Part:BBa_K4591009

T500-RFP-lox66-Hpall-lox71-XylSmut-tetO-sfGFP-T500

Usage and Biology

In order to observe the decomposition of PET by engineered bacteria, our team designed this plasmid. The plasmid mainly consists of RFP, lox66&lox71, PHpaII, Xylsmut, tetO and sfGFP genes. It is mainly used to detect and report the presence of PA and TPA, and visually monitor the expression through the GFP.When the TPA is detected by the Xylsmut, the downstream genes will be activated and transcribed——like the strain will express green fluorescence.

Fig 1. The expression level of the monellin.

What’s more, considering that in future product applications, the strain may not necessarily be used to degrade PET immediately after delivery, we designed the flipping system to cope with different use situations. During the strain is put into use to decompose the PET, we can regulate the flipping sequence to detect the presence of TPA and activate the expression of GFP. In other situations, flipping expresses the RFP and plays an alternative role. For example, when applied in a factory, in order to reduce adverse effects during fermentation production, the engineered bacteria express antibacterial products before turning over, and then turn over to express downstream degradation modules until they are put into use. Or in the field, the strain produces substances that increase fertility into the soil before the sequence is flipped. In turn, the engineered bacteria could have more purposes.

Fig 2. The expression level of the monellin.

Basic parts of the device

RFP：It is a strong reporter that has RFP expression that can be detected with a fluorescent plate reader or visualized by the unaided eye. 37°C was the optimal growth temperature for E. coli expressing the RFP and the expression/detection of the RFP increased over time with 48 hours showing the most robust red color. [Part:BBa_E1010]

Fig 3. Picture of visible RFP expression at different incubation times [Part:BBa_E1010]

XylS can bind benzoic acid and various derivatives, but it cannot recognize PA and TPA.[Part:BBa_K4591002(2-5)] So by directed evolution of a promiscuoustranscription factor, XylS from Pseudomonas putida, Jiawei Li and Mario Roque Huanca Nina successfully created twonovel variants, XylS-K38R-L224Q and XylS-W88C-L224Q, thatare able to bind PA and TPA.[1]

Such XylS mutants can be used to build PA and TPA biosensors that detect the presence of TPA and initiate the expression of the corresponding modules. This component is able to regulate the expression of downstream sfGFP gene to report the breakdown of TPA and the Pm promoter.

Fig 4. A kind of simple XylSmut-based fluorometric biosensors which can detect the TPA

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2580
  Illegal PstI site found at 2574
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2580
  Illegal PstI site found at 2574
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2580
  Illegal XhoI site found at 2261
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2580
  Illegal PstI site found at 2574
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2580
  Illegal PstI site found at 2574
  Illegal NgoMIV site found at 2255
  Illegal AgeI site found at 73
  Illegal AgeI site found at 185
  
• 1000
  COMPATIBLE WITH RFC[1000]