Difference between revisions of "Part:BBa K2505001"

Revision as of 07:42, 29 October 2017

pBad/araC-rbs-ahk4

Sequence and Features

This part produces AHK4, a receptor of iP(isopentenyl adenine: kind of cytokinin). This part was introduced to E.coli KMI002 strain and sensed iP synthesized by human cell. The gene(ahk4) is derived from A.thaliana and optimized for E.coli codon in reference to the codon usage. The AHK4 protein is transmembrane protein and toxic for E.coli cell. Thus, We regulated expression of AHK4 by P_BAD/araC: L-arabinose operon(tight transcriptionaly regulation system).

Characterization

The purpose of this experiment is to confirm that AHK4 can receive iP, a signal molecule produced by human cells, and AHK4→RcsD→RscB→cps pathyway will be activated in turn. To see the activation of the pathway we used KMI002 strain as a carrier of AHK4. This KMI002 possesses cps::lacZ fusion gene and the activation of AHK4→RcsD→RscB→cps::lacZ can be observed through the activity of β-galactosidase.

As a qualitative experiment we monitored if AKH4 carrying KMI002 develops blue color under the existence of iP and X-gal on agar plates.
As a quantitative experiment we cultured E. coli with various concentrations of iP in liquid medium and β-galactosidase activity was monitored by ONPG.

Result

Material and Method

Qualitative experiment

1.- LB agar plates containing chloramphenicol (34 µg/mL) were prepared.

2.- 50 µl of X-Gal (50 mg/ml), 10 µl of 100 mM iP or DMSO as a control, and 40 µl of LB medium was mixed in microtubes. Then the solutions were applied to the agar plates.

3.- Samples were inoculated and incubated at room temperature.

4.- Photographs were taken after sufficient blue color was developed.

Quantitative experiment

1.- Overnight culture of samples were prepared in 2 ml of LB medium containing chloramphenicol (34 µg/mL) at 25℃.

2.- Samples were diluted for 2000-fold in 1ml of fresh LB medium containing chloramphenicol (34 µg/mL) and various concentration of IP (10 nM-100 µM). Cells were also inoculated into medium containing DMSO instead of iP.

3.- Samples were cultured overnight at 900 rpm at 25℃.

4.- Cells were collected by centrifugation at 10,000 × g for 10min.

5.- All of supernatant was discarded and then cells were resuspended in 500 µL of PBS buffer containing 1 mM MgSO4 and 1 mM dithiothreitol (DTT). Also 500 µL of the same buffer in was prepared as a control for spontaneously splitting of ONPG.

6.- 20 µL of each suspension was added into 180µL of the buffer used above and Abs600 was measured and recorded by a microplate reader.

7.- 10µL of 0.1% SDS and 10 µL of chloroform was added into each tube including the control and vortexed for 15sec.

8.- Tubes were heated at 28℃ for 5min.

9.- 100 µL of ONPG (4 mg/mL) was added to each tube and incubated at 37℃ for 30min. ONPG was dissolved in the buffer used above.

10.- After 30min incubation, tubes were heated at 65℃ for 10min to inactivate β-galactosidase.

11.- All samples were centrifuged at 15,000 rpm for 10min.

12.- Abs420 of supernatant was measured and recorded by a microplate reader. The control was used as a blank.

13.- Relative β-galactosidase activity was calculated by following formula:

"Relative β-galactosidase activity = " Abs420/(Abs600∙10∙30min)