VICKI
Construction of LuxOD47A BBk with either J13002 promoter or B0015 terminator sequences
Purpose: We have LuxOD47A BBk on psB1AC3. We are going to attempt to insert either the J13002 promoter in front of it, or the B0015 terminator behind it.
Protocol:
The construction technique (restriction digest, Antarctic phosphatase treatment and ligation) was conducted in accordance with the procedure outlined on the protocol page. 6 sets were prepared where J13002 was treated as the insert and LuxOD47A BBk was treated as the recipient. Another 6 sets were prepared where LuxOD47A BBk was treated as the insert and B0015 was treated as the recipient.
Transformation of constructed plasmids into competent TOP 10 cells
Purpose: to insert the constructed plasmids into TOP 10 cells so that we can acquire more copies of the construct in an environment designed for long-term genetic stability in the freezer
Protocol: The transformation was conducted in accordance with the protocol outlined on the protocol page. As we already established that the cells are competent, pBluescript was not used. Once the transformation took place, overnight plates were prepared, with the J13002 + LuxOD47A BBk on psB1AC3 cultured on C-laced plates and the LuxOD47A BBk + B0015 on psB1AK3 cultured on AK-laced plates.
Sequence reading from yesterday’s results
We submitted the LuxOD47A BBk samples for which there was a successful NotI restriction digest for sequencing. The results arrived today and are included below. They confirm that we do indeed have LuxOD47A in biobrick form.
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