The first accumulator construct is a theoretical biobrick in which we attempted to make using BBa_K1355003 designed by the iGEM14_UFAM_BRAZIL team. Our goal was to introduce a Green Fluorescence Protein part before the terminator of the mercury accumulating device. The first problem that we encountered was that there were no restriction sites present inside the biobrick so we could not open the plasmid. In order to insert the GFP, we used the overlapping PCR protocol to insert the GFP. The way that it would have worked was by designing a primer that had an extra sequence attached that would hang freely when the primer is attached. This free hanging sequence would code for the GFP. We would use that GFP primer as a primer for the accumulator device so that when the DNA polymerase reads and copies the device sequence, it would add on the extra GFP sequence. Unfortunately, after three tried of this method, we were unable to get good results.
The Killer Red Construct is a double condition suicide sequence. The first condition is the presence of mercury. When the bacteria are in an environment containing mercury, the production of RBS (BBa_B0030) is initiated through the Promotor (BBa_K346002). Then, the Killer Red protein (BBa_K1184000) is produced. The second condition is the presence of light. In order for the Killer red to activate, light (540-585 nm) needs to be shined onto the bacteria to initiate the reaction.
Originally, we planned on using ligations to join the parts together to form our construct, but we encountered two problems along the way. One of them was the fact that except for the Killer Red, the other three parts were too small. Trying to isolate the individual parts was very difficult to do and each time we attempted to run them through a gel, we could not see bands. The second issue was that we ran out of SpeI enzyme. Being in Peru, getting supplies is more difficult than in other places. We asked all of the labs in Lima, and not a single laboratory had the specific enzyme. It is also very expensive to have it shipped here since the taxes are high, as well as the hipping fee.
To solve our issue, we asked IDT to synthesize the construct for us. The biobrick was made by copying the sequences of the components from their respective registry pages in the order: BBa_K346002, BBa_B0030, BBa_K1184000, and BBa_B0015. An XbaI/SpeI ligation scar was added in between each part to simulate a ligation, however, we replaced the ligation scars flanking the RBS with HindIII and ApaI restriction sites. These restriction sites serve two purposes:
1) To provide a method of checking whether or not the ligation of the biobrick to the PSB1C3 vector is successful
2) To facilitate future characterization of the part with different RBS in order to delay or promote the expression of KR.