G1 phase magic with cell synchronization device and CRISPRi induced alternative splicing device.
clb5 +d-box +GFP + src1 intron + tom40 + cca + gal + dCas9 + cca + snr52 + sgRNA(Hub1) + sup4
This part mainly contain a promoter of GAL, a gene of Sic1 and a terminator of ADH1. It can use to regulate cell cycle, prolong the cell cycle.
mutate the multi-phosphorylation site of Sic1.
clb5 Promoter + D-box + GFP + SRC1 Intron+GG + Targeting Peptide to Mitochondria outer membrane + T
Devices express dCas 9 protein and sgRNA targeting to gene HUBI.
Saccharomycescerevisiae TBY-1 terminator with stopcodon
Saccharomycescerevisiae terminator with stopcodon
Targeted to HUBI gene ATG downstream position of the 12 bp sgRNA, cooperate to dCas 9 protein, inhib
This part consists of promoter SNR52, sgRNA coding sequence and SUP4 terminator.
J63006(Gal I promoter)+k1051800(dCas 9)+k1051008(terminator)
TAACT AGGTG GTCCT TCCAT GAAAGTTTTTTTTTTTTGAAGATGTTCTTTTTTTTTTTTT GGGCCTCATTACAATGTAAGAGGTTATTATGTGTTACATGAGTAAAGCTAATTGACGCAA GCAGGTAATGAAGAAAGCCCTATATTACCGTATAGACAATGGGCGCGTCCCTAAAGAGGA TACCAAATAGCAGTTTTACGCGTACCACAAAATTTAACGACCAACGCGTATTCTTCGCGT TTTTTTTTTGCGACGGTAAACAAAAAAAGGGCCCTCTGCGGTAAAACAAGTGGGTCTACC ACAGGACACAGGACTTGGTTGTTTCTACTCTCCCTGTGTATCCTACAATTGGCAATTGGC AATTCGCAACCTGCGTTTAAATATTTACCGTTTTAGCACATTGATTACCTTTTTTGCTAG GGTTAAGTTTTTGCCTTTTTCTTATTAACATATATATAACTGCTTCATTTCATACAAGTG ACTTTTATCTTTTATTAAATAATAACAAGCTTCACTAATCTAGCGTTCATACAACATACA AATCAGTGTGTTGAAAATTATTATTCTCTGATATTCTCTCCCTCCTTTTAAATTTTTAAA
It is a promoter which promotes transciption in G1/S of the yeast cell cycle .If you want to express your proteins in G1/S of the yeast cell cycle ,you can add the sequence which is actived by clb6 before your gene sequence ,then put them in a yeast plasmid .After transformation ,you can get the protein you want from the metabolic product of the Bacterial you use for transformation .
We get the sequence which is actived by clb6 from a yeast database (http://www.ncbi.nlm.nih.gov/CBBresearch/Landsman/Cell_cycle_data/) .You can amplify it from the yeast gene using PCR .See the primers as follows : upstream:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTAACT AGGTG GTCCT TCCAT downstream:GTTTCTTCCTGCAGCGGCCGCTACTAGTATTTAAAAATTTAAAAGGAGG
5' - AATTC GCGGCCGC T TCTAGA GTACAAAGGTTGGCCTTAAACAATGTAACAAATACGACA ACTAGT A GCGGCCG CTGCA- 3' 5???- G CGGCCGC T ACTAGT TGTCGTATTTGTTACATTGTTTAAGGCCAACCTTTGTAC TCTAGAA GCGGCCGC G-3???
Mutate seven phosphorylation site of Sic1, stopping the cell cycle in the G1 phase.
Mutate seven phosphorylation site of Sic1 and one site of Spe1's restriction site.
S. cerevisiae CAA terminator with stopcodon
protein dCas9.Two mutations,D10A and H841A,from Cas9 gene without stop codon.
The Cas9 gene was got from Mr. Kai Tian,BGI Shenzhen China. The gene was splited into three parts to synthetize optimizing the codons in eucaryon.
Protein dCas9 is the key of the technology CRISPRi,and this part is the gene of the dcas9.Delete its ending codon is to provide an convenient way to add other protein domain to improve the function of dCas9.
To mutation four Restriction Enzyme cutting sites(SpeI) and two codons(D10A & H841A),we design PCR primers to mutate them by PCR using Phution DNA polymerase. Refrence http://www.ncbi.nlm.nih.gov/pubmed/?term=repurposing+CRISPR+as+an+RNA-Guided+Platfrom
TOM40;targeting peptide to mitochondria outer membrane
Saccharomyces cerevisiae genomics sequence
After added with 23 prefix and suffix, it can be used as a transporter to the mitochondria inner membrane. Also, when joining with different flourescent proteins, they can light the inner membrane using such proteins.
Intron from SRC1 and is added GG to form a Ser codon
Conversion of the iGEM parts registry to SBOL2.1