Part:BBa_K1668005:Design
CDS tcdA1
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7035
Illegal NheI site found at 7368 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 429
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 6469
Illegal NgoMIV site found at 7129
Illegal AgeI site found at 2305 - 1000COMPATIBLE WITH RFC[1000]
Source
The CDStcdA1 gene was amplified by PCR by three pieces to remove three enzyme site EcoRI in its original squnce.
The template is genomic DNA extracted from strain Photorhabdus luminescens TT01. We got the strain from Shandong University.
Design Notes
PCR
The CDStcdA1 gene was amplified by PCR by three pieces to remove three enzyme site EcoRI in its original squnce. The template is genomic DNA extracted from strain Photorhabdus luminescens TT01.
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, tcdA1-left, tcdA1-middle, tcdA1-right, and suffix sequence can be ligated seamlessly.
tcdA1-left F (F, 5’-3’): AATTCGCGGCCGCTTCTAGATGAACTCGCCTGTAAAAGAGA
tcdA1-left R (R, 5’-3’): AAATTCACAACAAGCGGATACATTACAC
tcdA1-middle F(F, 5’-3’): TATCCGCTTGTTGTGAATTTAATCCGG
tcdA1-middle R(R, 5’-3’): TAACATTAGCGGAAAATTCCATCACATAACCTGTTGC
tcdA1-right F(F, 5’-3’): GGAATTTTCCGCTAATGTTATGAATACCGAAGC
tcdA1-right R(R, 5’-3’): CTGCAGCGGCCGCTACTAGTATTATTATTTAATGGTGTA
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
