Part:BBa_K737010

Gvp cluster

This 1 kbp part contains 2 open reading frames coding for gas vesicle genes (gvpA gvpC-20) from Bacillus Planktothrix rubescens strain BC-Pl 9402. Promotion of the sequence results in expression of gas vesicles, organelles made entirely out of protein. These organelles contain gas and therefore provide bouyancy to the cell. This slows the rate of settling of the cells in different water layer from Lake Zurich by gas vesicles, see Walsby 2002 .

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Background

GVP gene cluster widely distributed in cyanobacteria, however As in other cyanobacteria, the gas vesicles of Planktothrix (Oscillatoria) spp from Lake Zurich comprise two proteins, the small, hydrophobic GvpA, which forms the ribs of the cylindricalstructure, and the larger, hydrophilic GvpC, which attaches to the outer surface.[1] 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.[2] Different GVPC gene can translate different diameter of gas vesicle, which can help cell adapted to different pressure and suspended in different liquid layer.[2]

Experimental Data

Figure 1 and 2 the expression situation of the Gas vesicle gene cluster in escherichia coli JM109,from the picture, we can see obviously gas vesicle due to the expression of bacteria produce clear separation phenomenon. The picture on the right of the experimental group were liquid surface layer and intermediate layer of fluid sampling results, we can see that the fluid layer surface bacteria volume density is obviously higher than that of the middle layer of fluid.

Figure 3 Trace stratified take liquid apparatus. It is made up of a 10 ml volumetric syringe and five a 1 ml volumetric syringe composition. It can be used as a culture cell containers and can be performed every layer of the synchronous sampling the aim is to avoid the sampling process caused by vibration caused by fluid layer surface thallus under. the whole device can make quantitative analysis more accurate.(ouc-china team)

Figure 4 the picture shows the gas vesicle in the cells after expressed in the overall trend. We in the cultivation of the cells into logarithmic phase after transferring them to test tube, quiet place culture 36 hours after using Trace stratified take liquid apparatus at the same time to each liquid layer for sampling. Then each layer of fluid samples of blood count the number of cells with statistics. Results show that gas vesicle due to the existence of the cell.

Figure 5 Graph of OD measurements at 600nm of both experience group and control group. From the experimental results can be seen in the liquid surface can see cell number have significant abrupt transformation.

Discussion and future work

In our project there have still some unfinished part for example in the genome of P.rubescens it has a 2kb length of gvp gene cluster which contain the gvpC-16 gene. However because competitive inhibition in the reaction of PCR, we can not get gene cluster and the gvpC-16 gene. On the other hand about the P.rubescens gene cluster There is also part of the structure are not clearly so we should try to use has been standardized GVP generator part in escherichia coli expressed in verified that we standardized part availability. As for experiment of Gas vesicle protein quantitative analysis, however we did not establish GFP protein expression quantity and the relationship between the Fluorescence Unit. So we can't make the relation curve of GFP fluorescence Unit and GVP expression. In addition to the above discussion and the project unfinished work we also believe that project in gas vesicle aspect also has lots to expand space for example through the cell floating in the sea surface and produce algal blue element to form a similar filter to restrain the effect of red tide algae growth, or cell floating in the sea surface release inhibition of red tide algae growth of toxins to restrain the growth of red tide algae. Even we can make cells in Marine thermocline and halocline gathering in some nutrient elements through the cell floatation will nutrient element to the sea surface. Standardizing the gas vesicle gene cluster and expression on Escherichia coli.

References

1. Spontaneous mutations in gas vesicle genes of Planktothrix spp. Affect gas vesicle production and critical pressure Steven J. Beard, Barbara A. Handley, Anthony E. Walsby FEMS Microbiology Letters 215 (2002) 189~195

2. Gas vesicles are strengthened by the outer-surface protein GvpC P. K. Hayes, B. Buchholz, and A. E. Walsby Arch Microbiol (1992) 157:229 234