Gas vesicles & Background

Gas vesicles

In our project, it’s our goal to make Escherichia coli buoyant in the presence of ocean concentrations of?the proportion of nitrogen and phosphorus. When our E. coli cells detect the change of the ratio of Nitrogen and Phosphorus before the Red Tide outbreaks, the gas vesicle gene inside will be transcribed and translate the gas vesicle protein to make the cell buoyant. In this section, we will mainly focus on the gas vesicle protein.
Gas vesicles are bacterial and cyanobacteria organelles consisting entirely of proteins that envelope a gas filled space. We had cloned the short gvp gene cluster form Planktothrix rubescens which only contain the gas vesicle structure gene gvpA and gvpC [1]. We created the new, shorter gene for floating, so that other teams can use this part and don’t have to think much about the length of plasmid (because the former Gas Vesicle Protein gene length is about 6Kbp, while ours is only 1Kbp).For further characterization of the gvp gene expression, we made the GvpC and GFP fusion protein and polycistron gene cluster. By detecting the expression of green fluorescent protein quantity we can quantify the expression of gvp genes.

Background

gvp gene cluster widely distributed in cyanobacteria, however as in other cyanobacteria, the gas vesicles of Planktothrix (Oscillatoria) spp.( Figure 1,2) from Lake Zurich comprise two proteins, the small, hydrophobic GvpA, which forms the ribs of the cylindrical structure, and the larger, hydrophilic GvpC, which attaches to the outer surface. [2] Although gvpA gene sequence had identified gvpC gene have three length variants genes gvpC-16 gvpC-20 and gvpC-28. In our project we using gvpC-20 gene which contain 99bp section beyond gvpC-16. [3] Different gvpC gene can translate different diameter of gas vesicle, which can help cell adapted to different pressure and suspended in different liquid layer. [3]
According to research, the different number of gvp copies may do a lot to the different buoyancy. In the iGEM history, several teams have involved in gas vesicles, but they all used the Bacillus megaterium’s gvp gene cluster (about 6kbp), which is much longer and more complex than Planktothrix rubescens’ gvp gene cluster. So this time, our team chooses the Planktothrix rubescens’ gvp gene as our source.[4]

Figure 1. The buoyancy of cultures of the Pla9402 of P.rubescens (taken by ouc-china)

Figure 2. Pla 9402 of P.rubescens cell in microscope (taken by OUC-China)

This year, our goal is to make the genetically modified Escherichia coli float after detecting the change of nitrogen/phosphorus ratio. So in this section, our aim is to make E.coli float both effectively and economically. In 2007 Melbourne iGEM Team, they had send Gas Vecicle Protein cluster to iGEM foundation. But it’s about 6Kbp. In consideration of the length of plasmid is limited, we decided to firstly make the gene cluster as short as possible, and then enhance the Gvp’s expression quantity. So we want to make it shorter. Also the artificial gene expression loop should be robust and can be insulated from other natural noises.
To make the gene cluster shorter and isolate it from other possible interference, we need to take out the gvpA and gvpC gene separately. Then, they will be assembled with other iGEM standard parts and tested through SDS-PAGE and other approaches in order to confirm its expression and assembly. It’s our ultimate goal to regulate the expression of GvpA/C and figure out the most appropriate gvpA/gvpC’s expression ratio and expression quantity.

Now, our goal of this project can be divided into different 4 levels.
(1)Clone gvp cluster from Planktothrix (Oscillatoria) spp , transform it into E.coli and make it buoyant.
(2)Clone gvpA and gvpC genes from genomic, ligate artificial cluster and make E.coli buoyant.
(3)Get GvpA and GvpC protein in transformed E.Coli.
(4)Ligate two inducible gene circuits with gvp genes, regulate the expression of Gvp protein and detect the expression of Gvp by detecting the fluorescence of GFP.

Figure 3 Project design flow chart