Difference between revisions of "Part:BBa K1969020"

Latest revision as of 15:29, 27 October 2016

ADRB2 with His-tag regulated by ADH1 promoter

The sequence of ADRB2 with a His-tag at its C terminus is cloned downstream the yeast ADH1 promoter and ended with an ADH1 terminator to generate a expression cassette in yeast Saccharomyces cerevisiae.

This composite part consists of the full length yeast ADH1 promoter BBa_K1969003, the human adrenergic beta 2 receptor with a his-tag at its C terminus, the terminator BBa_K1486025 and two artificially designed scars, BBa_K1969016 and BBa_K1969009.

Fig.1 The picture of mechanism. Upon binding with extracellular epinephrine, the Gpa2 protein will disassociate from ADRB2 (putatitive) and activates adenylyl cyclase, yielding cAMP surge which can be detected by our composite part (BBa_K1969007) product.

We expressed the human adrenergic beta 2 downstream the ADH1 promoter and transformed into strain SMT-pAN to generate a epinephrine sensing strain, SMT-pANpAA. Actually, the cds for ADRB2 has already been registered as a biobrick part (BBa_K209472), while it’s not biobrick compatitble, so we use site mutation to make it a biobrick compatitble one (BBa_K1969001) and added a His-tag to its C terminus (BBa_K1969002). We did immunocytochemistry experiments to test the localization of the membrane protein ADRB2. The results showed that the signal of the second antibody was mainly located on the cell membrane, which indicated that heterologously expressed ADRB2 was successfully located on cell membrane.

Fig.2 Detection of Membrane Localization of ADRB2 by Immunocytochemistry. The yeast cells were stabilized and stained with DAPI. As ADRB2 is expressed with a His-tag at C' terminal, thus anti-His mAb is used as the primary antibody and dunkey anti-mouse IgG 594 (Invitrogen) as the second antibody. Pictures were taken with Leica-SP5, 63 X.

Although the functional coupling of ADRB2 with yeast endogenous pheromone sensing pathway has been well studied in past few decades, the feasibility of functional signal transduction via ADRB2-Gpa2 has never been tested[4]. So we used our engineered strains SMT-pANpAA as biosensors to detect extracellular epinephrine concentration in PBS solution. The response of the epinephrine is detected within first 5 minutes after addition of epinephrine (dissolved in HAc then diluted using PBS) (Fig.7).

Fig.3 The luminescence triggered by epinephrine detected in three strains. Luminescence was collected at 30s after adding epinephrine. Data expressed as mean ± SD, significance was analyzed using Student-t test, n=3.

In this graph, we can tell that the constructed strain SMT-pANpAA was able to differentiate solution with relatively high concentration of epinephrine (75-100μM) with P value less than 0.05 by Student-t test. Although the difference between 100 μM and 75 μM treatment group was significant, the device can’t differentiate epinephrine solution at low concentration. And the expression of two exogenous protein under the same constitutive promoter ADH1 seems to interfere with each other as the overall luminescence decrease in strain SMT-pANpAA compared with strain SMT-pAN.

Sequence and Features