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 (microRNA), the enhanced green fluorescent protein (EGFP) or Gaussia luciferase (Gluc) expression was determined with or without co-expression of the vector expressing the key.

Vector construction: Gene fragments of EGFP or Gluc with or without the stem loop and the URA screening marker genes were inserted between HA1 and HA2 to construct the pCYC-stem loop-Gluc(or EGFP) 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 EGFP and Gluc expression, we can prove that the keys and corresponding locks can specifically and functionally interact.
The plasmids structure is shown in Figure 1.

Figure 1: A: pCYC-stem loop-EGFP B: pCYC-stem loop-Gluc

Functional verification:

1. Functional verification of stem loop Plasmids pCYC-stem loop-EGFP-Ura and pCYC-EGFP-Ura were transformed into yeast. The relative fluorescence intensity suggested that the lock can work as shown in Figure 2.

Figure 2: A, B: Yeast cell transformed by EGFP expression
plasmid with a stem loop upstream of the coding sequn-
ece. C, D: Yeast cell transformed by EGFP expression pla-
smid without a stem loop upstream of the coding sequence.

2. Qualitative verification of lock and key interaction Plasmids pCYC-stem loop-EGFP-Ura and pCYC-EGFP-Ura 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 3.

Figure 3: A, B: Yeast cell transformed by EGFP expression
plasmid with a stem loop upstream of the coding sequnece.
C, D: Yeast cell transformed by the stem loop EGFP expre-
ssion plasmid together with a plasmid expressing a small
RNA for the "key".

3. Quantitative verification of lock and key interaction Plasmid pCYC-stem loop-Gluc was 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 4, showing that the key and the lock indeed interacted.

Figure 4: Gluc activity of pCYC-stem loop-Gluc
vector with or without coexpressed key vector.
A: pCYC-Stemloop-Gluc B: pCYC-Stemloop-Gluc+Key
C: pCYC-Stemloop-Gluc+Negtive Key