Difference between revisions of "Part:BBa K1431301:Design"

Revision as of 20:09, 26 October 2014

TRE-3G promoter+SV40 PolyA, an ideal controller of mammalian gene expression with Tet-On 3G protein

Design Notes

For BbsI site, we have a series same design sequence. See the mechanism of BbsI site work below.

If you want to insert a coding sequence between promoter and polyA, the prefix of forward primer you design must contain GAAGACNNtaaaNNNN and reverse must contain GAAGACNNagttNNNN. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into promoter and polyA seamless. The PCR product must be such sequence, including the protect bases, shows below.

And the reason why we designed that because we want to make a seamless gather between promoter and protein or any number nucleotides. That depend on which will make the highest efficiency.

Protocol

1. Design the primers show below

TRE-3G promoter forward: T TCTAGA G TTTAAACTTTACTCCCTATC
TRE-3G promoter reverse: ACGCA GGATCC AT GAAGAC GATTTACGAGGGTAGGAAGTG
SV40 PolyA forward: ACGCG AGATCT AT GAAGAC CCAACTTGTTTATTGCAGCTTA
SV40 PolyA reverse: AAAACTGCAG CGGCCGC T ACTAGT A TCCATGCCGAGAGTGATGAA

Sequencing Results

We sent fresh bacteria broth for sequencing using standard Biobricks sequencing primer VF2/VR. The sequencing cooperation we used is Invitrogen Guangzhou filiale.

Sequence of sequencing primer we used:
VF2: tgccacctgacgtctaagaa
VR: attaccgcctttgagtgagc

The result shows the same sequence with our ideal design.

Source

The plasmid was from our instructor, Huangwei's lab. And we design primers to copy down by PCR.

References

[http://www.clontech.com/US/Products/Inducible_Systems/Tetracycline-Inducible_Expression/Tet-On_3G Clontech]