Difference between revisions of "Part:BBa K2615019"

Revision as of 11:16, 16 October 2018

MiniToe polycistron B: a new system using miniToe.

Background of 2018 OUC-China' project——miniToe family

This year, we design a toolkit named miniToe family focused on translational regulation, which is composed of a RNA endoribonuclease (Csy4) and a RNA module (hairpin). In our project, the cleavage function of Csy4 releases a cis-repressive RNA module (crRNA, paired with RBS) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. A Ribosome Binding Site (RBS) is an RNA sequence to which ribosomes can bind and initiate translation.

Fig.1 Csy4 and hairpin can form a stable structure.

We design miniToe family to meet the aim, "One system, diverse expression". This means by using one system we can even achieve flexable expression of target gene. So we further design four Csy4 mutants by point mutation( Csy4-Y176F, Csy4-H29A, Csy4-F155A, Csy4-Q104A). At the same time, we redesign 5 different hairpin mutants named miniToe(5 different types of Csy4 recognition sequence) which can be recognized and cleaved by Csy4 mutants. ( miniToe-1, miniToe-2, miniToe-3, miniToe-4, miniToe-5, miniToe-WT).

miniToe polycistron

Having designed different hairpin structures and finished experimental verification, we think it is a good idea to apply it to the polycistronic to achieve more functions by using miniToe. The polycistronic structure can meet the requirements of simultaneous and large-scale expression of different genes. We insert the hairpin structure(miniToe) into the polycistron and apply the interaction between the hairpin structure and the Csy4 enzyme to achieve the regulation of different ratio of multiple gene expression levels, meeting the requirements of practical application.

Fig.2 The working process of miniToe polycistron.

From now, we construct two polycistron(miniToe polycistron A and miniToe polycistron B) by inserting three miniToes before, between and behind two CDS. We use miniToe-WT(BBa_K2615020) before and behind miniToe polycistron B(BBa_K2615018), meanwhile the miniToe-6(BBa_K2615017) is used between two CDS. And we use sfGFP and mCherry to test our system. The result shows we can totally change the origin ratio of two genes.

Result

The setting of test groups and control groups

The miniToe polycistron is a new method designed by OUC-China this year. By inserting miniToe structure into circuits, more than one gene can be regulated. In this system, we focus on the ratio of gene expression. We tested polycistron system by two target genes, sfGFP and mCherry. Two kinds of groups have been set. One is the bicistron circuit without miniToe structures. The other group is for test which have miniToe system.

This year, we have two kinds of miniToe polycistron, miniToe polycistron-A and miniToe polycistron-B. In the future, we will test more polycistron based on miniToe family.

Fig.3 The two test groups. Group A is the control group without miniToe system. Group B is the test group with miniToe system.

Proof of functions

The result by microplate reader has been shown in the Fig.4. After culturing for 10 hours, the rate of fluorescence intensities by sfGFP/mCherry have been changed by miniToe family. The group A is a control group without miniToe family. The ratio of fluorescence intensities by sfGFP/mCherry is about 6.81 which means the gene near the promoter has much higher expression than the gene far from promoter in a normal polycistron. The test group-polycistron A has been changed by miniToe structure because the ratio of fluorescence intensities decrease to 4.38. To our surprise, the test group-polycistron B shows the significant change whose rate is about 2.82. It means the ratio of gene expression can be regulated by miniToe family. In the future, the miniToe family create more possibilities in regulating the ratio of gene expression.

Fig.4 The ratio of fluorescence intensities by sfGFP/mCherry. Error bars represent standard deviation of three biological replicates.

Sequence and Features