Part:BBa_K4245160:Experience

Lambert_GA 2022

The efficacy of the spacer was tested by cloning the T7 regulated miRNA1-broccoli-spacer. We ordered the T7 regulated miRNA1-broccoli-spacer in gBlocks Gene Fragments from Integrated DNA Technologies, Inc. (IDT). We then hydrated the part to 50 ng/uL. The insert and the backbone, a linearized pSB1K3, were digested with EcoRI-HF and PstI-HF. They were then ligated using T4 DNA ligase. The parts were cloned into NEB 10-beta electrocompetent cells using an electroporator.

Figure 1. The plates after the electrocompetent transformation.

Colony PCR was used to determine the colonies which took up the plasmid with the insert. The gel below shows hits at around 500 base pairs, consistent with the length of the insert, 216 bp, plus the length added by VF2 and VR. (see Fig. 2)

Figure 2. The gel showing the results of the colony PCR.

Liquid cultures of the working colonies were made, and we ran minipreps of the liquid cultures to obtain the plasmids with the inserts. We then nanodropped the samples (see Fig. 3)

Figure 3. Nanodrops for the selected colonies’ plasmids.

Once we got these plasmids, we used the cell free pellets from the Biobits Central Dogma kit and tested the fluorescence of the insert plasmid vs water (see Fig. 4)

Figure 4. Graph of the fluorescence of water in the biobits cell-free pellet, versus that of the plasmids in the biobits cell-free pellet, after fifteen minutes.

These results confirmed that the fluorescence of the insert, the T7 regulated miRNA1-broccoli-spacer, was not affected by the internal folding. This also eliminated internal folding as a factor affecting the fluorescence of the RCT product. Therefore, we had to go back and troubleshoot. However, because of time restraints, we decided not to continue RCT in the 2022 season.

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