Team:NEFU China/Demonstrate

          

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• PROJECT
  • BACKGROUND
  • DESCRIPTION & DESIGN
  • CODE BOOK
• EXPERIMENTS
  • LOCK & KEY
  • INFORMATION DESTRUCTION
  • Pre-RNA SPLICING
  • DEMONSTRATE

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  • KILLING MODEL
• SOFTWARE
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  • MISLEADING
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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 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

Information Destruction

(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(alpha) 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. And the constructed plasmid is shown in Figure 5.

Figure 5: A: pFig2C-EGFP B: pFig2C-Bax(alpha)

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 (Figure 6). In addition, we also did quantitative PCR for EGFP mRNA. The results showed that EGFP expression significantly increased at 12 h (Figure 7), compared with the control group. These results showed that α factor can induce expression of the Fig2C promoter.

Figure 6: 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.


Figure 7: Quantitative PCR for EGFP mRNA from Yeast with or without α factor.

Functional verification of Bax(alpha) gene
We transferred the constructed plasmid pFig2C-EGFP into yeast, we call it “Spy Yeast”, and induced them with α factor. And we define yeast that secretes alpha factor as “Killer yeast”. 10 ml of the spy yeast culture solution and 50ul of the killer yeast culture solution(OD600nm is about 1.4) were mixed for cocultivation.
Meanwhile, we used the cocultured spy yeast without integrated Bax gene and the killer yeast as a control group. Finally, we could determine that the spy yeast could be completely eliminated after 14 hours of the cocultivation (Figure 8).

Figure 8: OD(600nm) value of two experiment groups.

Pre-mRNA Splicing

Pre-RNA Splicing

Transformation of information
Although the stem loop can work as a “lock” to protect our ciphertext from being stolen, information burglar also can decode it by sequencing DNA directly. To overcome this situation, we were inspired by RNA splicing, a critical mechanism by which to modify transcriptome, it also can be used to realize the “misleading” part of the project. In our case, specific sequences contain misleading words are inserted into the information sequence we just designed by the code book as intron. Therefore, only after receivers tranform the “key plasmid” can cryptographic information be transcripted and spliced normally, and can the receiver get the correct information. As for information burglar, he will make a normal DNA sequncing only to obtain a misleading information with wrong meaning. Therefore, pre-mRNA splicing become a new method protecting our ciphertext from being decoded.

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 inserted between the two homologous arms, HA1 and HA2 to construct the pCYC-stem loop-information-Ura plasmid. HA1 and HA2 act as homology arms to promote the homologous recombination, which allows the integration of the DNA containing the stem loop lock sequence into the yeast genome. And the constructed plasmid is shown in Figure 1.

Figure 1： pCYC-stem loop-Information-Ura

Functional verification:

1. Verification of homologous recombination
   Information fragments with homology arms were transformed into yeast. In order to ensure that the information fragments were successfully integrated into the yeast genome, we picked the monoclonal culture and extracted the DNA for PCR verification. The results demonstrate that homologous recombination is successful（Figure 2）.
   

Figure 2： Results of PCR validation of homologous recombination.

2. Access to information
   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. The sequencing results back from the sequencing company showed that our information fragments were correctly expressed. And in the end we got the right information (Figure 3).
   

Figure 3: The sequencing results and information obtained nucleic acid to convert word.

Future plan:

At first we decided to use false information as an intron. In this way, though the thief gets the information sequence from the yeasts, he would get sentences with completely different meanings. And only the mature RNAs that has been spliced will carry the correct information. Therefore, the only way that we could get the correct meaning of the information is to reserve transcript the mature RNAs after getting it. But we didn't have much time to try out whether the introns we designed could be spliced, so we chose to use the natural introns in yeast. Unfortunately, our final result showed that the splice did not happen, either. So this is also the direction of our team's future efforts.

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