[ Parts ]
- T7 promoter - BBa_K3033000
- Constitutive promoter - BBa_J23100
- RBS+MerR - BBa_K1724002
- PcadA promoter - BBa_K1724000
- RBS - BBa_J34801
- mRFP - BBa_I13521
- TT - BBa_B0015
Our plasmid starts with a constitutive promoter upstream of a MerR protein, which is then followed by a PcadA promoter (BBa_K1724000) and Red Fluorescence Protein (BBa_I13521). The MerR protein represses the PcadA promoter. When cadmium is present, the MerR protein is released from the PcadA promoter site, allowing RFP to be expressed, signifying the presence of Cadmium.
Construct 2 & 3
According to The National Center for Biotechnology Information, in certain plasmids, there are non-coding regions of DNA present that can serve to enhance or repress certain gene expression mechanisms. These non-coding regions are referred to as “spacers”. Spacers were added before the PcadA promoter in Construct 2 and after the PcadA promoter in Construct 3 to test whether or not they serve to enhance our RFP expression rate.
Construct 4, 5 & 6
These plasmids are versions of constructs 1, 2, and 3 with the addition of BamHI and BgIII restriction sites surrounding the RBS. These restriction sites allow us to cut and insert the single RFP part into the plasmid. This is because in our previous experiment, having more RFP genes may have increased the intensity and the rate of expression of RFP. Therefore, we want to test whether more RFP genes lead to better RFP expression.
To conduct our cell-free experiments we are using the Sigma 70 Master Mix from our sponsor Arbor Biosciences. The master mix is compatible with our constitutive promoter but is said to “provide excellent protein yield” with the T7 promoter. Construct 7 is an alternate version of construct 1 with a T7 promoter instead. We are testing whether using a T7 promoter yields better protein expression when used in the cell-free system.