AarC

7.1

1.Use pcr to clone the AarC gene synthesized by Synbio Technologies Co., Ltd and added

restriction sites EcoR I & Xho I.

2.Use E.coli DH5a to amplified vector pET32.

7.2

1.Extract plasmid pET32 from E.coli DH5a.

2. Use EcoR I & Xho I to cut pET32a and pcr fragment. Line 2 is AarC fragment. Line 3 is pET32 cut

by EcoR I. Line 4 is cut by Xho I. Line 5 is cut by both enzymes.

3.Pure digestion products.

4.Connect these two fragments by T4 ligase.

7.3

1.Transform the connection product into E.coli DH5a.

7.4

1.Check positive results by colony PCR

2.Pick out colony and amplify them.

7.5

1.Extract recombinant plasmid.

2.Use PCR to check plasmid.

3.Transform the recombinant plasmid into E.coli BL21.

7.7

1. Induced protein expression by IPTG.

2.Check the expression by SDS-PAGE. Line 1 is supernate after cell disruption. Line 2 is precipitate.

Line 3 is whole cell. Line 4, 5, 6 are half loading.

7.8

1.We find that pET32 have type a, b and c. Our vector is pET32a. That means our protein have

frameshift mutation. We can not use it.

7.11-7.17

1.Use PCR to clone the AarC with His-tag and T7 terminator from recombinant pET32a.

2.Add promoter J23100, J23112, J23113, J23114 and RBS before the AarC by PCR.

3.Add restriction sites Xba I & Pst I.

4.Use PCR to product linear pSB1C3

5.Use Xba I and Pst I to cut the fragments. We found that AarC fragment was cut off.

7.19-7.23

1.We check the gene by gene sequencing. It has a Pst I restriction site.

7.25-8.5

1.Use overlap extension PCR to remove this restriction site:

(1)Long fragment: From start to Pst I.

(2)Short fragment: From Pst I to end.

(3)Two fragments have 30bp overlapping sequences.

(4)Mix two fragment equally and let them to be template for PCR.

(5)Find the best PCR condition.

8.6-8.10

1.Connect AarC after repair with pSB1C3 and transform it into E.coli BL21.

2. Check positive results by colony PCR.

3.It failed.

8.12

1.Restart OEPCR. Product two fragments.

2.Pure fragments.

8.13

1.Use two fragments as template to PCR

2.Pure the fragment

8.14

1.Use Pst I to cut the Fragment.

8.15-8.17

1.Add restriction sites Xba I & Xho I by PCR.

2.Cut the pET32a and AarC fragment by Xba I & Xho I.

3.Connect the fragment with pET32a for adding His-tag and T7 terminator.

4.Check positive results by colony PCR. There have one positive result.

5.Amplify the positive one.

8.18-8.20

1.Extract the plasmid of that positive one.

2.Add promoter J23100, 112, 113, 114 and RBS before AarC by PCR.

8.21-9.2

1.It was hard to connect the AarC expression fragment with pSB1C3, both of them are approximate

2000bp.

9.3-9.13

1.Wait for Gibson assembly master mix.

2.Use PCR to add extra fragments on AarC and pSB1C3 for Gibson assembly.

9.14-9.17

1.Use Gibson assembly to build recombinant plasmid.

2.Check the positive results by extract plasmids.

9.18

1.Use PCR to check the plasmids.

2.Transform the plasmids into E.coli BL21.

9.20

1.Extract plasmids from BL21 to check it.

9.25

1.Use HPLC to check the consistency of acetic acid in the LB media.