5 according to the protocol and add into 5ml LB liquid medium.

• 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.5 Induction of Mgfp-5

• 1.

  Add 1ml bacteria solution into 99ml K+ Lb.

• 2.

  Table concentrator in 37℃ for 1-1.5 hr.

• 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 5 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.

• 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.6 Denatured purification of Mgfp-5 and SDS-page

• 1.

  Purify the protein by denatured protocol.

• 2.

  Produce protein sample and load into the gel.

• 3.

  Gel electrophoresis.

• 4.

  Dye and wash the gel 1hr and scan using gel imager.

10.9 Transformation of Mgfp-5

• 1.

  Take 50ul competent cell into EP tube on the ice to melt.

• 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.

• 3.

  After that, heat shock them in 42℃ water for 90 seconds.

• 4.

  Put them into ice for 5 minutes.

• 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.

• 6.

  After the solution cools down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.10

• 1.

  Select monoclonal colonies of Mgfp-5 according to the protocol and add into 5ml LB liquid medium.

• 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.11 Induction of Mgfp-5

• 1.

  Add 1ml bacteria solution into 99ml K+ Lb.

• 2.

  Table concentrator in 37℃ for 1-1.5 hr.

• 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 5 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.

• 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.12 Denatured purification of Mgfp-5 and SDS-page

• 1.

  Purify the protein by denatured protocol.

• 2.

  Produce protein sample and load into the gel.

• 3.

  Gel electrophoresis.

• 4.

  Dye and wash the gel 1hr and scan using gel imager.

10.14 Transformation of Mgfp-5

• 1.

  Take 50ul competent cell into EP tube on the ice to melt.

• 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.

• 3.

  After that, heat shock them in 42℃ water for 90 seconds.

• 4.

  Put them into ice for 5 minutes.

• 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.

• 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.15

• 1.

  Select monoclonal colonies of Mgfp-5 according to the protocol and add into 5ml LB liquid medium.

• 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.16 Induction of Mgfp-5

• 1.

  Add 1ml bacteria solution into 99ml K+ Lb.

• 2.

  Table concentrator in 37℃ for 1-1.5 hr.

• 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 5 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.

• 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.17 Denatured purification of Mgfp-5 and SDS-page

• 1.

  Purify the protein by denatured protocol.

• 2.

  Produce protein sample and load into the gel.

• 3.

  Gel electrophoresis.

• 4.

  Dye and wash the gel 1hr and scan using gel imager.

Notes about Tyr

9.20

• Transform plasmid into BL21

• Culture for 16h

9.21

• Picking out single clone, inoculating into 5ml LB with K+ system x3

• Culturing for 16h

9.22

• Preserved the BL21 cells with tyrosinase plasmid

• *Due to the mistakes of plasmid synthesis, we do not know that whether PSBGAS, the replacement of the PET28a, has the site of lac. So we decide not to induce.

9.24

• Correct plasmid of tyrosinase arrived, new experiment started on 9.26

9.26

• The transformation of plasmid into BL21 cells

• Produced three plate with spectinomycin of BL21 cells and one of them scraped

• *Due to the mistakes of technician, the whole experiment is failed. 9.27 redo the experiment.

9.27

• The transformation of plasmid into BL21 cells

• Culturing 16h, 37°C, 220rpm

9.28

• Pick out the 3 monoclonal colonies into 5ml LB with K+

• Culture for 16h

9.29

• 12:25pm

  Inoculate 1ml solution into 100ml LB with K+ system x2

• 2ml solution into 200ml Lb with K+ system

• All cultured for 1.5h

• 14:00pm

  Add IPTG (100ul for 100ml; 200ul for 200ml)

• 14:08pm

  culture for 4h, 37°C, 220rpm

• 2x100ml were split into 2x50ml in 50ml centrifugation tube

• Give out 2 x dense 50ml E.coli solutions labeled 1and 2 x diluted 50ml E.coli solutions labeled 2

• centrifugate in 7400rpm, 4°C

• *The centrifugal machine does not support low temperature centrifugation.

• Preserve the solution in -20°C

• *Due to the changes of the laboratory, these solutions were scraped

10.2

• The transformation of plasmid into BL21 cells

• Culturing 16h, 37°C, 220rpm

10.3

• Pick out the 3 monoclonal colonies into 5ml LB with K+

• Culture for 16h

10.4

• 1. Inoculate 1ml solution into 100ml LB with K+ system x3 labeled 1-3 cultured for 1.5h

• Add IPTG 100ul for 100ml system

• culture for 4h, 37°C, 220rpm

The result solution from 1 were centrifuged

Tyr	Precipitate/g	Lysate/ml
1	0.53	2
2	0.51	2
3	0.48	2

• 2. Bacteria SDS-PAGE

Notes about mgfp5-Masp1

10.3 (For simplicity, all terms Masp1 refers to the part mgfp5-masp1 here). Transformation of Masp1

• 1.

  Take 50ul competent cell into EP tube on the ice to melt.

• 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.

• 3.

  After that, heat shock them in 42℃ water for 90 seconds.

• 4.

  Put them into ice for 5 minutes.

• 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.

• 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.4

• 1.

  Select monoclonal of mgfp5-Masp1 according to the protocol and add into 5ml LB liquid medium.

• 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.5 Induction of Masp1

• 1.

  Add 1ml bacteria solution into 99ml K+ Lb.

• 2.

  Table concentrator in 37℃ for 1-1.5 hr.

• 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 4 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.

• 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.6 Natural purification of Masp1 and SDS-page

• 1.

  Purify the protein by Natural protocol.

• 2.

  Producing protein sample and load into the gel.

• 3.

  Gel electrophoresis.

• 4.

  Dye and wash the gel 1hr and scan using gel imager.

10.9 Transformation of Masp1

• 1.

  Take 50ul competent cell into EP tube on the ice to melt.

• 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.

• 3.

  After that, heat shock them in 42℃ water for 90 seconds.

• 4.

  Put them into ice for 5 minutes.

• 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.

• 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.10

• 1.

  Select monoclonal of Masp1 according to the protocol and add into 5ml LB liquid medium.

• 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.11 Induction of Masp1

• 1.

  Add 1ml bacteria solution into 99ml K+ Lb.

• 2.

  Table concentrator in 37℃ for 1-1.5 hr.

• 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 5 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.

• 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.12 Denatured purification of Masp1 and SDS-page

• 1.

  Purify the protein by denatured protocol.

• 2.

  Producing protein sample and load into the gel.

• 3.

  Gel electrophoresis.

• 4.

  Dye and wash the gel 1hr and scan using gel imager.

10.13 Transformation of Masp1

• 1.

  Take 50ul competent cell into EP tube on the ice to melt.

• 2.

  Then add 1ul plasmid into the competent cell and put them on the ice for 30 minutes.

• 3.

  After that, heat shock them in 42℃ water for 90 seconds.

• 4.

  Put them into ice for 5 minutes.

• 5.

  Add 300ul nonreactive LB fluid medium in to the tube and use shaker in 37℃ 200rpm for 40-60min.

• 6.

  After the solution cool down, take 200ul solution from the tube and coat on an anti-culture medium in the clean bench. Incubate in 37℃ for 12-16 hr.

10.14

• 1.

  Select monoclonal colonies of Masp1 according to the protocol and add into 5ml LB liquid medium.

• 2.

  Table concentrator in 37℃ 200rmp 12-16 hr.

10.15 Induction of Masp1

• 1.

  Add 1ml bacteria solution into 99ml K+ Lb.

• 2.

  Table concentrator in 37℃ for 1-1.5 hr.

• 3.

  Add 100ul IPTG to 0.5M/L. Table concentrator in 37℃ for 5 hr. Preserve seed cells with 1:1 glycerol freeze in -80℃.

• 4.

  Centrifugation 400ul bacteria solution, eliminating the supernatant and store in 4℃.

10.16 Denatured purification of Masp1 and SDS-page

• 1.

  Purify the protein by denatured protocol.

• 2.

  Produce protein sample and load into the gel.

• 3.

  Gel electrophoresis.

• 4.

  Dye and wash the gel 1hr and scan using gel imager.