Part:BBa_K1467400:Experience

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Applications of BBa_K1467400

NRP-UEA 2015

At NRP-UEA we aim to produce a prebiotic of acylated/butrylated starch in plants. To do this, we had to infiltrate our constructs into plants. It was for this reason, along with the fact that we have a range of expertise on site, that we decided to work with GoldenGate cloning. The problem this posed, however, was when we wanted to send our constructs to the iGEM registry at the end of the project, they would be in the wrong backbone and not following the iGEM requirements. For all of our composite parts, we used the GoldenGate Module Flipper to easily convert our constructs into BioBrick compatible composite constructs.

We assembled parts into the MoCloFlipper pSB1C3 according to the GoldenGate one-step Digestion-Ligation. Putative clones were miniprepped and screened by digestion with NotI (Figure 1) and also with Ecor1/PstI(Figure 2) to confirm that cloning was successful and that our constructs had no internal BioBrick restriction sites.

Figure 1: The gel lanes are as follows – 1 = Ladder, 2&3 = BBa_K1618033, 4&5 = BBa_K1618029, 6&7 = BBa_K1618035, 8&9 = BBa_K1618031, 10&11 = BBa_K1618036, 12&13 = BBa_K1618032, 14&15 = BBa_K1618034, 16&17 = BBa_K1618030, 18 = ladder. These indicate that there are no unwanted internal Not1 restriction sites within our constructs.

Figure 2: The gel lanes are as follows – 1 = Ladder, 2&3 = BBa_K1618029, 4&5 = BBa_K1618031, 6&7 = BBa_K1618032, 8&9 = , 10&11 =BBa_K1618029, 12&13 = BBa_K1618031, 14&15 = BBa_K1618032, 16&17 = BBa_K1618030, 18 = Ladder. Lanes 2-9 were digested with Not1, while lanes 10-17 were digested with EcoR1/Pst1. The results indicate that the new tags allow our constructs to be BioBrick compatible.

These results indicate that the Golden Gate Module flipper was successful in converting our constructs into a BioBrick compatible composite part, ready to be shipped for the iGEM registry.

User Reviews

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