Difference between revisions of "Part:BBa K1378024"
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<partinfo>BBa_K1378024 short</partinfo>
 
<partinfo>BBa_K1378024 short</partinfo>
  
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MlrA coding sequence and terminator.
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This part is composed of MlrA (BBa_K1378001) gene and double terminator (BBa_B0015).
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MlrA is a 28kDa protease found in ''Sphingomonas'' sp which can cleavage microcystins(MCs).
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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.
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MlrA is sometimes referred as a metalprotease by inhibitor studies.
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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]
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<figure><img src="https://static.igem.org/mediawiki/2014/0/05/Peking2014jyj_2.png"/><figcaption>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]</figcaption></figure>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1378024 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1378024 SequenceAndFeatures</partinfo>
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=='''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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<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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</html>
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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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<html>
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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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</html>
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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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<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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</figcaption></figure>
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</html>
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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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</html>
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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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<html>
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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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</html>
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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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Revision as of 05:33, 15 October 2014

MlrA-Terminator

This part is composed of MlrA (BBa_K1378001) gene and double terminator (BBa_B0015).

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

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.

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)


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).


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.


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.

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.


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.


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.


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.

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.

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.


References

[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.