Part:BBa_K1668009:Design
plu0840-device
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Source
The device is composed of arabinose inducible promoter pBad, CDS plu0840 and reporter mCherry. As the mCherry is already in pSB1C3, we linearize the part with enzyme PstI as backbone. The other two genes are amplified by PCR separately from parts BBa_I0500 (pBad), BBa_K1668006 (CDS plu0840).
We got pBad from Part Registry and the CDS plu0840 is a part we have constructed ourselves.
Design Notes
PCR
We use primer pBad F / pBad R to amplify pBad and standard F / plu0840-right R for CDS plu0840.
Seamless assembly
We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol. By this way prefix sequence, pBad, CDS plu0840 and front twenty bases of mCherry sequence can be ligated seamlessly.
pBad F (F, 5’-3’): ATTCGCGGCCGCTTCTAGAGTTATGACAACTTGACG
pBad R (R, 5’-3’): GCTAGCCCAAAAAAACG
standard F (F, 5’-3’): CCGTTTTTTTGGGCTAGCAGAAAGAGGAGAAATACTAG
plu0840 R (R, 5’-3’): CTAGTATTTCTCCTCTTTCTTTATTTCGATGGGGTCAAAG
Transformation and confirmation
After seamless assembly, standard plasmid pSB1C3 containing plu0840 gene was transformed into E.coli DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used mCherry F(a primer on the beginning of mCherry)/ VR as the universal primers for the plu0840 is too long to amplify all the parts sequence with VF2/VR .Primers are shown below. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.
tcdA1_M1(F, 5’-3’): CTGCCAATCTATGCCACACC
VR(F, 5’-3’): ATTACCGCCTTTGAGTGAGC
Plasmid map
