Team:TzuChiU Formosa/Project

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Background

RNAi RNAi, also known as RNA interference is a type of method that uses small fragments of RNA to interfere the molecular expression of mRNA. When inside an eukaryotic organism (RNAi), it transcribes a pre-shRNA (a strand of RNA that forms a hairpin when folded) and is then cleaved via a dicer (removing the loop) to form smaller fragments of double-stranded 20 bps RNA. The RNAi function is then performed through the use of RISC(RNA-induced silencing complex). [Through the use of RISC (RNA-induced silencing complex) to recognize the complimentary mRNA, the function of RNAi is then performed]. Through the artificial modifications, the 20 bps of RNA is then sent into the cell to achieve the function where we may control the expression of gene translation. When RNAi recognizes its complimentary strand, three things may happen: 1. mRNA simply gets degraded, 2. Blocks mRNA transcription, 3. It controls the promoter and related enzymes after this stand of RNA is transcribed. The main advantage of RNAi is that it is very easy and handy to use. You do not need to go through transgenic procedures to achieve gene regulation. The cons of RNAi on the other hand is that it “knocks down” a gene instead of “knockout” hence, meaning that it cannot completely suppress certain genes. The mechanism of RNA interference is probably originated form an organism that is infected by a virus. The RNA of the virus enters into the cell and is then recognized and cleaved by the dicer into small fragments. These small fragments possess the function of RNAi hence, inhibiting the original physiological mechanism of the cell.

Overview

Our idea RNAi does not possess a RISC system inside a prokaryotic cell. In place of the RISC is its peculiar CRISPR system. CRISPR is an immunization gene in a prokaryotic cell that fights against the exogenous DNA. CRISPR is divided into two major components namely the CAS gene and the repeat-spacer array. When an exogenous DNA enters into the prokaryotic cell, the CAS protein captures part of this exogenous DNA and inserts a section of this DNA into the repeat-spacer array to perform transcription. We will then end up having a strand of repetitive RNA with partial sections being the complimentary fragments of the exogenous gene. CAS ll will then cleave these repetitive RNA fragments and form many small fragments with the antisense RNA. Lastly, CAS lll will carry these fragments and recognize the complimentary exogenous DNA therefore, decreasing / suppressing the function of the exogenous gene. We aim to us this mechanism as a basis and send our desired RNA sequence into the cell, to activate the CRISPR system in order to interfere specific genes with our fragments. Our ultimate goal is to use the technique of RNAi, interfering the antibiotic resistance gene of the bacteria. This would then result in the bacteria losing the function of being resistant to antibiotics. IPTG inducible regulation After the discovery of the Lac operon in E.coli, the expression system became one of the earliest applied methods. The Lac operon is composed of three main components namely the Lac promoter, Lac operator and the structural gene. Transcription of the Lac promoter is regulated by Lacl, (type of repressor protein) and then combines with the Lac operator sequence thereby inhibiting the transcription process. IPTG is a lactose analog where it can combine with the end product of Lacl thus changing the structure of Lacl. This change in structure results in the detachment of the Lac operator hence activating transcription. The reason why we have chosen to use the IPTG induction is because this inducible system complies with the need of our experiment. After IPTG is added, it will combine with LacI thus detaching from the sequence from the Lac operator. This will then activate transcription to our antisense gene and the transcribed antisense mRNA will bind to resistant gene CamR. If we have successfully knocked down CamR, and it cannot be cultured onto the LB plate, then it proves that our idea has worked! Our plan The figure below is the composite we plan to use in our project:                         We intend to make use of the iGEM biobricks and the parts we constructed to compose an IPTG inducible system, via the Biobrik Assembly Kit. The T7 promoter(BBa_I719005)、T7 terminator(BBa_K731721) and Lac promoter(BBa_I14032) are igem biobricks. Joining the biobrick with our Lac operator+CamR antisense (BBa_K1222984), (BBa_K1222983), (BBa_K1222982), (BBa_K1222981) and LacI (BBa_K1222003) would be the IPTG inducible system part (BBa_K1222000) we are going to submit. The figure below is the biobrick assemble kit overview:

Result

Group one: pSB1C3 We have successfully obtained the pSB1C3 vector and the insert we have designed (BBa_K1222987、BBa_K1222986、BBa_K1222985、BBa_K1222984、BBa_K1222983、BBa_K1222982、 BBa_K1222981). After ligation, we transformed the plamid think DH5α.                                 We have Picked a single colony of the bacteria cultured and performed streaking. According to each quadrant of single colony, we have done a colony PCR but only a primer dimer band was shown. We conjecture that this is because the specificity of the primer is not high enough.                      While performing the experiment above, we have also attempted to clone lac I. Firstly, we designed a primer with regard to lac I on the pET11d and used TGradient Thermocycler to test several annealing temperatures (Tm) but unfortunately, we did not succeed. We assume that this is because the sequence for lac I is too large hence, it cannot be cloned out at once. We then tried overlapping PCR and designed several groups of primers. Again, we did not manage to succeed. Group two: pET11d Since the pET11d plasmid contains the lac I gene, so to make up for the previous group (group 1), we have decided to use pET11d. Successfully we managed to get the pET11d vector and our designed antisense insert (BBa_K1222994, BBa_K1222995, BBa_K1222996, BBa_K1222997, BBa_K1222998, BBa_K1222988, BBa_K1222989), which includes GFP and Ampicillin. After ligation, we transformed it into BL21 but nothing grew on the agar plate. The control group (Ampicillin free) proved that there is nothing wrong with the competent cell and in fact we have obtained a complete plasmid from digestion. Therefore, we assume the reasons could be the following: a.     Unsuccessful ligation b.     The size of the plasmid is too large c.     Probably BL21 already contains a plasmid (T7 polymerase sequence) therefor the chances of it         would not accept another plasmid One of the main reason is that there is not enough time on hand so the result displayed is our experiment for now. We will still continue working on the experiment to complete our idea and design.                                                      Due to the problems above, we have already carried out some solutions: We have redesigned a new higher specificity check primer for the pSB1C3 colony PCR. According to pSB1C3 the lac I for this group: a.     We have once again designed another overlapping primer, repetitively trying to clone the lac I         sequence from the pET11d b.     Not only did we attempt cloning the lac I off pET11d, we also tried cloning other plasmids with         the lac I sequence such as PQE80L. This is to increase the probability of us getting the lac I gene. In accordance to pET11d, we have tried some minor alterations to the transformation protocol in this experiment such as: a.     Increasing the time of heat shock and b.     Make use of electroporation to prepare competent cell. We plan to give it a try, carry out these         alterations and complete the experiment in the near future.

Brand New Design

At first, we started off this project baring the thought that " Are organisms able to perform an easier mechanism to carry out our desired plan? " Now that the competition is right around the corner, it seems like we still need a little extra time to prove the feasibility of the experiment. A few weeks ago, we have seen a paper called "DNA targeting specificity of RNA-guided Cas9 nucleases" which gave us a moment of epiphany! We have then realized that if we could add the CRISPR/Cas9 mechanism to our experiment, the results would probably be much closer to our expectation than it is now. Our team members then spent some extra time redesigning the proposal by adding both conjugation and CRISPR to our original plan. In doing this we hope to design a more powerful and effective system that complies more with our expectations.