Team:NEFU China/Lock Key

          

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Lock & Key

Yeast: a-type yeast.

Function: In order to verify the interaction between the lock (stem loop) and the key (small RNA), the enhanced green fluorescent protein (EGFP) or Gaussia luciferase (Gluc) expression was determined with or without coexpression of the vector expressing the key.

Vector construction: Gene fragments of EGFP or Gluc with the stem loop and the URA screening marker genes were inserted between HA1 and HA2 to construct the pesc-ura-stem loop-Gluc(or EGFP)-URA plasmid. HA1 and HA2 act as homology arms to facilitate homologous recombination, which allow the integration of the DNA with the stem loop lock sequence into the yeast genome. By verifying Gluc expression, we can prove that the keys and corresponding locks can specifically and functionally interact.

Functional verification:

1. pesc-ura-stem loop-Gluc-URA plasmids were transformed into yeast. Meanwhile, plasmids with Gluc fragments and plasmids with keys were also transformed into the same yeast. Then, we measured Gluc expression using the GloMax 20/20 Luminometer to evaluate whether a lock and its corresponding key have functional interaction. In another word, we wanted to determine whether the key could specifically resolve the stem loop structure of a lock to enhance Gluc expression. Our data is presented in Figure 1, showing that the key and the lock indeed interacted.

Figure 1. Gluc activity of pCYC-stem loop-Gluc vector
with or without coexpressed key vector.

2. pesc-ura-stem loop-EGFP-URA plasmids were transformed into yeast. Meanwhile, plasmids with EGFP fragment and plasmids with keys were transformed into the same yeast. The relative fluorescence intensity suggested that the key and the lock had specific interaction as shown in Figure 2 and Figure 3.

Figure 2. Plasmids with stem loop.

Figure 3. Plasmids with EGFP fragments and plasmids
with keys were co-transformed into the yeast.

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