Team:Kyoto/Workflow

Team:Kyoto/Project - 2018.igem.org

Table of contents

1) Creation of KO yeast strains
2) Plasmid construction
3) Assesment of halotorelance
4) Assesment of the amount of absorbing Na+
5) Assesment of aggregation

At a first step, we worked on creation of KO yeast strains which uptake more sodium and therefore show salt-sensitivity. It is because we need to use salt sensitive yeast strains in order to assess functions of proteins which contribute salt torelance, and also Na+を外に漏らさずため込ませたかったからだ。作り方はSpecial protocolを見てください。(リンク) Based on Aachen 2017's result, we created ΔNHA1, ΔENA1ΔNHA1,ΔENA1 at first, and 実験を進めるにあたって東北大学の魚住先生にいただいたG19株(ΔENA1,2,3,4)がよく塩を吸収し、高い塩感受性を示すことがわかり、ENA1だけでなく同じタンデムにあるENA2,3,4もノックアウトする方がいいことがわかった。またNHA1をノックアウトすることも塩吸収に貢献していたため、最後にΔENA1,2,5ΔNHA1も作成しました。以下が私たちが作成した変異株です。実験においては、上記のG19株と、渡部先生にいただいた醤油酵母も用いた。

・ΔNHA1
・ΔENA1ΔNHA1
・ΔENA1
・ΔENA1,2,5ΔNHA1

次に、私たちはプラスミドのコンストラクションを行いました。デザインページにあるように、塩耐性のためにMangrin, ZrGPD1,ZrFPS1を、塩の回収のためにAtHKT1,AVP1, AtNHXS1, SseNHX1の作成をしました。

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Figure 2-e Time course of the rate of eGFP(+) nematodes.
Nematodes were grown on eGFP(+) yeast and examined by fluorescence microscopy at the indicated time. (n=18)

6) Improve transport of mRNA to cytosol

As shown in the figure (Figure 6-a), the diameter of the stylet is very small, about a fraction of a single cell of yeast. For this reason, B. xylophilus seemed to draw out the cytoplasmic fraction, but large cellular componentns such as the nucleus may not be efficiently consumed by B. xylophilus.

Figure 6-a Nematode’s stylet, diploid yeast, and haploid yeast. Scale bar : 10μm

A number of studies have been done on the nuclear export of mRNA, and the basic mechanism has been elucidated. It is known that various RNAs such as mRNA, rRNA, tRNA, etc. are recognized by transporters specific to each type and pass through the nuclear pore complex[4]. However, since the dsRNA as prepared this time does not exist in nature, it is not known whether there is a transport factor that recognizes this RNA or whether it is efficiently transported out of nucleus.

In order to prepare remedies for this problem, we tried experiments utilizing the REV factor of HIV-1 RNA, which is known to have the function of improving the efficiency of nuclear export of RNA.

As shown in the figure, REV plays the role of carrying an unspliced RNA genome to the cytoplasm in the life cycle of HIV-1 (Figure 6-b). In the case of ordinary mRNA, there is a retention mechanism that prevent molecules retaining introns from transferring out of the nucleus, thus preventing the transport of immature mRNA. REV binds to a specific part (RRE: Rev responsive element) of the intron on the HIV-1 RNA genome and binds itself to the nuclear export factor CRM1, and overcomes such a retention mechanism and transports RNA to the cytoplasm[5]. Even if the dsRNA is not recognized as a nuclear export factor or even if it is retained in the nuclear retention factor, we thought that it is possible that the efficiency of nuclear export can be improved by inserting REV-RRE system, and provide these new parts to the iGEM community (BBa_K2403000, BBa_K2403002)

Figure 6-b Rev protein induces nuclear export of RRE-containing RNAs.

The results of microinjection of RI-labeled dsRNA with RRE into the nucleus of Xenopus oocytes are shown in the figure (Figure6c ~ Figure6f). Nuclear and cytoplasm were separated after a certain period of time following injection, RNA was recovered from each and analyzed .

* Dr. Taniguchi, Institute for fronter life and medical science, Kyoto university, conducted an experiment on the process of treating RI on and after in vitro transcription.

Figure 6-c Outline of Xenopus oocyte microinjection

Figure 6-d RNAs produced by in vitro transcription
U6 and U6-RRE are used as controls.

Figure 6-e Microinjection of dsGFP with or without RRE into Xenopus oocyte
Indicated RNAs with or without Rev protein were injected into Xenopus oocyte nucleus. The oocytes were dissected at the indicated time points (t=0, 60 min). The nuclear RNAs (N) and the cytoplasmic RNAs (C) were extracted and analyzed by PAGE. The left pannel shows dsGFP with RRE (GFP-RRE + GFPrev), the right pannel shows dsGFP without RRE (GFPfwd + GFPrev).

Figure 6-f Long exposure

As expected, when U6-RRE is injected together with buffer without Rev, U6-RRE remains in the nucleus, whereas U6 - RRE is injected with buffer containing Rev, U6-RRE was remarkably transported outside the nucleus. This result demonstrated that nuclear export of RNA is promoted depending on both the RRE sequence and the Rev protein in the case of U6 RNA originally staying in the nucleus. These effects indicate that these parts are promising as devices for efficiently transporting highly structured RNA, which is often used in synthetic biology, to the cytoplasm.(BBa_K2403000 BBa_K2403002) Unfortunately, transcription products of GFP-RRE were too thin to understand whether they responded to Rev. However, from the figure on the right of Figure 6-f, it was also found that dsRNA remained in the nucleus a lot. It is suggested that implementation of a system to promote nucleocytoplasmic transport is effective. Compared to the signal at T = 0, since many signals are lost at 60 minutes after injection, there may be a mechanism for degrading dsRNA in cells. In addition to promoting transportation efficiency, there will be room for improvement to improve stability.

This is the Result obtained in this project. We would like to discuss Discussion on interpretation of Result and Future plan.

Reference

[1] X. rong Wang, X. Cheng, Y. dong Li, J. ai Zhang, Z. fen Zhang, and H. rong Wu, “Cloning arginine kinase gene and its RNAi in Bursaphelenchus xylophilus causing pine wilt disease,” Eur. J. Plant Pathol., vol. 134, no. 3, pp. 521–532, 2012.
[2] A. Sigova, N. Rhind, and P. D. Zamore, “A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe,” genes Dev., 2004.
[3] R. Esteban and R. B. Wickner, “A new non-mendelian genetic element of yeast that increases cytopathology produced by M1 double-stranded RNA in ski strains.,” Genetics, 1987.
[4] M. T. B. Sloan, Katherine E, Pierre-Emmanuel Gleizes, “Nucleocytoplasmic Transport of RNAs and RNA–Protein Complexes,” J. Mol. Biol., vol. 428, no. 10, pp. 2040–2059, 2016.
[5] V. W. Pollard and M. H. Malim, “the Hiv-1 Rev Protein,” Annu. Rev. Microbiol., vol. 52, no. 1, pp. 491–532, 1998.