Difference between revisions of "Part:BBa K4083006"

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===Usage and Biology===
 
===Usage and Biology===
  
The P. aeruginosa is gram-negative bacillus, and opportunstic pathogen. It secretes rhamnolipids - the rhamnose containing glycolipid biosurfactant. This biosurfactants are used by P. aeruginosa to emulsify the oil sunstances for easy digestion. Thus, rhamnolipids can increase the availability of fats wchich can be important in many different areas like petrleum, bioremediation, cosmetics, food, agroculture, etc. [1] However, due to the toxicity and infectiousness of P. aeruginosa, other alternative organisms are tested. Currently, he organism that showed the best results is Pseudomonas putida.  
+
The P. aeruginosa is gram-negative bacillus, and opportunstic pathogen. It secretes rhamnolipids - the rhamnose containing glycolipid biosurfactant. This biosurfactants are used by P. aeruginosa to emulsify the oil sunstances for easy digestion. Thus, rhamnolipids can increase the availability of fats wchich can be important in many different areas like petrleum, bioremediation, cosmetics, food, agroculture, etc. [1] However, due to the toxicity and infectiousness of P. aeruginosa, other alternative organisms are tested. Currently, genetically engineered Pseudomonas putida has more proising results than others. P. putida only lacks two enzymes for mono-rhamnolipid production: RhlA and RhlB. These enzymes are encoded by rhlA and rhlB coding regions in rhlAB operon. It was previously thought that rhlA and rhlB forms heterodimer, however, further research showed that they act independently from each other [2].
  
Our team planned to extract rhLA and rhlB genes from P.aeruginosa and to insert them into pRGPDuo2 plasmid obtained from Gauttam, R. [2] We developed the new approach to increase the P. putida's rhamnolpid synthesis by adding nadE gene which encodes NAD synthetase. This way, we hoped to see more rhamnolipid production in engineered P. putida.
+
Our team planned to extract rhLA and rhlB genes from P.aeruginosa and to insert them into pRGPDuo2 plasmid obtained from Gauttam, R. [3] We developed the new approach to increase the P. putida's rhamnolpid synthesis by adding nadE gene which encodes NAD synthetase. This way, we hoped to see more rhamnolipid production in engineered P. putida.
  
 
The rhamnosyltransferase B (RhlB) catalyzes the reaction between 3-(3-hydroxyalkanoyloxy)alkanoic acid and dTDP-L-rhamnose which forms mono-rhamnolipid [1].
 
The rhamnosyltransferase B (RhlB) catalyzes the reaction between 3-(3-hydroxyalkanoyloxy)alkanoic acid and dTDP-L-rhamnose which forms mono-rhamnolipid [1].
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[1] Chong, H., & Li, Q. (2017, August 5). Microbial production of rhamnolipids: opportunities, challenges and strategies. Microbial Cell Factories. https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-017-0753-2
 
[1] Chong, H., & Li, Q. (2017, August 5). Microbial production of rhamnolipids: opportunities, challenges and strategies. Microbial Cell Factories. https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-017-0753-2
  
[2] Gauttam, R., Mukhopadhyay, A., & Singer, S. W. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid, 110. https://doi.org/10.1016/j.plasmid.2020.102514
+
[2] Wittgens, A., Kovacic, F., Müller, M. M., Gerlitzki, M., Santiago-Schübel, B., Hofmann, D., Tiso, T., Blank, L. M., Henkel, M., Hausmann, R., Syldatk, C., Wilhelm, S., & Rosenau, F. (2016). Novel insights into biosynthesis and uptake of rhamnolipids and their precursors. Applied Microbiology and Biotechnology, 101(7), 2865–2878. https://doi.org/10.1007/s00253-016-8041-3
 +
 
 +
[3] Gauttam, R., Mukhopadhyay, A., & Singer, S. W. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid, 110. https://doi.org/10.1016/j.plasmid.2020.102514
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 12:44, 20 October 2021


rhlB with SacI and SalI sites

The rhlB gene is responsible for the production of rhamnosyltransferase called RhlB in Pseudomonas aeruginosa.

Usage and Biology

The P. aeruginosa is gram-negative bacillus, and opportunstic pathogen. It secretes rhamnolipids - the rhamnose containing glycolipid biosurfactant. This biosurfactants are used by P. aeruginosa to emulsify the oil sunstances for easy digestion. Thus, rhamnolipids can increase the availability of fats wchich can be important in many different areas like petrleum, bioremediation, cosmetics, food, agroculture, etc. [1] However, due to the toxicity and infectiousness of P. aeruginosa, other alternative organisms are tested. Currently, genetically engineered Pseudomonas putida has more proising results than others. P. putida only lacks two enzymes for mono-rhamnolipid production: RhlA and RhlB. These enzymes are encoded by rhlA and rhlB coding regions in rhlAB operon. It was previously thought that rhlA and rhlB forms heterodimer, however, further research showed that they act independently from each other [2].

Our team planned to extract rhLA and rhlB genes from P.aeruginosa and to insert them into pRGPDuo2 plasmid obtained from Gauttam, R. [3] We developed the new approach to increase the P. putida's rhamnolpid synthesis by adding nadE gene which encodes NAD synthetase. This way, we hoped to see more rhamnolipid production in engineered P. putida.

The rhamnosyltransferase B (RhlB) catalyzes the reaction between 3-(3-hydroxyalkanoyloxy)alkanoic acid and dTDP-L-rhamnose which forms mono-rhamnolipid [1].

800px-BBa_K4083007_rhamnolipid_Metabolic_pathway.png

Part functionality

Reference

[1] Chong, H., & Li, Q. (2017, August 5). Microbial production of rhamnolipids: opportunities, challenges and strategies. Microbial Cell Factories. https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-017-0753-2

[2] Wittgens, A., Kovacic, F., Müller, M. M., Gerlitzki, M., Santiago-Schübel, B., Hofmann, D., Tiso, T., Blank, L. M., Henkel, M., Hausmann, R., Syldatk, C., Wilhelm, S., & Rosenau, F. (2016). Novel insights into biosynthesis and uptake of rhamnolipids and their precursors. Applied Microbiology and Biotechnology, 101(7), 2865–2878. https://doi.org/10.1007/s00253-016-8041-3

[3] Gauttam, R., Mukhopadhyay, A., & Singer, S. W. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid, 110. https://doi.org/10.1016/j.plasmid.2020.102514

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1172
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 65
    Illegal NgoMIV site found at 786
    Illegal NgoMIV site found at 899
  • 1000
    COMPATIBLE WITH RFC[1000]