Difference between revisions of "Part:BBa K401000"

Revision as of 02:22, 6 November 2010

PM2 (Glycine max)

Function

PM2 corresponds to the LEA3 protein from soybean (Glycine max). Late Embryogenesis Abundant (LEA) proteins are known for their roles in plant embryogenesis, as important dessication-resistance factors. It has also been shown that they confer tolerance under several stress conditions in Escherichia coli. You can read more about it in our wiki and in several references, as, for example, the following: Liu et al. (2010).

Three dimensional representation of the LEA14 protein from Arabidopsis thaliana similar to LEA3.(EPDB)

PM2 can be useful as a general stress-resistance factor. It could improve survival under low or high temperatures, water stress and maybe other atypical conditions.

Original source

Our PM2 was inserted into the pET28a vector and transformed into E. coli BL21 Star cells. We sincerely thank professor Yizhi Zheng and Yun Liu (College of Life Science, Shenzhen University. Guangdong, China) for sending us the plasmid with the gene, and, in consequence, let us develop our project.

Sequence

The sequence of the part is the corresponding to the PM2 gene, but this sequence is not reported and we have not sequenced it. The amplification previous to the clonation into the pSB1C3, was made using the primers previously employed in Liu and Zheng(2005):

• Forward actagtagcggccgctgcagATGGCGTCCAAGAAAC
• Reverse tctagaagcggccgcgaattcTGCGTCTATATATAC

(capital letters indicate the region of the sequence that pairs with the coding sequence of PM2).

Verification of LEA protection against extreme temperature

Temperatures tested

In order to demonstrate that PM2 (LEA3) protein is useful to protect the cells during a part of terraforming process we tried to choose representative temperatures for the treatments.

The chart below shows the maximum and minimum temperatures on Mars along a year, depending on the latitude at 0 longitude. Treatment temperatures were selected focusing on the equator area (around latitude 0). In this area current temperatures range, during most of the year, are between -80ºC and 20ºC approximately. And, in a partially terraformed Mars, these temperatures would be warmer.

The considered temperatures are as follows:

• -80ºC, as the minimum temperature on the equator area,
• 20ºC, as the maximum temperature on the equator area,
• -20ºC, as the minimum temperature on the equator area in a partially terraformed Mars,
• 50ºC, as the upper limit of temperature on Mars during the terraforming process.

These experiment temperatures are realistic for the equator area. In colder areas we can use the Red-House device to reach them. Thus, if we demonstrate the cell resistance at these temperatures we might consider these cells be able to live in other areas (and also to grow) because of the temperature rise achieved with the Red-House.

Moreover, in order to mimic the cycle temperatures during a solar day on Mars (sol), the treatments for the experiments consists of varying the temperature from maximum value to a minimum. So, the result of combining these temperatures are four different treatments. In addition, we also carried out this assays with cells in a 8% of glycerol solution, due to its protective effect against freezing temperatures. The reason why we used glycerol was because we were afraid no cells at 0% glycerol could survive on such harsh temperatures.

Verification protocol

The experiments were carried out both in E. coli expressing LEA and in others transformed only with the backbone.

Schematic representation of expressing LEA protein cells (left) and backbone transformed cells (right).

The figure below summarizes all the treatments carried out in LEA and not LEA cells.

Different combinations of conditions (temperature cycles and glycerol presence) in the experiment.

We got, for each case, three replicates in order to obtain three independent events for our results. Later, both results were compared to demonstrate that LEA really helps E. coli to survive. Liquid medium was chosen to the treatments, and, after that, the cells were spread on plates in order to calculate their concentration.

You can see the whole protocol at the Protocols section.

Results

The measured output is the survival ratio calculated as follows:

Bacterial concentration has been calculated from the number of colonies counted on the plates before and after the treatment (taking into account initial OD, glycerol concentration and dilutions).

Obtained data has been analyzed by means of a non-parametric Kruskall-Wallis analysis. This analysis compares survival ratio obtained in the different conditions: with and without LEA. The confidence value has been set at 0,05.

An independent analysis has been done for each combination of conditions.

The chart below represents in logarithmic axes the mean of survival ratio for all different conditions. This chart clearly shows that bacteria expressing LEA survive (and even some times grow) while non-expressing LEA cells yield a very reduced survival ratio. So, a highly protective effect of LEA is visible on this figure. LEA is having in all cases but at -80/50 combination a higher protective action than glycerol's.

Results of the experiment. Logaritmic survival ratio of four different cases: E.coli growing with and without LEA, and cultures with 0% and 8% of glycerol.

For every analysis we obtained a p-value lower than 10^-4. These results point out the protective effect of LEA as statistically significant.

Finally, it is noteworthy that the chart shows a synergic interaction between glycerol and LEA presence. When the expression of PM2 and the presence of glycerol in the growth medium are combined, the survival ratio results in a much higher protective effect.

References

• Liu, Y., Zheng, Y. (2005). PM2, a group 3 LEA protein from soybean, and its 22-mer repeating region confer salt tolerance in Escherichia coli. Biochem. Biophys. Res. Com. 331: 325-332.

• Liu, Y., Zheng, Y., Zhang, Y., Wang, W., Li, R. (2010). Soybean PM2 protein (LEA3) confers the tolerance of Escherichia coli and stabilization of enzyme activity under diverse stresses. Current Microbiology. 60: 373-378.

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Sequence and Features