Difference between revisions of "Part:BBa K1919001:Design"
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Two steps were needed in order to obtain BBa_K1919001. | Two steps were needed in order to obtain BBa_K1919001. | ||
Step 1, BBa_K1919000 was inserted into pET32a(+). The primers used for PCR are as following: | Step 1, BBa_K1919000 was inserted into pET32a(+). The primers used for PCR are as following: | ||
| − | F: 5'-CCGGGATCCATGAACTTCGCTAAAATCCTGTCTT-3' (BamH I site was added) | + | * F: 5'-CCGGGATCCATGAACTTCGCTAAAATCCTGTCTT-3' (BamH I site was added) |
| − | R: 5'-CCGCTCGAGTTATTATTTACCGATAGCTTTA-3' (Xho I site was added) | + | * R: 5'-CCGCTCGAGTTATTATTTACCGATAGCTTTA-3' (Xho I site was added) |
BamH I/Xho I Double enzymatic cutting was used for both pET32a(+) and BBa_K1919000. | BamH I/Xho I Double enzymatic cutting was used for both pET32a(+) and BBa_K1919000. | ||
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Step 2, RBS and 5 downstream tags as well as original CecropinXJ was inserted at the downstream of promotor J23105 with the same procedure as step 1. The primers used for PCR are as following: | Step 2, RBS and 5 downstream tags as well as original CecropinXJ was inserted at the downstream of promotor J23105 with the same procedure as step 1. The primers used for PCR are as following: | ||
| − | F: 5'-GCTCTAGAGCGAAGGAGATATACATATGAGCG-3' (Xba I site was added) | + | *F: 5'-GCTCTAGAGCGAAGGAGATATACATATGAGCG-3' (Xba I site was added) |
| − | R: 5'-TGCACTGCAGTGCAGACTAGTCGTTATTGCTCAGCGGTGG-3'(Pst I site was added) | + | *R: 5'-TGCACTGCAGTGCAGACTAGTCGTTATTGCTCAGCGGTGG-3'(Pst I site was added) |
Xba I/Pst I double enzymatic cutting was used for PCR product and Spe I/Pst I double enzymatic cutting was used for BBa_J23105. After enzymatic linkage, the construction of BBa_K1919001 was finished. Then this part was added to pSB1C3 backbone. | Xba I/Pst I double enzymatic cutting was used for PCR product and Spe I/Pst I double enzymatic cutting was used for BBa_J23105. After enzymatic linkage, the construction of BBa_K1919001 was finished. Then this part was added to pSB1C3 backbone. | ||
Latest revision as of 10:16, 12 October 2016
Constitutive promotor J23105 and downstream CecropinXJ
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 506
Illegal BamHI site found at 549
Illegal XhoI site found at 750 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Two steps were needed in order to obtain BBa_K1919001. Step 1, BBa_K1919000 was inserted into pET32a(+). The primers used for PCR are as following:
- F: 5'-CCGGGATCCATGAACTTCGCTAAAATCCTGTCTT-3' (BamH I site was added)
- R: 5'-CCGCTCGAGTTATTATTTACCGATAGCTTTA-3' (Xho I site was added)
BamH I/Xho I Double enzymatic cutting was used for both pET32a(+) and BBa_K1919000.
Step 2, RBS and 5 downstream tags as well as original CecropinXJ was inserted at the downstream of promotor J23105 with the same procedure as step 1. The primers used for PCR are as following:
- F: 5'-GCTCTAGAGCGAAGGAGATATACATATGAGCG-3' (Xba I site was added)
- R: 5'-TGCACTGCAGTGCAGACTAGTCGTTATTGCTCAGCGGTGG-3'(Pst I site was added)
Xba I/Pst I double enzymatic cutting was used for PCR product and Spe I/Pst I double enzymatic cutting was used for BBa_J23105. After enzymatic linkage, the construction of BBa_K1919001 was finished. Then this part was added to pSB1C3 backbone.
Source
The sequence of constitutive promotor J23105 comes from biobrick BBa_J23105. RBS and 5 downstream tags come from commercial plasmid pET32a(+). The sequence of CecropinXJ comes from biobrick BBa_K1919000.