Difference between revisions of "Part:BBa K401000"

Latest revision as of 19:37, 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 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).

The sequence of the part, the PM2 gene, was first reported by Z.-Y. Chen, .Y-I.C. Hsing and T.-Y. Chow and it is available at the NCBI Nucleotide database (accession number AF532313). The complete sequence is pasted below:

       1 cacaaaagtg ttccacttga gtgaaaagta gtgtgttaag aactaaacaa tttttcaatg
      61 gcgtccaaga aacaagagga gcgagctgag gcagctgcga aagttgctgc caaagaactc
     121 gaacaagtca acagagaaag aagagaccgt gatttcggtg ttgttgctga acaacaacaa
     181 caacatcatc aggaagatca acaaaaacgt ggtgtaatcg ggtccatgtt taaggcggtg
     241 caagacacct acgagaacgc caaggaagct gtcgttggca agaaagaagc tactaataac
     301 gcgtacagta atacagaggt tattcacgat gttaacattc agcccgatga cgtgtcggca
     361 acgggggaag taagggacat atcagccaca aagactcatg atatctacga ttctgccacg
     421 gacaacaaca acaacaaaac cggttccaag gtcggagagt acgcagatta cgcttctcag
     481 aaggccaagg aaacaaaaga tgcaacgatg gaaaaagctg gagagtacac agattatgct
     541 tcgcagaaag cgaaggaagc gaagaagacg accatggaga agggtggaga atacaaggat
     601 tactctgcgg agaaagctaa ggagagaaaa gatgctactg tgaataagat gggagagtat
     661 aaggactatg ctgcggagaa agccaaagag gggaaagatg ctactgtgaa taaaatggga
     721 gagtataagg actatgctgc ggagaaaacg aaagagggga aagatgccac tgtgaataag
     781 atgggagagt ataaggatta cactgcggag aaggcgaaag aggggaaaga tacgacgttg
     841 gggaagcttg gggagctgaa ggacacggct tcggatgcgg cgaagagggc cgtgggttac
     901 ttgagcggca agaaagagga aactaaagag atggcttcgg agaccgccga ggcgacggcg
     961 aataaggcag gggagatgaa ggaggcaaca aagaaaaaga cggcggagac cgcggaggcg
    1021 gcgaagaata aggcggggga gatcaaggac agagccgcgg agacggcgga ggccgcgaaa
    1081 aacaagaccg cggagaccgc ggaagtgacg aagaataagg ctttggagat gaaggatgca
    1141 gcgaaggaca ggaccgctga gacaacggat gcggcgaagc agaaaactgc acaggcaaag
    1201 gagaacacca aggaaaatgt gagtggtgca ggtgaaactg caaggaggaa aatggaagag
    1261 ccaaagcttc aaggtaaaga agggtatggg ggccgtggag acaaggtggt ggtgaaagtg
    1321 gaagagagtc gaccaggggc aattgcggaa acgctgaaag ccgccgacca gattgcggga
    1381 cagaccttca acgatgtagg acgcttcgat gaagagggtg tcgtcaatgt ggagcgccgc
    1441 aagaaataat taaaacgtga tctatgatac aacaatatta gtatatatag acgcatgcag
    1501 tttatatagt atatattgtc atgttgtatg tttttacatt ttggtttgct tgtttacatt
    1561 ctcttcaaaa aaaaaaaaaa tgtgtagtac gtgtaaggtt ttgaagattg gttctaggct
    1621 ccgtgggaac catttcaaca ataaacattt tgcgcgttct tgtacacgta gtgatgagaa
    1681 gagatgcctt atgggcagta tcatctaaaa cttattttca tccatcatag aatttggatc
    1741 t

Verification of LEA protection against extreme temperature

Temperatures tested

In order to demonstrate that PM2 (LEA3) protein is useful to protect cells against extreme temperatures in a terraforming context 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 [http://2010.igem.org/Team:Valencia/RH Redhouse] 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 Redhouse.

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 of resistance with LEA

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.

Protocol description

• Preculture
  • Take two tubes of 25 ml and add 5 ml of LB in both of them.
  • Add 5µl of kannamicine in each tube.
  • Inoculate in each tube a colony corresponding E. coli (with LEA and without LEA).
  • Maintain the tubes during 8 hours at 37 ºC and 250 rpm.

• Culture overnight of liquid medium
  • Take two flasks of 500 ml and add 50µl of kannamicine in both flasks.
  • Inoculate 500µl of corresponding preculture.
  • Maintain the flask incubator at 37ºC and 250 rpm.

• Measure of OD of culture and dilution to exponential growth zone
  • Measure of the OD of each culture.
  • Dilution the cultures with LB fresh to OD of 0.5.
  • Grow the cultures to OD of 0.6.

• Induction with IPTG
  • Add IPTG to 1mM final concentration.
  • Incubate, both cultures, during 4 hours at 37 ºC and 250 rpm.

• Adjust of OD
  • Measure the OD of each culture.
  • Adjust the OD of both cultures at 0.4.

• Add glycerol
  • Extract three aliquots, for each culture, corresponding to the different concentrations of glycerol (the volumes are calculated for glycerol to 99,5%):
    • 0% glycerol: take 50 ml of culture and nothing of glycerol.
    • 8% glycerol: take 4 ml of glycerol and 56 % and 46 ml of culture.
    • 50% glycerol: take 25.3 ml of glycerol and 24.7 ml of culture.
  • Prepare these premixes with tubes of 50 ml (only for mixing).

• Separate the cultures for the different conditions
  • Extract twelve 1 ml aliquots from each stock of 50 ml.

• Spread to count initial colonies
  • Take 1 ml of each 50mL stock.
  • Prepare serial dilutions of each 1 ml of the samples:
    • 1/1,000
    • 1/5,000
    • 1/25,000
    • 1/125,000
  • Spread (with glass beads)LB plates (without antibiotic) with 100 µl for each dilutions.

• Stress by extremely conditions
  • Low temperatures during 2 hours and high temperatures during 45’
  • Take 1 ml of sample of each culture after each completed cycle.
  • Prepare serial dilution of each sample:
    • 1/1,000
    • 1/5,000
    • 1/25,000
    • 1/125,000

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 scale the mean of survival ratio for each of the four different conditions. This chart clearly shows that bacteria expressing LEA survive (and even sometimes 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 each of the four treatments 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.

Sequence and Features