Part:BBa_K4216040

LacZ gene under p59m promotor

Output

DpB.014 is an Ouput device that can receive signals from the processor device. LuxRQ (transcription factor produced in the Processor) activate pLux78B in the presence of OC6 (its inducer). Signals are outputed as electronically readable information. (See Electrical resistanceas a measure of OD) They are can be used to report signals from computation upstream in our computation pipeline or as a standalone reporter They can also be used as standalone for prototyping and debug of for specific applications

Use

Terrell_et_al. demonstrated the use of redox modality in bacterial cells to generate an electro-readable signal, and we wished to integrate this system in our collection as an Output component (1). The LacZ gene produces the enzyme β-galactosidase, a well known enzyme in molecular biology for its use in the common blue-white screen test. This enzyme has many catalytic activities, one of which is to cleave 4-aminophenyl-β-d-galactopyranoside (PAPG) to the electronically detectable p-aminophenol (PAP) (2). Using the PAPG redox substrate as a signal mediator, the expression of LacZ can be read using differential pulse voltammetry (DPV) with a potentiostat.

Figure: Illustration taken from Terrell_et_al.. Instead of the Plas promoter, we used the Plux promoter to make LacZ expression dependent on the activity of the upstream Processor plasmid.

Materials and methods

Design

Here an overview of our design for BBa_K4216036 :

Cloning

We ordered the constructs from IDT as gBlocks and used Golden Gate cloning to insert them into the V35 vector plasmid (Bba_K4216046). We confirmed our constructs via sequencing.

Further Work

We did not have time to perform experiments with this construct and to characterize it, but we added it in our collection because we believe this system is a strong candidate for an effective Output system. To demonstrate the LacZ gene as a electroreadable output system, we would use our potentiostat to perform squarewave voltammetry and retrieve the DPV values. Once we confirmed this part functions in the V35 plasmid, we would insert this construct in the pCDFDuet plasmid (Bba_K4216044) and use a Processor plasmid to create a modular biosensor

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]