Team:OUC-China/Project/GVP/DiscussionandFutureWork

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Revision as of 04:37, 26 October 2012

Discussion

The results showed that some of our speculations include that the gvp genes in prokaryotes of generality. The Cyanobacteria gvp gene exists corresponding identification σ factor in escherichia coli in causing gvp gene can be in E.coli to the normal expression. (Wipa Chungjatupornchai) On the other hand when we transformed the BBa_K737006 and BBa_K737007 into the Expression strains Top10 it can also make floating bacteria in the layer surface.( Figure20). The results show that although gvp gene cluster has some unknown control sequence, however these control sequence will not effect on gas vesicle structure protein assembly. When we prove the gvp gene is relying on electrostatic interactions, salt bridges and van der Waals forces to maintain assembly. [7] Through our experiment once again proven that the gas vesicle structure between the two structural protein assembly don't need some control sequence exist in the genome. In conclusion the regulation that greatly enhanced the gas vesicle gene scalability in applied field. In most cases majority of foreign protein expression in E. coli expression system may cause metabolic burden to form some insoluble aggregate termed “inclusion”.[8] Through our experiment also proven that the gas vesicle protein could not in E.coli expression system.



Future Work

Gas Vesicle


Though we have done a lot of work, there still are some works to do in the future. We want to regulate the ratio of GvpA and GvpC to make E.coli floating better. In one hand, we 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. On the other hand, we make fusion protein GvpC:GFP to detect the expression of GvpC.

Next, we want to get the 2kb length of gvp gene cluster which contain the gvpC-16 gene in addition. We think it may make E.coli buoyant better. As for experiment of Gas vesicle protein quantitative analysis, we will establish GFP protein expression quantity and the relationship between the Fluorescence Unit. Now, we have ligated the inducible gene circuits. Later, we will examine them by inducing with IPTG or aTc.

In addition to the above discussion and the project to do, we also believe that project in gas vesicle aspect 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.

Phosphate Accumulation

Background


Inorganic phosphate (Pi) is recognized as one of the major nutrients contributing to the outbreak of red tide. So we want our RNA comparator not only to be a receptor but also an actuator that can accumulate excess Pi in the form of polyphosphate (polyP) from water when the E.coli senses the concentration of phosphate over the warning limit. An enzyme responsible for polyP synthesis is the homotetrameric polyP kinase (PPK). PPK polymerizes the terminal phosphate of ATP into poly. [9]Acetate kinase (ackA) was employed as an ATP regeneration system for polyphosphate synthesis. Researchers have demonstrated Strain MV1184 containing pEP03 (ppk and ackA ) showed the highest rates of Pi removal from the medium.



This recombinant strain removed approximately 90% of the Pi from the medium within 4 h. The data of this result is very satisfactory. Although another recombination strain contain pEP022+pBC29 (ppk and pst operon) has better effect after six hours [10], the pst operon is too long to be used, so we choose to create the recombination strain containing ppk and ackA that we standardize by ourselves.







Design and Experiment

To get standardized parts, an entire ackA gene fragment was amplified by PCR with the primers designed by ourselves from the mutation of E.coli K12 strain, DH5αand inserted into the PSB1C3 vector. ppk contains an EcoRI enzyme site need to be mutanted. So we got it from the E. coli DH5αchromosome and inserted it into pGEM-Tvector, then cut it in XbaI and PstI and inserted the fragment into PSB1C3 cutting by XbalI and PstI sites first. We ligated the two parts with strong promoter and terminator and transferred them into high-copy-number vector to test the ability of recombination strain to accumulate inorganic phosphate (Pi).We would like to monitor the change of Pi in the medium using an autoanalyser AA3(Bran+Luebbe)applying the standard colorimetric procedures(Koroleff, 1983).




Future Work

We also want to control the gene dosage of the two parts by ligating them with Lac promoter and Tet promoter, so we can monitor whether the ability of E.coli to absorb the Pi in the medium would have change and how would it change by adding different amount of IPTG and arabinose.



References

1. A.E. Walsby (March 1994). "Gas Vesicles". Microbiological reviews 58(1): 94-144
2. Steven J. Beard, Barbara A. Handley, Anthony E. Walsby Spontaneous mutations in gas vesicle genes of Planktothrix spp. Affect gas vesicle production and critical pressure FEMS Microbiology Letters 215 (2002) 189~195
3. P. K. Hayes, B. Buchholz, and A. E. Walsby Arch Microbiol Gas vesicles are strengthened by the outer-surface protein gvpC (1992) 157:229 234
4. P. K. Hayes. R. S. Powell Arch Microbiol The gvpA/Ccluster of Anabaena flos-aquae has multiple copies of a gene encoding gvpA (1995) 164 :50-57
5. Jeff Hasty , David McMillen, & J. J. Collins Engineered gene circuits NATURE VOL 420 14 NOVEMBER 2002
6. oliver scholz, Anja Thiel, wolfgang Hillen and Michael Niederweis Eur.J.Biochem, Quantitative analysis of gene expression with an improved geen fluorescent protein 267,1565-1570 (2000) FEBS 2000
7. Hussein M. Ezzeldin a, Jeffery B. Klauda b, Santiago D. Solares Modeling of the major gas vesicle protein, gvpA: From protein sequence to vesicle wall structure Journal of Structural Biology 179 (2012) 18–28
8.Vasina JA, et al, Expression of aggregation prone recombinant, parteins at lour temperatures a comparative study of the Escherichia coli cspA and tac promoter systems protein Expr purify 1997.9: 211-218
9. Gregory D. Davis,* Claude Elisee, Denton M. Newham, Roger G. Harrison New Fusion Protein Systems Designed to Give Soluble Expression in Escherichia coli BIOTECHNOLOGY AND BIOENGINEERING, VOL. 65, NO. 4, NOVEMBER 20, 1999

9. Ahn, K., and A. Kornberg. 1990. Polyphosphate kinase from Escherichia coli. J. Biol. Chem. 265:11734-11739.
10. Unichi Kato, Katsufumi Yamada, Ayako Muramatsu, Hardoyo, Hisao Ohtake.1993. Genetic Improvement of Escherichia coli for Enhanced Biological Removal of Phosphate from Wastewater. Microbiology, 59(11):3744-3749