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From 2011.igem.org

Introduction

The Ribozyme Project aims to establish a novel way to make fusion proteins, therefore, it is important to ensure that the intron components will effectively remove an interrupting sequence and effectively re-ligate together, ensuring a functional protein product. Consequently, an easily detectable protein product must be interrupted with a sequence that is definite to either stop translation or alter the protein enough to render it ineffective. In our design, GFP is interrupted with a stop codon-containing sequence, which is flanked by the two intron parts. The parts submitted for this design were sequence and subsequently ligated into PSB1C3.

Making the Construct with RFC53

Figure 6 is a flow chart of the general work-flow involved in the construction of our experimental plasmid, as per RFC53 conventions. The enzymes used are SacI, EarI and SapI, however, Bg1II could have been used if ligation were done in the opposite direction.

• (1) The insert is isolated through a series of enzyme digestions. As shown, the plasmid is initially cut by SacI. One Intron (in blue) is shown here as a representation. Note that SacI is the first enzyme to cut in the meta-prefix, instead of EarI because of the TCGA complementary overhand needed for ligation in (3). Next, EarI cuts at the meta-suffix to produce the CCA overhang also necessary for the step (3) ligation. The desired intert (containing the intron) is isolated for subsequent use.

• (2) Similarly, the PSB1C3 vector is isolated through enzyme digestion. Note that "N" indicates that this is the vector portion. SapI cuts in the meta-prefix region to produce the GGT overhang (complimentary to CCA in (1)). Next, SacI further cuts in the meta-prefix to produce the other sticky end (AGCT). The vector is also isolated for the ligation step.

• (3) The two components (insert and vector) are ligated together to produce the final construct.

• (4) According to the experimental design, the final construct will contain self-excising ribozymes, which in the last step result in a non-disruptive ligation scar and, therefore, the expression of GFP.