Our characterisation of existing BioBrick K1090000:

AHL signal sender with RFP reporter under lac promoter control

Aims and Rationale

BioBrick BBa_K1090000 on a pSB1C3 backbone is a composite part encoding a quorum sensing (QS) sender driving production expression of AHL (N-acyl homoserine lactone), while also constitutively expressing red fluorescent protein (RFP).

We aimed to confirm the constitutive RFP-expression, but also the ability of this composite part to direct the production of biologically active AHL. In combination with an AHL-responsive QS receiver BioBrick (T9002), we also aimed to investigate how the efficiency of QS signalling was positively or negatively affected by the physical proximity of this QS sender to any QS receiver (responder to homoserine lactone), through coincident surface-binding of the sender and receiver bacteria.

Materials and Methods

K1090000 E. coli transformants were grown in conjunction with other E. coli transformed with the AHL ‘Receiver’ plasmid BBa_T9002. BBa_K1090000 codes for AHL-synthesising enzymes, and also constitutively expresses RFP.

Poly-L-lysine Cell Adhesion
To investigate QS signalling, and how it is affected by binding of sender and receiver bacteria to a solid surface, we immobilised our cells on a surface coated with poly-lysine:

1. 50μl 0.01% Poly-Lysine was added to each well of a 96 well glass-bottom Plate (Whatman), and incubated at room temperature for 2 hours

2. Excess poly-lysine was removed and the plate washed three times with sterile water and allowed to dry at room temperature.

3. E. coli cultures were grown overnight in a 37C shaking water bath, and diluted to an optical density-600nm(OD600) of 0.02.

4. K1090000 ‘Sender’ transformants washed by centrifugation at 13000rpm for 1minute and resuspension in phosphate buffered saline (PBS), three times.

5. Dual Sender-Receiver wells were diluted and combined in a colorimetric ratio to achieve a total OD600 of 0.02, with a range of 1:1 to 1:10,000,000 Sender:Receiver. 50μl total volume in Liquid Broth (LB) used per well. Incubated at 4C for 1 hour.

6. Unbound cells removed by lightly shaking over waste and washed three times with Phosphate-Buffered Saline (PBS). 50μl fresh LB medium was added to each well.

7. A FluoSTAR OPTIMA Fluoresence Plate Reader was used to measure Red(Excitation 544nm/Emission 612nm) and Green(Excitation 485nm/Emission 520nm) Fluorescence was recorded every 5 minutes for 7 hours; this incubates the samples at 37 degrees C and shakes (1mm double-orbital) for 30 seconds before each read.

Results

K1090000 RFP expression RFP expression of K1090000 transformed E. coli was compared against untransformed XL1-Blue E. coli negative control and an RFP pSB1C3 plasmid positive control. Analysis of red fluorescence using a fluorimeter plate reader clearly shows that K1090000 constitutively expressed moderate amounts of RFP (Figure 1).