Team:Shenzhen BGIC ATCG/stories

The Magic

Life, the most brilliant magic in the universe, celebrated with the ability of reproduction and revolution. And the magic, is based on a sophisticated mechanism, called cell cycle. Most genetic reactions in a cell are regulated, directly or indirectly, by cell cycle. As we wish to build artificial lives, it is worthwhile to learn from the exquisite design of creature, to make use of cell cycle. As a pioneer study, we try to performing a "Cell Magic", by capturing cell cycle with fantastic reporters. By grasping the usage of cell cycle tools, we are promised to direct refined actions in a cell. For instance, when producing Paclitaxel (harmful to centrosome in S phase) with a cell factory, we may able to transport it out before it comes detrimental.

So this is our project, the "Cell Magic", to engineer two versions of cell cycle based magic in both budding yeast and E. coli. We will present the blueprint of our stories by the yeast version.

Version #1, Degradation Tag X, Targeting Peptides X, Cell Cycle Regulator X

Promoters of cyclins, which can initial transcription in specific phase in a cell cycle, are selected to produce different fluorescence proteins in G1/SG2/M phases respectably. However, in our first version of story, with low natural degradation rate, fluorescence proteins would remain in cell after their promoters stop working. So colors for each phase cannot be distinguished, as the figure shows.

Version #2, Degradation Tag √, Targeting Peptides X, Cell Cycle Regulator X

In the second version, degradation tags are added to the reporters to solve this problem. All fluorescence proteins are thought to be degraded in 15 minutes which is far less than the period of a cell cycle.

Version #3, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator X

While we are not sure if degradation tags can work as expected, we try to turn the time magic into a time-space one. Reporters are located to different cell structures by targeting peptides. Different phase with different color shines in different organelle, that is the third version.

Version #4, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator √

To capture the magic macroscopically, we developed a microfluidic device to synchronize all cells into the same phase (G1). So synchronization devices was added to the forth version.

To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device (a translational unit of a cyclin regulator), will splice the targeting peptide tail when cell are being synchronized to G1 phase, so all cells turn green; while synchronization device not working, targeting peptide remains in the reporter so only mitochondria turn green. In this way, the cell phase and statues can be reported by fluorescences.

Cyclin Promoters

Yeast Version

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

Previous study (Wolfsberg et al., 1999) 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 Spellman et al., 1998)

E. coli Version

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.The well-regulated cell cycle in E.coli consists of three key events: DNA replication, nucleon segregation and the initiation of cell division. The replication initiator is DnaA protein; the second step relates to MukB protein and the cell division activator is the FtsZ protein. In our project, we take full aadvantage of the two initiators DnaA and FtsZ - using their promoter to control the transcript of specific genes binding behind them in the first step or last step of E. coli cell cycle.

DnaA protein

As mutation strain demonstrated, dnaA gene is absolutely required for initiation at oriC. When the concentration of DnaA increases, there is increased initiation?, indicating that DnaA protein is the switch for initiation (Atlung, Løbner-Olesen, & Hansen, 1987). Biochemical studies have shown that, with the help of accessory proteins, DnaA protein binds to five sites within oriC, therefore, leading to strand opening of a region containing three 13-mer repeats. This opening results in the begin of bidirectional replication (Atlung, Løbner-Olesen, & Hansen, 1987).

FtsZ

The cell itself seems to regard FtsZ gene expression as the commitment to division, because of the fact that endogenous division inhibitors have FtsZ as target(Bi & Lutkenhaus, 1990). Increasing FtsZ concentration resulted in excess cell division, with the cell size decreasing (Ward & Lutkenhaus, 1985), vice verse. Thus the critical role of FtsZ in division is similar to that of DnaA in replication initiation. FtsZ protein exists randomly throughout cytoplasm during cell elongation, while polymerizes into a membrane-associated ring at the precise site of cell division (Bi & Lutkenhaus, 1991). Just before the appearance of a visible constriction. The ring decreasing along with the septation and FtsZ is dispersed again at completion. Such ring formation is presumed to be essential for cell division, since it cannot be observed in the presence of the cell division inhibitors (Bi & Lutkenhaus, 1993) but is always present when and where septa are formed.

Reporter Modification

The XFP, as reporter in our project has been modified through

1)removed the stop codon in them, 2) modified with 23# prefix and suffix， and then 2)added the terminator within stop codon to them.

Furthermore, all biobricks were added with 23# prefix and suffix, which ensures it to be standard.

Degradation Tags

Yeast Version

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 proteas