Team:Aberdeen Scotland/Parts/ 0000

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Revision as of 23:35, 17 October 2014

Team:Aberdeen Scotland/Parts - 2014.ogem.org




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.
Since the obvious way to audit the effectiveness of the QS Sender K1090000 BioBrick was to use a QS Receiver, we combined our experimental assessment of K1090000 and T9002 in a series of joint experiments. Therefore some of the experiments reported under K1090000 are the same as those reported for T9002 (see adjacent Experience tab, this section of the Wiki).

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). K109000 is an efficient producer of QS-active AHL
K1090000 transformants were grown overnight as a source of AHL. Medium from this culture was taken after the K1090000 cells had been removed by centrifugation and filter sterilisation. E. coli T9002 transformants (QS Receiver producing GFP in response to a QS signal) was resuspended in conditioned growth medium containing AHL, derived from the filter sterilised K1090000 suspension. The T9002 transformants in this medium as expected displayed a slow, exponential increase in GFP, indicating the T9002 receiver cells were responding to the QS signalling caused by AHL in the medium. However, QS responses by the T9002 receivers were even more marked when T9002 transformants were 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 K1090000 ‘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. This clearly shows the the K1090000 QS Senders were effective and efficient producers of AHL.


Figure 2 – Production of GFP by T9002 Receiver, driven by either by filtered AHL-containing culture medium derived from K1090000 cultures, 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.
• Poly-Lysine surface-immobilisation of T9002 and K1090000 E. coli extends the longterm production rate of GFP expression.