Team:Shenzhen BGIC ATCG/stories

The Magic

This year, our project named "Cell Magic". We engineered the budding yeast in time level as well as space level. "Cell Magic", like a movie,can be divided into two parts: the actor related and the time regulation related one. In the first part, our actor, signal peptides, were all made up with colorful clothes by fluorescent proteins. Furthermore, the intron and degradation biobricks as make-up artists can decide how the clothes be matched. Through these steps, our actors may wearing in green, yellow or even mixed, appear in their specific sub-locations of yeast cell. With respect to the time level, we took advantages of the natural cell cycle in budding yeast to improve the time process to be more suitable for our movie.In general, the movie director, the promoters from five cycline gene, can decide the expression of downstream gene in G1 and G2 phase phase, respectively. Also, freezer Sic1 help our movie stay a longer time in the G1 phase which is important for the actor performing their stories. And the last tools we utilized is the microfluidics, through which numerous cells can project our "Cell Magic", thus ensuring our observation by naked eyes.

Cyclin Promoters

Cell cycle is a complex process and can be separated into G0, G1, S, G2, M phase. In each phase, distinct transcription factors help the phase-specific gene express through recognizing their promoters. Thus, these promoters are phase-specific too and can be fused with other genes in order to express such gene in a defined cell cycle phase. As we all know, different proteins are planned to be expressed along the cell cycle. And their mRNAs are transcripted through the specific promoters. These promoters locate at their upstream sequences and can be recognized by the RNA polymerase, which can initiate such process. There are databases containing the upstream 600 amino acid sequence in the upstream of DNA, which can be transcript in the G1, S, G2 and M phase, respectively [http://www.genome.jp/kegg-bin/show_pathway?org_name=sce&mapno=04111&mapscale=&show_description=hide]. Previous study 【Candidate regulatory sequence elements for cell】demonstrated that cell cycle specific promoters posses their conserved five to six base pairs. Thus, we found the high-confidence 600 promoter-containing sequences in database that harbor the paper-mentioned 5/6 bp sequence. Consequently, we pick up the upstream 600bp sequence of cln2, cln3, clb2, clb5 and clb6. The Clb2 gene is highly expressed in G2 phase, and the genes are very strongly induced by GAL-CLB2, whereas GAL-CLN3 appears somewhat repressive 【Comprehensive Identification of Cell Cycle–regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray Hybridization】

Reporter Modification

Degradation Tags

=== Make-up Artists - Degradation Peptide === In yeast cell, protein degraded through several mechanisms, a major one is the ubquitin- pathway. In detail the signal in substrates can be recognized by the enzyme E3, which transport the ubiqutin from E2 to such protein. And through such way, the ubiquited protein would finally be degraded by the proteasome. [http://upload.wikimedia.org/wikipedia/commons/5/5b/Ubiquitylation.svg] ***PEST There are two types of cyclins in the budding yeast. One kind contains PEST sequence at C-terminal: Cln1, Cln2, Cln3, Pcl1, Pcl2. Another kind is Clb1~6 which posses 9 conserved amino acid sequence in N-terminal named D-box (destruction box). The D-box are necessary for later kind cyclins degradation in M phase and can function in same way when combined with other proteins. In our study, we utilized the D-box, PEST sequence and ubiquitin-mediated pathway for fluorescent proteins degradation. 【蛋白质的磷酸化作用和泛肽化降解作用与芽殖酵母的调控】 ***Poly-ubiquitin Ubiquitin, a highly conserved 76-residue protein, was involved in the ubiquitin–proteasome pathway of protein proteolysis, which is a fundamental way for protein turnover, signal transduction and cell cycle control. For example, the degradation of CDC34 through such way is necessary for S phase start; another instance is that APC\C(anaphase-promoting complex\cyclosome) is degraded through ubiquitination, which is the foundation of M phase beginning. And both activated by Cdc28-cyclins. The mechanism of ubiquitin-mediated protein degradation can be separated into three continuous enzyme catalyze steps. First 1) a ubiquitin is activated by the E1 (ubiquitin-activating enzyme) through a thioester linkage，and then 2)added into the a small ubiquitin-carrier E2. Finally,3) through the E3 ubiquitin protein ligase, this ubiquitin complex was conjugate to the ε-amino group of lysine residues in substrate proteins, forming a glycyllysine isopeptide bond【Roles of ubiquitin-mediated proteolysis in cell cycle control.】In some conditions, even E3 enzymes themselves carry ubiquitin as a thioester 【A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase】 In our project, we design a poly-ubiquitin(5 ubiquitin combined) biobricks which can directly degrade the proteins fused with it. ***-D-box The best studied substrates of ubiquitin- and APC/C-mediated proteolysis are the mitotic cyclins【The role of cyclin synthesis and degradation in the control of maturation promoting factor activity 】Mitosis-exit inducer, a cyclin B, are highly dependent on its 90 residues for ubiquitin-mediated proteolysis. Analysis of its N-terminal region demonstrated the sequence essential for cyclin proteolysis, called ‘destruction box’(D-box). Meanwhile, when cyclin destruction started, substrates containing D-boxes were rapidly poly-ubiquitinated. The consensus motif in B-type cyclins is RXALGXIXN. For much of the cell cycle, the D-box may not be recognized with high affinity, and when it is recognized, the ‘bait’ construct becomes highly unstable. 【The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor】 When the D-box exists in the protein N-terminal, this protein is recognized more easily by the E1 and then captured into the ubiquitin-mediated degradation pathway. Thus, we combined it to the fluorescent proteins’ N-terminal to fasten their degradation and avoid veiling the following fluorescence.

Targeting Peptides

Cell Cycle Regulator

Alternative Splicing Device

Microfluidic Device

Cell Synchronization