Team:Aberdeen Scotland/Parts/ 9002

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<p>Antigen 43 (Ag43), the product of the </i>flu</i> gene, is a cell-surface autotransporter protein found in <i>Escherichia coli</i>. It is expressed at about 50,
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000 copies/cell and is initially synthesised as a precursor of 1039 amino acids. Upon removal of the signal peptide, the protein is transported to the cell surface
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and is composed of an α subunit (499 amino acids) at the N-terminus and a β subunit (488 amino acids) at the C-terminus. Ag43 is mainly known to induce cell-to-cell
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aggregation and be involved in biofilm formation. However, as the necessary information required for auto transportation resides in the protein itself, the main of
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<h3>Aberdeen iGEM team Characterisation of existing BioBrick T9002: GFP Producer Controlled by 3OC6HSL Receiver Device</h3>
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our project was to use it as a platform for displaying specific peptides on the surface of <i>E. coli</i>.</p>
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<br>
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<img src="http://2014.igem.org/wiki/images/2/2e/Ag43.jpg" alt="Ag43">
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<b>Aims and Rationale</b>
 +
 
 +
<br>
 +
 
 +
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 described to exhibit increased GFP expression in the presence of AHL (homoserine lactone).
 +
<br>
 +
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.
 +
<br>
 +
<br>
 +
 
 +
<h3>Materials and Methods</h3>
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<br>
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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.
 +
<br>
 +
 +
<b>Poly-L-lysine Cell Adhesion</b>
 +
<br>
 +
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:
 +
 
 +
<br>
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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
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 +
<br>
 +
 
 +
2. Excess poly-lysine was removed and the plate washed three times with sterile water and allowed to dry at room temperature.
 +
 
 +
<br>
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 +
3. E. coli cultures were grown overnight in a 37C shaking water bath, and diluted to an optical density-600nm(OD600) of 0.02.
 +
 
 +
<br>
 +
 
 +
4. K1090000 ‘Sender’ transformants washed by centrifugation at 13000rpm for 1minute and resuspension in phosphate buffered saline (PBS), three times.
 +
 
 +
<br>
 +
 
 +
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.
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 +
<br>
 +
 
 +
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.
 +
 
 +
<br>
 +
 
 +
7. A FluoSTAR OPTIMA Fluoresence Plate Reader was used to measure Red(Excitation 544nm/Emission 612nm) and Green(Excitation 485nm/Emission 520nm) Fluoresence every 5 minutes for 7 hours, this incubates the samples at 37C and shakes (1mm double-orbital) for 30 seconds before each read.
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<h3>Results</h3>
 +
<br>
 +
K1090000 RFP expression
 +
 
 +
<br>
 +
 
 +
RFP expression of K1090000 transformed E. coli was compared against untransformed XL1Blue E. coli negative control and an RFP pSB1C3 plasmid positive control; Figure 1. K1090000 constitutively expressed moderate amounts of RFP.
 +
T9002 with AHL
 +
 
 +
<br>
 +
 
 +
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).
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<br>
 +
<br>
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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 XL1Blue E. coli (dashes).
 +
T9002 with K1090000
 +
 
 +
<br>
 +
<br>
 +
 
 +
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 XL1Blue E. Coli (X) acted as control.
 +
 
 +
<br>
 +
 
 +
T9002 Receivers in the presence of K1090000 Sender exhibit significantly higher GFP response than T9002 with a control (non-AHL expressing untransformed XL1Blue E. coli); Figure 4.
 +
 
 +
<br>
 +
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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 Green fluorescence of E.coli free in 50μl suspensions incubated at 37C, 1mm double-orbital shaking for 30 seconds every 5 minutes for 355 minutes.
 +
 
 +
<br>
 +
<br>
 +
 +
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 XL1Blue 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.
 +
 
 +
 
 +
<br>
 +
<br>
 +
 +
Figure 4 – K1090000 Sender E. coli induction increased GFP expression in T9002 Receivers. Growth started in colorimetric ratios of aliquots totalling OD600 0.02 densities, suspended in liquid LB. Untransformed XL1Blue:Receiver 1:100 ratio (Filled Diamonds), Sender:Receiver 1:100 ratio (Open circles). Values are blank corrected.
 +
 
 +
 
 +
<br>
 +
<br>
 +
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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.
 +
 +
<br>
 +
<br>
 +
 +
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 colorimetric ratio.
 +
 
 +
<br>
 +
<br>
 +
 
 +
<h3>Conclusions</h3>
 +
 
 +
BBa_K1090000 ‘Sender’ is an effective AHL expressor that is able to activate BBa_T9002 ‘Receiver’ GFP production.  
 +
<br>
 +
BBa_K1090000 has moderate constitutive RFP expression.
 +
<br>
 +
BBa_T9002 is an AHL receiver coupled to a GFP reporter, it has ‘leaky’ GFP expression.
 +
<br>
 +
GFP production significantly increases in the presence of AHL.
 +
<br>
 +
Poly-Lysine surface-immobilisation of T9002 and K1090000 E. coli extends the longterm production rate of GFP expression.
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
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Revision as of 21:57, 17 October 2014

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



Background to Parts Design


Aberdeen iGEM team 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 described to exhibit increased GFP expression in the presence of AHL (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) Fluoresence every 5 minutes for 7 hours, this incubates the samples at 37C 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 XL1Blue 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 XL1Blue 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 XL1Blue 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 XL1Blue 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 Green 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 XL1Blue 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 colorimetric ratios of aliquots totalling OD600 0.02 densities, suspended in liquid LB. Untransformed XL1Blue: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 colorimetric 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.