Difference between revisions of "Part:BBa K3699001"
Line 25: Line 25:
  
 
<p>These bacteria all have mlr gene clusters. On the gene cluster, four mlr genes are located sequentially as mlrC, A, D and B, where mlrA and mlrD are transcribed in forward direction while mlrC and mlrB in the reverse.</p>
 
<p>These bacteria all have mlr gene clusters. On the gene cluster, four mlr genes are located sequentially as mlrC, A, D and B, where mlrA and mlrD are transcribed in forward direction while mlrC and mlrB in the reverse.</p>
−
<figure><img src="??"/><figcaption><b>Figure 1. Sketch map of mlr gene cluster responsible for MC-biodegradation. The relative localization of each gene is shown. The direction of the outline borders embracing gene name represents the transcription direction of respective gene (adapted from Bourne et al. (1996, 2001)).</figcaption></figure>
+
<figure><img src="https://static.igem.org/mediawiki/parts/8/80/T--BUCT--mlr_gene_cluster.jpg"/><figcaption><b>Figure 1. Sketch map of mlr gene cluster responsible for MC-biodegradation. The relative localization of each gene is shown. The direction of the outline borders embracing gene name represents the transcription direction of respective gene (adapted from Bourne et al. (1996, 2001)).</figcaption></figure>
  
 
<p>Among them, MlrA is the most important enzyme. The first enzyme encoded by mlrA (i.e., MlrA) gene can hydrolyze cyclic MC-LR at Adda-Arg bond. Linearized MC-LR was reported to be 160-fold less toxic than cyclic MC-LR.[2]</p>
 
<p>Among them, MlrA is the most important enzyme. The first enzyme encoded by mlrA (i.e., MlrA) gene can hydrolyze cyclic MC-LR at Adda-Arg bond. Linearized MC-LR was reported to be 160-fold less toxic than cyclic MC-LR.[2]</p>

Revision as of 13:22, 22 October 2020

MlrA from Novosphingobium sp. THN1

Introduction

This part is microcystin enzyme (MlrA) from Novosphingobium sp. THN1.

MlrA is an enzyme that degrades cyanobacterial toxins, especially microcystin-LR (MC-LR).

By comparing the ability of Sphingomonas sp. ACM-3962, Novosphingobium sp. THN1 and other bacteria’s data, we found Novosphingobium sp. THN1 shows a stronger activity. So we characterize it.

To promote enzyme expression, we replaced the commonly used strong promoter J23110 with the stronger J23119 in its family. This part successfully expressed MlrA enzyme and showed activity on degrading MC-LR.

Figure 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]

Usage and Biology

Background

Early in 1994, Sphingomonas sp. ACM-3962 was identified as the first bacterium capable of degrading MCs as sole carbon and nitrogen source for its growth. [1] Afterwards, other bacteria of Sphingomonas sp., Sphingopyxis sp., Novosphingobium sp., Stenotrophomonas sp. and Bacillus sp. were verified as able to degrade MCs, including Novosphingobium sp. THN1.

These bacteria all have mlr gene clusters. On the gene cluster, four mlr genes are located sequentially as mlrC, A, D and B, where mlrA and mlrD are transcribed in forward direction while mlrC and mlrB in the reverse.

Figure 1. Sketch map of mlr gene cluster responsible for MC-biodegradation. The relative localization of each gene is shown. The direction of the outline borders embracing gene name represents the transcription direction of respective gene (adapted from Bourne et al. (1996, 2001)).

Among them, MlrA is the most important enzyme. The first enzyme encoded by mlrA (i.e., MlrA) gene can hydrolyze cyclic MC-LR at Adda-Arg bond. Linearized MC-LR was reported to be 160-fold less toxic than cyclic MC-LR.[2]

Figure 3. Degradation process of MC-LR

By comparing the ability of Sphingomonas sp. ACM-3962, Novosphingobium sp. THN1 and other bacteria’s data, we found Novosphingobium sp. THN1 shows a stronger activity. So we characterize it.

Figure 4. MlrA degradation data from different strains

Construction

Expression

Activity measurements

References

[1] Li J , Li R , Li J . Current research scenario for microcystins biodegradation - A review on fundamental knowledge, application prospects and challenges[J]. ence of the Total Environment, 2017, 595(OCT.1):615.

[2] Gehringer M M , Milne P , Lucietto F , et al. Comparison of the structure of key variants of microcystin to vasopressin[J]. Environmental Toxicology & Pharmacology, 2005, 19(2):297-303.