Part:BBa_K1201001:Design
Derived from serratia marcescens
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 186
Illegal BamHI site found at 1005 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 111
Illegal NgoMIV site found at 550
Illegal NgoMIV site found at 556
Illegal NgoMIV site found at 649
Illegal NgoMIV site found at 1359 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 67
Illegal SapI.rc site found at 757
Design Notes
ChiB was received in an IPTG inducible expression plasmid, pQE-60. The parts contain a pstI site. This site was removed via a site directed mutagenesis using overlapping primers. In the gel below lane 5 shows one half of ChiB which was amplified using a forward primer and a reverse muatgenic primer. Lane 6 shows the second half of ChiB which was amplified using a reverse primer and a forward mutagenic primer.
In the gel below in lane 3 is the full gene (ChiB). The two halves were mixed together and allow to self primer giving a template. This template was then amplified using the outer primers. ChiB was then gel extracted and ligated to pSB1C3.
Characterization
We used the Chitin Azure Assay to test the activity of the chitinase genes. In the diagram on the left is the whole cell culture of E. Coli Top10 strain expressing IPTG induced ChiA in pQE-70, ChiB in pQE-60 and ChiC in pQE-60. 200uL of each culture was added to 600uL of chitin azure assay substrate. The reaction was incubated at 37C for 72 hours. 400ul of ChiB and 400uL of chinin azure assay substrate were also incubated at 37C for 72 hours. ChiB did not produce a reaction when tested on its own, but a positive result was received when all three Chi genes were in one reaction mixture.
Source
Serratia marcescens; gifted by Dr. Frank Sargent from The University of Dundee