Template:SBB12 part sbb1224

From OpenWetWare

Jump to: navigation, search
Partname:     sbb1224
Featurename:  DmrB/FKBP
Genename:     dmrB
Source:       synthetic

You will need to synthesize this one using PCA with full E. coli codon usage. What it encodes is the FKBP domain from DmrB protein. The DmrB binding protein binds to a ligand called B/B Homodimerizer (or AP20187). The sequence is available as part of the iDimerize Inducible Homodimer System optimized for mammalian codon usage. You'll use their sequence info at this site to get the peptide sequence, and then synthesize it for use in E. coli.

This part encodes a homodimer domain

You are going to put the homodimer on the C-terminus of a truncated ToxR. For this part, you're going to go off BioBricks. Kindov. Your final product will be a BioBrick, but you are going to use an ad hoc procedure to create it. You should examine this illustration which refers to two sequences: pBca9525-Bca1834 and pBca9525-Bca1839. The important thing here is that you think through the translation frame of the final product, and make sure that you add some linker between your sequence and the sequence 5' to it. You should also remove the start methionine from you homodimer, unless your project description explicitly says not to. You should include the stop codon.

The product of your construction file should resemble pBca9525-Bca1839 in terms of it encoding a full expression cassette containing a ToxR fusion protein with your designed feature. If you were given part sbb1293, your product would be called pBca9525-sbb1293.


Construction File

PCA1 on dmrb.01.o01-dmrb.01.o14           (pca1)
PCA2 with dmrb.01.o01/dmrb.01.o14 on pca1 (367 bp, pca2)
Digest pca2                               (NheI/BamHI, L, 1224dig)
Digest pBca9525-Bca1834                   (NheI/BamHI, L, vectdig)
Ligate 1224dig + vectdig, product is pBca9525-sbb1224
----
>dmrb.01.o01	CCATAGCTAGCGGCAGTGGATCTGCTTCTCGTGGTGTTCAGGTTGAAAC
>dmrb.01.o02	GACCGTCACCCGGAGAGATGGTTTCAACCTGAACACCACGAGAA
>dmrb.01.o03	ATCTCTCCGGGTGACGGTCGTACCTTCCCGAAACGTGGTCAGAC
>dmrb.01.o04	CCAGCATACCTGTGTAGTGAACAACGCAGGTCTGACCACGTTTCGGGA
>dmrb.01.o05	GTTCACTACACAGGTATGCTGGAAGACGGTAAAAAAGTTGATTCTTCTCGTG
>dmrb.01.o06	CATGAATTTGAACGGTTTGTTACGGTCACGAGAAGAATCAACTTTTTTACCG
>dmrb.01.o07	ACCGTAACAAACCGTTCAAATTCATGCTGGGTAAACAGGAAGTTATCCG
>dmrb.01.o08	CTGAGCAACACCTTCTTCCCAACCACGGATAACTTCCTGTTTACCCAG
>dmrb.01.o09	TTGGGAAGAAGGTGTTGCTCAGATGTCTGTTGGTCAGCGTGCTAAAC
>dmrb.01.o10	GTAAGCGTAGTCCGGAGAGATGGTCAGTTTAGCACGCTGACCAACAG
>dmrb.01.o11	ATCTCTCCGGACTACGCTTACGGTGCTACAGGTCATCCGGGTATCATC
>dmrb.01.o12	AAAACCAGGGTAGCGTGCGGCGGGATGATACCCGGATGACCTGTA
>dmrb.01.o13	CGCACGCTACCCTGGTTTTCGACGTTGAACTGCTGAAACTGGAATAAGGAT
>dmrb.01.o14	CACTGGGATCCTTATTCCAGTTTCAGCAGTTC
>pca2
CCATAGCTAGCGGCAGTGGATCTGCTTCTCGTGGTGTTCAGGTTGAAACCATCTCTCCGGGTGACGGTCGTACCTTCCCGAAACGTGGTCAGACCTGCGTTGTTCACTACACAGGTATGCTGGAAGACGGTAAAAAAGTTGATTCTTCTCGTGACCGTAACAAACCGTTCAAATTCATGCTGGGTAAACAGGAAGTTATCCGTGGTTGGGAAGAAGGTGTTGCTCAGATGTCTGTTGGTCAGCGTGCTAAACTGACCATCTCTCCGGACTACGCTTACGGTGCTACAGGTCATCCGGGTATCATCCCGCCGCACGCTACCCTGGTTTTCGACGTTGAACTGCTGAAACTGGAATAAGGATCCCAGTG
Personal tools