Our characterisation of existing BioBrick T9002:

GFP Producer Controlled by 3OC6HSL Receiver Device

Aims and Rationale

BioBrick BBa_T9002 on a pSB1C3 backbone is a composite part encoding a quorum sensing (QS) receiver driving expression of green fluorescent protein. T9002 was previously reported to exhibit increased GFP expression in the presence of AHL (N-acyl homoserine lactone).

We aimed to confirm GFP-expression responsiveness and dependence of AHL, and how the physical proximity of this QS receiver to any QS sender (producer of homoserine lactone) is facilitated by surface-binding affects quorum signalling.

Materials and Methods

T9002 E. coli were grown in conjunction with other E. coli transformed with the AHL ‘Sender’ plasmid BBa_K1090000. 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;

Figure 1. K1090000 constitutively expressed moderate amounts of RFP.

T9002 with AHL
T9002 E. coli resuspended in AHL derived from a filter sterilised K1090000 suspension displayed a slow, exponential increase in GFP.

Figure 2. Accumulation of GFP occurred at a more rapid exponential rate when co-cultured with K109000 QS sender transformants (Figure 2).

Figure 1 - K1090000 E. coli constitutively expresses RFP. Red (Excitation 544nm/Emission 612nm) fluorescence of K1090000 E. coli (triangles), RFP positive control pSB1C3 (Circles), untransformed XL1-Blue E. coli (dashes).

T9002 with K1090000 We observed that optimal conditions for absolute green fluorescent production by T9002 receiver transformants were a ‘sender’ (S) to ‘receiver’ (R) ratio of 1:100; Figure 3. It was inferred that ratios greater or smaller than this resulted in too little AHL or undesirable competition effects, preventing optimal GFP fluorescence. An untransformed XL1-Blue E. coli (X) acted as control.
T9002 Receivers in the presence of K1090000 Sender exhibit significantly higher GFP response than T9002 with a control (non-AHL expressing untransformed XL1-Blue E. coli); Figure 4.

Figure 2 – Production of GFP by T9002, induced either by filtered AHL-containing culture medium, or by actively growing K1090000 sender transformants. AHL derived from culture medium is sufficient for slow rate T9002 Receiver GFP production (-), although T9002 GFP production was more efficient when paired with actively-growing K1090000 senders (circles). Mean GFP fluorescence of E.coli free in 50μl suspensions incubated at 37C, 1mm double-orbital shaking for 30 seconds every 5 minutes for 355 minutes.

Figure 3 – Greatest absolute fluorescence by T9002 transformants is observed in a Sender (S) 1:100 Receiver (R) cell number initial ratio. T9002 Receiver-produced GFP was compared with that produced when paired with an untransformed XL1-Blue E. coli (X) control or K1090000 Senders (circles), in triplicate. 50μl suspensions were incubated at 37C, 1mm double-orbital shaking for 30 seconds every 5 minutes for 355 minutes.

Figure 4 – K1090000 Sender E. coli induction increased GFP expression in T9002 Receivers. Growth started in cell number ratios of aliquots totalling OD600 0.02 densities, suspended in liquid LB. Untransformed XL1-Blue:Receiver 1:100 ratio (Filled Diamonds), Sender:Receiver 1:100 ratio (Open circles). Values are blank corrected.

  T9002 and K1090000 bound to a Poly-Lysine surface; Overall, immobilised Sender/Receiver pairs exhibited a greater extended rate of GFP production response, and higher absolute response after 7 hours than sender-receiver pairs free in suspension; Figure 5.

Figure 5: Surface-immobilisation of sender-receiver QS tranformants results in improved GFP expression. Poly-L-lysine wells (triangles) had greater rate of response compared to cells free in suspension (circles). This was performed in a Sender 1:10 Receiver cell number ratio.

Conclusions

• BBa_K1090000 ‘Sender’ is an effective AHL expressor that is able to activate BBa_T9002 ‘Receiver’ GFP production.
• BBa_K1090000 has moderate constitutive RFP expression.
• BBa_T9002 is an AHL receiver coupled to a GFP reporter, it has ‘leaky’ GFP expression.
• GFP production significantly increases in the presence of AHL.
• Poly-Lysine surface-immobilisation of T9002 and K1090000 E. coli extends the longterm production rate of GFP expression.