Team:Cambridge/Notebook/Week10

= Week 10 =

Threshold Devices
Confirmed successful transfer of 40 (pBad followed by GFP) into the low copy plasmid pSB3K3. Transformed 40 in pSB3K3 into arabinose strain.

Confirmed successful construction of pBad + activator (50, 51, and 52) + B0015.

Transformation of TOP10 with activator constructs 50, 51, 52 and the part B0015 in preparation for ligation to pigment parts.

Wet Work
Transformants from yesterday put into overnight culture.

Wet Work
Activator constructs 50, 51, 52 and the part B0015 were mini-prepped from the overnight culture and run on a gel for extraction and purification. Ligations between each of the activators and B0015 were then carried out.

Wet Work
Following the ligation, the constructs 50, 51, 52 ligated to B0015 were transformed into the arabinose strain. As a control, the products of the digest and gel extraction including all of the parts pre-ligation were also transformed into the same strain in order to check whether the ligation had worked.

The products of the digest and extraction are to be found in Box 1, labelled 50d, 51d, 52d, B0015d 1.5 and B0015d 2.0 (as there were two bands for this part following the digest, both were extracted).

Wet Work
The control transformations from yesterday worked, in that no bacteria could be seen on the plates (undiluted and x10 dilution). The transformations with the ligated constructs did not work, however, indicating that the ligation procedure did not work. Today we aim to use an improved protocol.

A ligation mix containing just B0015 was also made, marked as NI (no insert) in the freezer. Transformations with the 50, 51, 52 and B0015 ligation mixes were carried out. Because yields of DNA from the gel extraction were so low, 1.5ul of DNA were used in the transformations.