Team:Hong Kong-CUHK/BIOBRICKS CHAR

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Characterization

We did characterization of our biobricks to confirm the biobricks contain the sequence we want and protein expression for our functional application.

I. biobrick DNA length verification

In order to check the inserted biobrick DNA length, PCR was performed with standard biobrick vector sequencing primers (BBa_G00100 and BBa_G00101) / restriction digestion by EcoRI and PstI, targeting on biobrick standard RFC10 site.

1. BBa_K786001

2. BBa_K786002

3. BBa_K786003

5. BBa_K786010

II. Protein expression verification

Negative Phototactic Construct for Blue Light Detection (BBa_K786001)
We verified whether BBa_K786001 was expressed in E. coli. After transformation of BBa_K786002 into 3 different E. coli strains (BL21, TOP10 and DH5α), cells were collected and lyzed. Cell lysates were resolved on SDS-PAGE. Western blot with anti-His antibody was conducted. Bands were detected in the cell lysates, indicating the fusion protein was expressed in all 3 bacterial strains.

Since the fusion proteins were also put under the same constitutive promoter in other two constructs BBa_K786002 and BBa_K786003, it is believed that these constructs can also express the corresponding fusion proteins in above 3 bacterial strains.

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