Difference between revisions of "Part:BBa K1378001"
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=='''Characterization'''==
 
=='''Characterization'''==
  
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To test the degradation efficiency, we need to measure the concentration of MCs after MlrA treatment. We introduce Protein Phosphatase 1(PP1) inhibition assays. MCs can inhibit the activity of PP1 effectively. Thus we constructed a standard curve reflecting the relation between the concentration of MC and the relative activity of PP1. Therefore, the concentration of MCs in any solution could be quantified by measuring corresponding PP1 relative activity.
 
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p-Nitrophenyl phosphate (pNPP) is a widely used non-specific substrate to test protein phosphatase activity and it can be hydrolyzed to p-Nitrophenyl(pNP) with characteristic absorption at 405nm. The measurement of PP1 activity is based on the accumulation of pNP. Considering the microcystin(MC) is the inhibitor of PP1 and MlrA can disrupt MC’s structure to disrupt its inhibitory effect, the MlrA activity can be detected by quantification of absorption at 405nm. (Fig. 2)
 
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<h3 id="degradation0301">1. Constructing method for analysis of MC concentration</h3>
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<p>p-Nitrophenyl phosphate (pNPP) is a widely  used non-specific substrate to test protein phosphatase activity and it can be  hydrolyzed to p-Nitrophenyl(pNP) with characteristic absorption at 405nm. The  measurement of PP1 activity is based on the accumulation of pNP. Considering  the microcystin (MC) is the inhibitor of PP1 and MlrA can disrupt MC’s  structure to disrupt its inhibitory effect, the MlrA activity can be detected  by quantification of absorption at 405nm <b>(Fig. 4)</b>. So the concentration of MCs  after degradation can be finally measured by absorption spectrophotometry  method with all the calibration curves for all the interactions above.</p>
  
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<figure><img src="https://static.igem.org/mediawiki/2014/2/26/Peking2014jyj_4.png"/><figcaption>Fig. 2 Measurement of MlrA activity. The OD405 indicates the concentration of pNP, and the change of pNP level could reflect the PP1 activity(a). MC can strongly inhibit the PP1 activity(b), and the MlrA can cleave the MC and dampen its toxicity(c).</figcaption></figure>
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<figure><img src="https://static.igem.org/mediawiki/2014/f/f8/Peking2014jyj_3.png"/><figcaption><b>Figure 3.</b> Measurement of MlrA activity. The OD405 indicates the concentration of pNP, and the change of pNP level could reflect the PP1 activity(a). MC can strongly inhibit the PP1 activity(b), and the MlrA can cleave the MC and dampen its toxicity(c).</figcaption></figure>
  
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</html>
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<p>Firstly a calibration curve of PP1 activity  was generated. The concentration of substrate pNP is sufficient overall so the  PP1 enzyme is saturated and proportion to the accumulation rate of product  pNPP. We could select a proper working concentration of PP1 in the range of  nearly linear relationship between PP1 and change rate of 405nm absorption.</p>
  
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<figure><img src="https://static.igem.org/mediawiki/2014/2/26/Peking2014jyj_4.png"/><figcaption><b>Figure 4.</b> Calibration curve of PP1. p-Nitrophenyl Phosphate solution is treated with different concentration of PP1 solutions. Absorbance at 405nm was measured after 80 minutes. The absorbance increases in direct proportion to PP1 concentration between 0.02-0.1 unit/ul.</figcaption></figure>
  
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Firstly a calibration curve of PP1 activity was generated. The concentration of substrate pNP is sufficient overall so the PP1 enzyme is saturated and proportion to the accumulation rate of product pNPP. We could select a proper working concentration of PP1 in the range of nearly linear relationship between PP1 and change rate of 405nm absorption.
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<p>Based on the premise of linear relationship  between product and absorbance, we choose 0.05unit/ul as the working  concentration of PP1 and then test the inhibition efficiency of MC-LR. As a result, PP1 activity decreases after the addition of MC-LR and there is a  positive correlation between the reduction of absorbance and concentration of MC-LR.</p>
  
 +
<figure><img src="https://static.igem.org/mediawiki/2014/d/d4/Peking2014jyj_5.png"/><figcaption><b>Figure 5.</b> Inhibition efficiency of MC-LR. Working concentration of PP1 is 0.05 unit/ul. Different concentration of MC-LR samples are added to the reaction system. MC-LR shows strong inhibition of PP1 activity and a rapid change of PP1 activity is observed between 10ug/L to 30 ug/L of MC-LR concentration.</figcaption></figure>
  
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<html>
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<h3 id="degradation0302">2. Verifying the degradation effect of MlrA</h3>
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  <figure><img src="https://static.igem.org/mediawiki/2014/d/d4/Peking2014jyj_5.png"/><figcaption>Fig. 3 Calibration curve of PP1. p-Nitrophenyl Phosphate solution is treated with different concentration of PP1 solutions. Absorbance at 405nm was measured after 80 minutes. The absorbance increases in direct proportion to PP1 concentration between 0.02-0.1 unit/ul.</figcaption></figure>
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<p>To test the degradation efficiency of MlrA  expressed by <i>E. coli</i>, MlrA expression plasmid has been constructed and  transformed into <i>E. coli</i> strain BL21(DE3) <b>(Fig. 6a)</b>. After induction, the  bacteria are lysed by lysozyme and incubated with MC solution. Judged by PP1 activity treated by the mixture, the activity in experiment group expressing  MlrA is much higher than strain carrying blank vectors, suggesting that MC-LR  is degraded <b>(Fig. 6b)</b>. Therefore, it could be concluded that MlrA function well  in <i>E. coli</i> expression system.</p>
  
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</html>
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<figure><img src="https://static.igem.org/mediawiki/2014/3/35/Peking2014jyj_6.png"/><figcaption><b>Figure 6.</b> Plasmid construction and results of degradation assays. (a) In our expression plasmid, MlrA is expressed in expression vector pET-21a, while blank vector is used as a negative control. (b) The result shows that MlrA expressed by <i>E. coli</i> has obvious function in degrading MC, which significantly reducing the inhibition effect of MC to PP1.</figcaption></figure>
  
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We choose 0.05unit/ul as the working concentration of PP1 and then test the inhibition efficiency of MC-LR because in this region absorbance displays a nearly linear relationship with PP1 concentration less than 0.05 unit/uL. As a result, PP1 activity decreases after the addition of MC-LR and there is a positive correlation between the reduction of absorbance and concentration of MC-LR.
 
  
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<h3>References</h3>
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<html>
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<p>[1] Gehringer, M. M., Milne, P., Lucietto, F., &amp; Downing, T. G. (2005). Comparison of the structure of key variants of microcystin to vasopressin. <i>Environmental toxicology and pharmacology, 19</i>(2), 297-303.</p>
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<p>[2] Runnegar, M., Berndt, N., Kong, S. M., Lee, E. Y., &amp; Zhang, L. F. (1995). <i>In vivo</i> and <i>in vitro</i> binding of microcystin to protein phosphatase 1 and 2A. <i>Biochemical and biophysical research communications, 216</i>(1), 162-169.</p>
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<figure><img src="https://static.igem.org/mediawiki/2014/3/35/Peking2014jyj_6.png"/><figcaption>Fig. 4 Inhibition efficiency of MC-LR. Working concentration of PP1 is 0.05 unit/ul. Different concentration of MC-LR samples are added to the reaction system. MC-LR shows strong inhibition of PP1 activity and a rapid change of PP1 activity is observed between 10ug/L to 30 ug/L of MC-LR concentration.
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<p>[3] Bourne, D. G., Jones, G. J., Blakeley, R. L., Jones, A., Negri, A. P., &amp; Riddles, P. (1996). Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR. <i>Applied and environmental microbiology, 62</i>(11), 4086-4094.</p>
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</figcaption></figure>
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<p>[4] Choi, J. H., & Lee, S. Y. (2004). Secretory and extracellular production of recombinant proteins using <i>Escherichia coli</i>. <i>Applied Microbiology and Biotechnology, 64</i>(5), 625-635.</p>
  
 
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To test the efficiency, a degradation assay is performed. MlrA coding sequence is inserted into the pET-21a(+) plasmid. This plasmid is transformed into ''E. coli'' strain BL21(DE3) as a secretion vector. Bacteria carrying a blank vector are used as control.
 
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<figure><img src="https://static.igem.org/mediawiki/2014/6/66/Peking2014jyj_9.png"><figcaption>Fig. 5 Expression vector for degradation assays. Vector (a) is our secretion system. Blank Vector (b) is used as a negative control of MlrA expression system.</figcaption></figure>
 
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MC-LR is co-cultivated with the bacteria and the sample was measured as before to test the degradation efficiency. The MC-LR rest can be tested by spectrophotometry described above. The absorbance of bacteria carrying vector(b) is higher than that bacteria carrying blank vectors, suggesting that MlrA exhibits some activity towards MC-LR.
 
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<figure><img src="https://parts.igem.org/File:Peking2014jyj_8.png"/><figcaption>Fig. 6 Degradation efficiency of MC-LR. MC-LR is added to ''E. coli'' culture at a final concentration of 100ug/L. After 12, 36 and 72 hours of co-cultivation, the sample is sterilized, diluted to 25ug/L and added to the reaction system as describe before. Vector(b) described in Fig. 5 are used as control experiment. The result shows that MlrA has some degradation activity toward MCs.</figcaption></figure>
 
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References
 
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[1] Bourne, D. G., Jones, G. J., Blakeley, R. L., Jones, A., Negri, A. P., & Riddles, P. (1996). Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR. Applied and environmental microbiology, 62(11), 4086-4094.
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1378001 parameters</partinfo>
 
<partinfo>BBa_K1378001 parameters</partinfo>
 
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Revision as of 21:37, 17 October 2014

MlrA

Introduction

MlrA is a 28kDa protease found in Sphingomonas sp which can cleavage microcystins(MCs).

MlrA is one part of the gene cluster responsible for the ability of MC degradation. The cluster includes four ORFs, mlrA, mlrB, mlrC and mlrD, which can hydrolyze MCs and facilitate absorption of the products as carbon source. MlrA is sometimes referred as a metalprotease by inhibitor studies.

MlrA can cleavage the Adda-Arg bond and causes ring opening.(Fig. 1) The first-step linearized product shows much weaker hepatoxin compared with MCs. In the experiment of mouse bioassay, up to 250 mg/kg of linearized MC-LR shows no toxicity to mouse, much higher than 50% lethal dose 50mg/kg of cyclic MC-LR. Furthermore, the linearization also raise the median inhibition concentration to 95nM, around 160 times higher than original 0.6nM. [1]

Fig. 1 First step of biodegradation of MC-LR. MlrA mediates breaking peptide bond between Adda and Arg, which leads to significant decrease of toxicity.[1]



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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 244
    Illegal AgeI site found at 373
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

1. Constructing method for analysis of MC concentration

p-Nitrophenyl phosphate (pNPP) is a widely used non-specific substrate to test protein phosphatase activity and it can be hydrolyzed to p-Nitrophenyl(pNP) with characteristic absorption at 405nm. The measurement of PP1 activity is based on the accumulation of pNP. Considering the microcystin (MC) is the inhibitor of PP1 and MlrA can disrupt MC’s structure to disrupt its inhibitory effect, the MlrA activity can be detected by quantification of absorption at 405nm (Fig. 4). So the concentration of MCs after degradation can be finally measured by absorption spectrophotometry method with all the calibration curves for all the interactions above.

Figure 3. Measurement of MlrA activity. The OD405 indicates the concentration of pNP, and the change of pNP level could reflect the PP1 activity(a). MC can strongly inhibit the PP1 activity(b), and the MlrA can cleave the MC and dampen its toxicity(c).

Firstly a calibration curve of PP1 activity was generated. The concentration of substrate pNP is sufficient overall so the PP1 enzyme is saturated and proportion to the accumulation rate of product pNPP. We could select a proper working concentration of PP1 in the range of nearly linear relationship between PP1 and change rate of 405nm absorption.

Figure 4. Calibration curve of PP1. p-Nitrophenyl Phosphate solution is treated with different concentration of PP1 solutions. Absorbance at 405nm was measured after 80 minutes. The absorbance increases in direct proportion to PP1 concentration between 0.02-0.1 unit/ul.

Based on the premise of linear relationship between product and absorbance, we choose 0.05unit/ul as the working concentration of PP1 and then test the inhibition efficiency of MC-LR. As a result, PP1 activity decreases after the addition of MC-LR and there is a positive correlation between the reduction of absorbance and concentration of MC-LR.

Figure 5. Inhibition efficiency of MC-LR. Working concentration of PP1 is 0.05 unit/ul. Different concentration of MC-LR samples are added to the reaction system. MC-LR shows strong inhibition of PP1 activity and a rapid change of PP1 activity is observed between 10ug/L to 30 ug/L of MC-LR concentration.

2. Verifying the degradation effect of MlrA

To test the degradation efficiency of MlrA expressed by E. coli, MlrA expression plasmid has been constructed and transformed into E. coli strain BL21(DE3) (Fig. 6a). After induction, the bacteria are lysed by lysozyme and incubated with MC solution. Judged by PP1 activity treated by the mixture, the activity in experiment group expressing MlrA is much higher than strain carrying blank vectors, suggesting that MC-LR is degraded (Fig. 6b). Therefore, it could be concluded that MlrA function well in E. coli expression system.

Figure 6. Plasmid construction and results of degradation assays. (a) In our expression plasmid, MlrA is expressed in expression vector pET-21a, while blank vector is used as a negative control. (b) The result shows that MlrA expressed by E. coli has obvious function in degrading MC, which significantly reducing the inhibition effect of MC to PP1.

References

[1] Gehringer, M. M., Milne, P., Lucietto, F., & Downing, T. G. (2005). Comparison of the structure of key variants of microcystin to vasopressin. Environmental toxicology and pharmacology, 19(2), 297-303.

[2] Runnegar, M., Berndt, N., Kong, S. M., Lee, E. Y., & Zhang, L. F. (1995). In vivo and in vitro binding of microcystin to protein phosphatase 1 and 2A. Biochemical and biophysical research communications, 216(1), 162-169.

[3] Bourne, D. G., Jones, G. J., Blakeley, R. L., Jones, A., Negri, A. P., & Riddles, P. (1996). Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR. Applied and environmental microbiology, 62(11), 4086-4094.

[4] Choi, J. H., & Lee, S. Y. (2004). Secretory and extracellular production of recombinant proteins using Escherichia coli. Applied Microbiology and Biotechnology, 64(5), 625-635.