Part:BBa_K1668008:Design
tcdA1-device
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
Illegal NheI site found at 8257
Illegal NheI site found at 8590 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1651
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 7691
Illegal NgoMIV site found at 8351
Illegal AgeI site found at 979
Illegal AgeI site found at 3527 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Source
The device is composed of arabinose inducible promoter pBad, CDS tcdA1 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 tcdA1 is a part we have constructed ourselves.
Design Notes
PCR
We use primer pBad F/pBad R to amplify pBad and standard F/ tcdA1-right R for CDS tcdA1.
We use primer tcdA1-left L and tcdA1-left R to amplify the left side of gene., tcdA1-middle L and tcdA1-middle R to amplify the middle part and tcdA1-right L and tcdA1-right R to amplify the right side. Primers are shown below.
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 tcdA1 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
tcdA1 R (R, 5’-3’): CTAGTATTTCTCCTCTTTCTTTATTTAATGGTGTAGCGAA
Transformation and confirmation
After seamless assembly, standard plasmid pSB1C3 containing tcdA1 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 tcdA1_M1(a primer on the right part of tcdA1)/VR as the universal primers for the CDStcdA1 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
