Part:BBa_K4083008

RPGDuo2 plasmid with nadE and rhlBA genes Our team added rhlA/rhlB and nadE genes to pRGPDuo2 plasmid. The pRGPDuo2 plasmid (https://parts.igem.org/Part:BBa_K4083000) was obtained from Gauttam, R. [1] We planned to insert rhlA and rhlB genes to MCS1 and nadE gene to MCS2 of pRGPDuo2 plasmid. Thus, the rhlA and rhlB genes are controlled by the IPTG-inducible Ptac promoter, while the nadE gene is controlled by aTc-inducible PtetR/teA promoter. Those promoters are regulated by lacI and tetR repression systems

Structure of Plasmid

This plasmid consists of four parts: pRGPDuo2 plasmid; nadE, rhlA, and rhlB coding sequences (https://parts.igem.org/Part:BBa_K4083000, https://parts.igem.org/Part:BBa_K4083004, https://parts.igem.org/Part:BBa_K4083006, https://parts.igem.org/Part:BBa_K4083007). However, for Biobricks construction purposes, we segmented pRGPDuo2 plasmid into smaller fragments:

pTetR/TetA promoter: https://parts.igem.org/Part:BBa_K2800025

tetR: https://parts.igem.org/Part:BBa_K4083012

pRO1600 origin of replication: https://parts.igem.org/Part:BBa_K4083013

LacI reverse: https://parts.igem.org/Part:BBa_K4083010

Laciq reverse promoter: https://parts.igem.org/Part:BBa_K4083011

Ptac promoter: https://parts.igem.org/Part:BBa_K864400

KanR: https://parts.igem.org/Part:BBa_J31003

ColE1 origin of replication: https://parts.igem.org/Part:BBa_K2560036

By inserting this plasmid, we predicted the effective rhamnolipid production by Pseudomonas putida. Since Pseudomonas putida has all genes required for rhamnolipid production except for rhlAB, the rhlA and rhlb coding sequences inserted in plasmid can allow engineered P. putida to synthesize mono-rhamnolipid by itself. [2]

Reference

[1] 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] Tiso, T., Sabelhaus, P., Behrens, B., Wittgens, A., Rosenau, F., Hayen, H., & Blank, L. M. (2016b). Creating metabolic demand as an engineering strategy in Pseudomonas putida – Rhamnolipid synthesis as an example. Metabolic Engineering Communications, 3, 234–244. https://doi.org/10.1016/j.meteno.2016.08.002

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 5565
  Illegal BamHI site found at 6125
  Illegal XhoI site found at 1757
  Illegal XhoI site found at 2024
  Illegal XhoI site found at 6301
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1949
  Illegal NgoMIV site found at 2019
  Illegal NgoMIV site found at 2240
  Illegal NgoMIV site found at 4400
  
• 1000
  COMPATIBLE WITH RFC[1000]