Part:BBa_K1331005

Modified rhlABRI operon genes from Pseudomonas aeruginosa SQ6

Introduction

Rhamnosyltransferase operon genes rhl are essential for the regulation of rhamnolipid synthesis which is originally found in Pseudomonas aeruginosa, a kind of bacterium genetically related with Pseudomonas stutzeri, our recipient bacterium. Rhl includes three enzyme-coding genes rhlA, rhlB, rhlC, and two regulatory genes rhlR, rhlI. rhlA and rhlB encode the two subunits of rhamnosyltransferase I, respectively, while rhlC encodes the rhamnosyltranferase II that facilitate the ligation of mono-rhamnolipid into di-rhamnolipids. RhlR and rhlI are two necessary regulatory genes for transcription. RhlI encodes acyl-homoserine-lactone synthase, which catalize the synthesis of C4-HSL. C4-HSL is a autoinducer molecule of rhlR and is essential for the regulation of transcription of rhlAB. In the presence of C4-HSL, rhlR is activated through binding to its autoinducer, and activated rhlR binds to the promoter region and activates transcription of rhlAB.

Figure 1: Regulation model of rhlABRI.

In previous work of our lab, 9 different engineered E.coli strains had been constructed based on 9 different combinations of the five genes, and the production of rhamnolipid as well as the oil recovery rate of each strain had been tested respectively to select the best gene combination.

Figure 2: Different combinations of gene rhlA, rhlB, rhlR, rhlI, rhlC.

According to the testing results, we found that rhlA and rhlB are necessary for rhamnolipid production, because they encode proteins that together make up of rhamnosyltranferase I in rhamnolipid synthesis. Moreoever, the oil recovery rate of the fermentation broth from bacteria with rhlR and rhlI are significantly higher than those of the bacteria without. Thus, we selected the rhlABRI gene combination for our project.

Transformation and Amplification of rhlABRI in E.coli DH5α

We transformed pBBR1-2rhl, the constructed plasmid with rhlABRI genes, into E.coli DH5α by CaCl2-heat shocking transformation. To test the transformation result, we did the bacterial liquid PCR with the primer of rhlR gene. According to the result, the PCR product is around 900 bp, which is exactly the size of rhlR gene. This proves that the plasmid with rhlABRI gene had been successfully transfected and amplified in E.coli DH5α.

Figure 3: PCR result of transformed E.coli DH5α bacterial liquid

Determination the Component of the Fermentation Product

Next, we performed the high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) analysis to determine the component of the fermentation product. As is shown in the figure, the product has the same TLC result as purified rhamnolipid, and the HPLC result proves that the component of hydrolysis product is rhamose.

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Figure 4: Results of thin layer chromatography (TLC, up), and high performance liquid chromatography (HPLC, down).

Characterization of the Properties of the Fermentation Product in Water-oil Mixture

We cultured 3 types of equivalent bacteria E. coli DH5α, wildtype, pBBR1MCS-2 and pBBR1-2rhl, in the blank culture medium (7.5%NaNO3, 1%NaH2PO4, 1%K2HPO4, 0.1%Fe2(SO4)3, 0.1%Na2MoO4, 0.1%MgSO4, 0.1%CaCl2, 1%sucrose, 5%glycerinum) , and measured several parameters of each group respectively. According to the result, after 72 hours culture, the growth of E.coli DH5α carrying pBBR1-2rhl had no obvious difference from wildtype and pBBR1MCS-2 empty vector, while the surface tension of its fermentation broth has significantly reduced, and the emulsifying ability has remarkably improved.

Figure 3: The characterization of properties of the fermentation product of different groups. *The number of “+” reflects the degree of each parameters.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 3133
  Illegal BamHI site found at 69
  Illegal BamHI site found at 629
  Illegal BamHI site found at 2614
  Illegal XhoI site found at 805
  Illegal XhoI site found at 2099
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 992
  Illegal NgoMIV site found at 1713
  Illegal NgoMIV site found at 1826
  Illegal NgoMIV site found at 3113
  Illegal NgoMIV site found at 3117
  Illegal NgoMIV site found at 3264
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 294
  Illegal BsaI site found at 1342
  Illegal BsaI site found at 3396
  Illegal BsaI.rc site found at 478