Team:NEFU China/Suicide

Information Destroyed


Information Destroyed

α factor induced apoptosis
Yeast: a-type yeast.
Function: The expression of the Fig2c promoter can be induced by adding a mating factor. We want to use this promoter to express the Bax gene. We used the enhanced green fluorescent protein (EGFP) to test the effect of the Fig2c promoter. When the Fig2c promoter is induced, EGFP is expressed. In this way, we can detect the strengthen of the Fig2c promoter using EGFP as a reporter.
Vector construction:
We first inserted the Fig2c promoter and EGFP coding sequence into the pesc-ura plasmid. The EGFP coding sequence fragment was obtained from pEGFP-N2 by PCR, and then purified, followed by visualization by electrophoresis (Figure. 1A). The predicted sizes of EGFP cDNA is 750 bp, which matches our experimental results. The vector that expresses EGFP is shown in Figure. 1B.
A:
B:
Figure 1. Electrophoresis of the PCR fragments for the EGFP coding sequence (A) and the diagram of pFig2c-EGFP vector (B).
Functional verification:
In order to verify whether α factor induces the expression of the Fig2c promoter, we transferred the constructed plasmids into yeast, and induced them with α factor. We observed the fluorescence in the transformed yeast cells under a fluorescence microscope. In addition, we also did quantitative PCR for EGFP mRNA. The results showed that EGFP expression significantly increased at 12 h (Figure. 2), compared with the control group. These results showed that α factor can induce expression of the Fig2c promoter.
A:



B:

Figure 2. Fluorescence image of transformed yeast cells at 12h time point after cultivation with (A) and without (B) 0.4 g of α factor dry powder.

After confirming that α factor can induce expression of the Fig2c promoter, we replaced the EGFP cDNA with the Bax suicide gene. We wanted to determine the expression of Bax suicide gene by detecting density of cultured yeast cells. If the Bax suicide gene is expressed, the OD value of yeast should decrease.
Verification of lock vector expression

Yeast: a-type yeast.
Function:
The stem loop acts as a lock to protect our encrypted information to be transmitted. EGFP, or Gaussia luciferase (Gluc), is used as a reporter for our encrypted information system to verify the function of our lock part. Only when a specific small RNA (designated as a “key”) binds to the stem loop and resolve its secondary structure, EGFP transcription can be initiated and EGFP protein can be expressed.
Vector construction:
We inserted stem loop, EGFP or Gluc coding sequence and URA screening gene into the pesc-ura plasmid, as shown in Figure. 3. HA1 and HA2 act as two homology arms at both ends to achieve homologous recombination and allow the DNA with the lock structure sequence integrated into the yeast genome. The vector is depicted in Figure. 3.


Figure 3. The diagram of a vector with pesc-trp-Backbone-Stemloop-EGFP-URA.
Functional verification:
We used the EGFP or Gluc to test the effect of the stem loop. After the plasmids of pesc-trp-Backbone-Stemloop-EGFP(or Gluc)-URA were transformed into yeast, we detected EGFP expression from the yeast genome by RT-PCR, suggesting that homologous recombination of transformed DNA was successful. Plasmids with the stem loop and plasmids without it were introduced into yeast followed by detection of gene expression to verify the function of stem loop. (Figure. 4A, Figure. 4B)

A:


B:


Figure 4. Microscopy to detect EGFP expression in yeast cells transformed by plasmids without (A) or with (B) the stem loop lock.



Lock and key interaction
Yeast: a-type yeast.
Function: In order to verify the interaction between the lock (stem loop) and the key (small RNA), the EGFP or 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-Backbone-Stemloop-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-Backbone-Stemloop-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 3A, showing that the key and the lock indeed interacted.

Figure 5. Gluc activity of pCYC-Stemloop-Gluc vector with or without coexpressed key vector.
2.pesc-ura-Backbone-Stemloop-EGFP-URA plasmids were transformed into yeast. Meanwhile, plasmids with EGFP fragments 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 4B and Figure 6.

Figure 4. Plasmids with stem loop (B).

Figure 6. Plasmids with EGFP fragments and plasmids with keys were co-transformed into the yeast.

Transformation of information
Every letter has its own frequency in English words, so does each codon in organisms. Therefore, we created a coding list, in which each letter corresponds to several codons with the same frequencies. In addition, we composed a program that can easily convert English sentences into DNA sequences.
We have made the transition from nucleotide sequences to DNA barcode.
Information expression verification
Yeast: a-type yeast.
Function: Using the specific “key-lock” interaction, information can be obtained when expressing a specific key.
Vector construction:
Gene fragments of information with a stem loop and URA screening genes were ration inserted between the two homologous arms, HA1 and HA2 to construct the pesc-ura-Backbone-Stemloop-information-URA plasmid. HA1 and HA2 act as homology arms to promote the homologous recombination, which allows the integration of the DNA containing the steem loop lock sequence into the yeast genome. (配图)
Functional verification:
Plasmids with information fragments were transformed into yeast. Then we extracted the total RNA of yeast and carry out reverse transcription. DNA sequencing will be used to verify the correct expression of the information fragment is expressed, which will be compared with the original information.