したっぱ昆虫細胞研究者のメモ

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2016年 06月 16日

6x8x8 人工イクラ細胞アレイ

産業用の動物細胞の研究開発では、増殖や生産性が良くなることを期待して培地を改造することがあります。アミノ酸、ビタミン、乳酸、グルタミン酸など多数の培地成分について濃度を振って培養試験を行うためサンプル数が多くなりがちなので、どうやって捌くかが悩みの種になっています。
タンパク質製造のための細胞の培養試験では、細胞数、生産タンパク質濃度の他、糖や乳酸などの代謝物の測定を行うことが多いのですが、細胞数の測定の自動化の点で特に問題がありました。例えば、最も普及している自動細胞数測定装置であるvicellは、一度の測定に500μLのサンプルを要求します。同時に多くの試験を行うにはひとつひとつの培養を小さくしたいのですが、小さくし過ぎると細胞数が数えられなくなってしまうのです。
そこで著者らは、侵襲性のある方法ではあるものの細胞数を測定可能な小さな培養系を用いることにしました。

PMID 24227746
Microarray platform affords improved product analysis in mammalian cell growth studies

培養系の構築は下記の通りです。
polystyrene-co-maleic anhydride でコートしたガラス板の上にpoly-L-Lysineと塩化バリウムの混合液をスポットします。スポットにはMicrosSys 5100-4SQ microcontact microarray spotter という装置を用いるようです。
スポットが十分に乾いたら、200細胞を含んだ60nlの培養液をその上にスポットします。ここで、培養液には、アルギン酸が1%溶かされています。元のスポットに含まれるバリウムがアルギン酸を架橋してアルギン酸バリウムゲルになります。ちなみに人工イクラはアルギン酸カルシウムゲルです。
このようにして固定した細胞を含むゲルたちを、培地で封入して乾燥を防ぎます。

実験終了後はガラス板上に固定したままインビトロジェンのlive/dead viability/ cytotoxicity kit for mammalian cellsを用いて生細胞数を測定することができます。

論文中ではこの系を用いて培地の改変を行っています。



ひとつのガラス板には6x8個の培養をつくることができ、ひとつの装置にはガラス板が8枚乗るそうなので、6x8x8で384の培養をつくることができます。10日間の培養中に4回測定するとしてn=5でやっても20。ひとつの装置で20ほどの条件を調べることができる計算です。
著者らが引用していたambr15は15mL程度のバイオリアクターを24個並列に実験可能ではありますが、ambr15はクリーンベンチひとつ独占するので単位ラボ面積あたりでは利点がありそうです。

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96穴プレート振るのと比べて果たして買うかなと思いました。
一方で、培地の最適化で使う材料はなるほどなと思いました。
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# by koretoki | 2016-06-16 11:43
2014年 09月 09日

ダーウィンは一回だけevolved と書いた

wiki見てたら"ダーウィンは、『種の起源』の中で、evolution ではなく、Descent with modification という単語を使っている。これは Evolution という語が進歩や前進を意味しており、ダーウィンは進化にそのような意味を込めていなかったからである。" とあったので The Origin of Species を眺めてみた。

The Origin of Species
http://www.literature.org/authors/darwin-charles/the-origin-of-species/index.html

evolve 一個だけ
"There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved."

一方、変化を伴う由来(Descent with modification)の方はそこら中にでてきた。

-----

descent の数を数えた。少ない時は引用した。

http://www.literature.org/authors/darwin-charles/the-origin-of-species/preface.html

In 1846 the veteran geologist N. J. d'Omalius d'Halloy published in an excellent though short paper ("Bulletins de l'Acad. Roy Bruxelles,' tom. xiii. p. 581) his opinion that it is more probable that new species have been produced by descent with modification than that they have been separately created: the author first promulgated this opinion in 1831.

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-01.html

May not those naturalists who, knowing far less of the laws of inheritance than does the breeder, and knowing no more than he does of the intermediate links in the long lines of descent, yet admit that many of our domestic races have descended from the same parents may they not learn a lesson of caution, when they deride the idea of species in a state of nature being lineal descendants of other species?

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-02.html

The term 'variety' is almost equally difficult to define; but here community of descent is almost universally implied, though it can rarely be proved.

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-03.html

There is no exception to the rule that every organic being naturally increases at so high a rate, that if not destroyed, the earth would soon be covered by the progeny of a single pair. Even slow-breeding man has doubled in twenty-five years, and at this rate, in a few thousand years, there would literally not be standing room for his progeny. Linnaeus has calculated that if an annual plant produced only two seeds and there is no plant so unproductive as this and their seedlings next year produced two, and so on, then in twenty years there would be a million plants. The elephant is reckoned to be the slowest breeder of all known animals, and I have taken some pains to estimate its probable minimum rate of natural increase: it will be under the mark to assume that it breeds when thirty years old, and goes on breeding till ninety years old, bringing forth three pairs of young in this interval; if this be so, at the end of the fifth century there would be alive fifteen million elephants, descended from the first pair.

So it is when we travel northward, but in a somewhat lesser degree, for the number of species of all kinds, and therefore of competitors, decreases northwards; hence in going northward, or in ascending a mountain, we far oftener meet with stunted forms, due to the directly injurious action of climate, than we do in proceeding southwards or in descending a mountain. When we reach the Arctic regions, or snow-capped summits, or absolute deserts, the struggle for life is almost exclusively with the elements.

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-04.html

"descendants" "descends" 等54カ所

Natural Selection * its power compared with man's selection * its power on characters of trifling importance * its power at all ages and on both sexes * Sexual Selection * On the generality of intercrosses between individuals of the same species * Circumstances favourable and unfavourable to Natural Selection, namely, intercrossing, isolation, number of individuals * Slow action * Extinction caused by Natural Selection * Divergence of Character, related to the diversity of inhabitants of any small area, and to naturalisation * Action of Natural Selection, through Divergence of Character and Extinction, on the descendants from a common parent * Explains the Grouping of all organic beings

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-05.html

14カ所

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-06.html

15カ所

Difficulties on the theory of descent with modification * Transitions * Absence or rarity of transitional varieties * Transitions in habits of life * Diversified habits in the same species * Species with habits widely different from those of their allies * Organs of extreme perfection * Means of transition * Cases of difficulty * Natura non facit saltum * Organs of small importance * Organs not in all cases absolutely perfect * The law of Unity of Type and of the Conditions of Existence embraced by the theory of Natural Selection

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-07.html

No complex instinct can possibly be produced through natural selection, except by the slow and gradual accumulation of numerous, slight, yet profitable, variations. Hence, as in the case of corporeal structures, we ought to find in nature, not the actual transitional gradations by which each complex instinct has been acquired for these could be found only in the lineal ancestors of each species but we ought to find in the collateral lines of descent some evidence of such gradations; or we ought at least to be able to show that gradations of some kind are possible; and this we certainly can do.

For no amount of exercise, or habit, or volition, in the utterly sterile members of a community could possibly have affected the structure or instincts of the fertile members, which alone leave descendants. I am surprised that no one has advanced this demonstrative case of neuter insects, against the well-known doctrine of Lamarck.

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-08.html

10カ所

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-09.html

14カ所

"revolution"
He who can read Sir Charles Lyell's grand work on the Principles of Geology, which the future historian will recognise as having produced a revolution in natural science, yet does not admit how incomprehensibly vast have been the past periods of time, may at once close this volume.

From these and similar considerations, but chiefly from our ignorance of the geology of other countries beyond the confines of Europe and the United States; and from the revolution in our palaeontological ideas on many points, which the discoveries of even the last dozen years have effected, it seems to me to be about as rash in us to dogmatize on the succession of organic beings throughout the world, as it would be for a naturalist to land for five minutes on some one barren point in Australia, and then to discuss the number and range of its productions.

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-10.html

25カ所

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-11.html

13カ所

"revolution"
Several facts in distribution, such as the great difference in the marine faunas on the opposite sides of almost every continent, the close relation of the tertiary inhabitants of several lands and even seas to their present inhabitants, a certain degree of relation (as we shall hereafter see) between the distribution of mammals and the depth of the sea, these and other such facts seem to me opposed to the admission of such prodigious geographical revolutions within the recent period, as are necessitated in the view advanced by Forbes and admitted by his many followers.

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-12.html

6カ所

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-13.html

79カ所

"revolve"

Would it be thought sufficient to say that because planets revolve in elliptic courses round the sun, satellites follow the same course round the planets, for the sake of symmetry, and to complete the scheme of nature? An eminent physiologist accounts for the presence of rudimentary organs, by supposing that they serve to excrete matter in excess, or injurious to the system; but can we suppose that the minute papilla, which often represents the pistil in male flowers, and which is formed merely of cellular tissue, can thus act?

http://www.literature.org/authors/darwin-charles/the-origin-of-species/chapter-14.html

33カ所

"revolution"
When the views entertained in this volume on the origin of species, or when analogous views are generally admitted, we can dimly foresee that there will be a considerable revolution in natural history.

"evolved"
There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved.




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# by koretoki | 2014-09-09 12:20
2014年 05月 23日

genome.fa とtranscripts.gtf でtranscripts.fa をつくる


genome.fa とtranscripts.gtf でtranscripts.fa をつくる

編集 | 削除
tophat-cufflinks を使ってtranscripts.gtf は手に入れたもののtranscripts.fa がないから遺伝子見るの大変、とか、何でか知らないけどtranscripts.gtf しか貰えなかったパターンのときにtranscripts.fa を作らないといけない状況になります。

#transcripts.gtf とgenome.fa からtranscripts.fa を作る。
#transcripts.gtf を加工1
more transcripts.gtf | awk -F "\t" '{print $1"\t"$3"\t"$4"\t"$5"\t"$9}' | grep exon | awk '{print $1"\t"$3"\t"$4"\t"$6"\t"$8"\t"$10}' | tr -d "\"" | tr -d ";" | awk '{print "transcript_id_"$5"\t"$1"\t"$2"\t"$3"\t"$6}' | awk '{print $1"\t"$2"\tsubstr($2,"$3","$4-$3+1")\t"$5/100"0"}' | awk '{print $1"\t"$2"\t"$3"\tn"substr($4,3,2)}' | sed -e '/n01/s/subs/@subs/g' | awk '{print $3}' | tr -d "\n" | tr "@" "\n" | grep substr > temp1.txt
#transcripts.gtf の加工2
more transcripts.gtf | awk -F "\t" '{print $1"\t"$3"\t"$4"\t"$5"\t"$9}' | grep exon | awk '{print $1"\t"$3"\t"$4"\t"$6"\t"$8"\t"$10}' | tr -d "\"" | tr -d ";" | awk '{print "transcript_id_"$5"\t"$1"\t"$2"\t"$3"\t"$6}' | awk '{print $1"\t"$2}' | uniq > temp2.txt

#genome.fa を1行にしておく
more genome.fa | sed -e '/>/s/$/@/g' | tr -d "\n" | tr ">" "\n" > line_genome.txt

#transcripts.gtf の情報を使って
paste temp2.txt temp1.txt | head -n 10
transcript_id_CUFF.1.1NODE_1000_length_89123_cov_46.018040substr($2,2,2497)
transcript_id_CUFF.2.1NODE_1000_length_89123_cov_46.018040substr($2,3291,1003)substr($2,4409,646)

#こういうコマンドをつくりたい
more line_genome.txt | grep NODE_1000_length_89123_cov_46.018040 | awk -F "@" '{print ">transcript_id_CUFF.1.1@"substr($2,2,2497)}' | tr "@" "\n" >> transcripts.fa
>transcript_id_CUFF.1.1
CGTAGTAGTATCGATCGTATCTACTATCGTACGTAGCTAGTCGATGCTAGTCGTAGCTAACTCGATCGATCGTAGTCTGTTAGCTAGCTGATCGTAGCTAGCTGATCGTAGCTAGCTGATCGTAGCTAGCTGATCGTAGCTGATCGTAGCTGATGCTGTAGCTGATCGTAGCTGATCGTAGCTAGTCTAGCTGATGCTAGCTGATCGTAGCTAGCTGATCGTAGCTAGCTGATGCTAGCTGATCGTAGCTGATCGTAGCTAGCTGATCgTAGCTAGCTTATCGTGTAGTCGTAGCTAGCTGATCGTAGCTAGATAAAAACGGGGATCGATCGTAGCTAAAAAGCTTTTTTTGAAAAATCGTAGCTGATGCTAGCTGATGCTGTAGTGTGTGATTTATATTACTAGCTAGCCCCATGCTAGTCGATCACACAC

#transcripts.gtf の情報を使ってコマンドを作る
paste temp2.txt temp1.txt | awk '{print "more line_genome.txt | grep "$2" | awk -F \"@\" %{print \">"$1"@\""$3"}% | tr \"@\" \"\\n\" >> transcripts.fa"}' | tr "%" "'" > make_transcripts.sh

#準備ができたら実行
sh make_transcripts.sh

#できる
ls

genome.fa
transcripts.gtf
temp1.txt
temp2.txt
line_genome.txt
make_transcripts.sh
transcripts.fa <- NEW!!!


終わってから気づいたけどこれでいいじゃん

http://cufflinks.cbcb.umd.edu/gff.html

Extracting transcript sequences
The gffread utility can be used to generate a FASTA file with the DNA sequences for all transcripts in a GFF file. For this operation a fasta file with the genomic sequences have to be provided as well. For example, one might want to extract the sequence of all transfrags assembled from a Cufflinks assembly session. This can be accomplished with a command line like this:

gffread -w transcripts.fa -g /path/to/genome.fa transcripts.gtf

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# by koretoki | 2014-05-23 15:00
2014年 01月 09日

makeblastdb

makeblastdb -in ref.fa -dbtype nucl -hash_index
blastn -db ref.fa -query que.fa -outfmt 6 > blastout.txt

makeblastdb -in ref.pep.fa -dbtype prot
blastp -db ref.pep.fa -query que.pep.fa -outfmt 6 > blastout.txt
[PR]

# by koretoki | 2014-01-09 12:25
2014年 01月 07日

メタボデータをくっつけた

メタボローム解析の反復データをひとつにまとめたい。
n=3のものとn=1のものを比べたいのだけど、できればn=3全部使いたい。
(a1+a2+a3)/3 ってやっていいのかわからなかったから調べてみた。

#もともと持っているのはこんなデータ。いわゆるMartixとして得られたデータである。
name a1 a2 a3
CYT001 0.0007330321 0.0004886212 0.0008590029
CYT002 0.1763202000 0.1228157000 0.1730612000
CYT003 0.0137760700 0.0123575800 0.0164345100
CYT004 0.0121694300 0.0103101300 0.0095795930
CYT005 0.0008732506 0.0006902750 0.0008904565
CYT006 0.0007769990 0.0005277728 0.0005936377
CYT007 0.0010050660 0.0005964599 0.0010336850
CYT008 0.0000000000 0.0000000000 0.0000000000
CYT009 0.0000000000 0.0000000000 0.0000000000
CYT010 0.0005402510 0.0004378932 0.0003912269
CYT011 0.0000000000 0.0000000000 0.0000000000
CYT012 0.0000000000 0.0000000000 0.0000000000
#(これがずっと続いてる)

#R様に読んでいただく。
> data<-read.table("data.txt",header=T,row.names=1)
> attach(data)
> summary(data)
a1 a2 a3
Min. :0.0000000 Min. :0.0000000 Min. :0.0000000
1st Qu.:0.0003296 1st Qu.:0.0002691 1st Qu.:0.0000955
Median :0.0015751 Median :0.0015310 Median :0.0010649
Mean :0.0696770 Mean :0.0660012 Mean :0.0445601
3rd Qu.:0.0200691 3rd Qu.:0.0198329 3rd Qu.:0.0128484
Max. :2.4163290 Max. :2.2341410 Max. :1.5474730

#読んでいただけた。実際にはデータが全部表示されるが省略。
> data
a1 a2 a3
CYT001 0.0007330321 0.0004886212 0.0008590029
CYT002 0.1763202000 0.1228157000 0.1730612000
CYT003 0.0137760700 0.0123575800 0.0164345100
CYT004 0.0121694300 0.0103101300 0.0095795930
CYT005 0.0008732506 0.0006902750 0.0008904565
CYT006 0.0007769990 0.0005277728 0.0005936377
CYT007 0.0010050660 0.0005964599 0.0010336850

#総和がこんなにズレてる...
> sum(a1)
[1] 15.53798
> sum(a2)
[1] 14.71827
> sum(a3)
[1] 9.936896

#これをそのままプロットして1:1のところに線引いてみる。
> plot(a1,a3)
> abline(0,1,col="red")
e0160319_1135217.png

#ずれずれっぽいけどよく見えないからlog
> plot(log(a1),log(a3))
> abline(0,1,col="red")
e0160319_114586.png


#とりあえず総和で割ればいけそう
> plot(log(a1/sum(a1)),log(a3/sum(a3)))
> abline(0,1,col="red")
e0160319_1152369.png


(a1/sum(a1)+a2/sum(a2)+a3/sum(a3))/3
すればとりあえず物質間の比については使えるデータになりそう。
a1*x+a2*y+a3*zの形にしたい。
総和でも良さそうだけど怖いから回帰で比を出してもらうことにした。

#それぞれ回帰してもらう
#回帰モデルする
> fit_12<-lm(a1~a2)
> fit_23<-lm(a2~a3)
#中見る1vs2
> fit_12

Call:
lm(formula = a1 ~ a2)

Coefficients:
(Intercept) a2
-0.0005735 1.0643829
#中見る2vs3
> fit_23

Call:
lm(formula = a2 ~ a3)

Coefficients:
(Intercept) a3
0.006123 1.343758

#調節する時にかける値は
x:y=1:1.0643829
y:z=1:1.343758
x:y:z=1:1.0643829:1.430273=0.2861512:0.3045745:0.4092743

0.2861512*a1+0.3045745*a2+0.4092743*a3 で欲しい値が取れる。

#総和の比でやると
> sum(a1)
[1] 15.53798
> sum(a2)
[1] 14.71827
> sum(a3)
[1] 9.936896
> sum(a1)/sum(a2)
[1] 1.055693
> sum(a2)/sum(a3)
[1] 1.481174
> #x:y=1:1.055693
> #y:z=1:1.481174
> 1.055693*1.481174
[1] 1.563665
> #x:y:z=1:1.055693:1.563665
> 1*1/(1+1.055693+1.563665)
[1] 0.2762921
> 1*1.055693/(1+1.055693+1.563665)
[1] 0.2916796
> 1*1.563665/(1+1.055693+1.563665)
[1] 0.4320283
> #x:y:z=1:1.055693:1.563665=0.2762921:0.2916796:0.4320283

総和の方が強い?補正になってるのは、値の大きな子が引っ張ってるのかなー?

#回帰で求めた比がちゃんとハマるか心配
> plot(a1,a2)
> abline(0,1/1.0643829,col="red")
> plot(a2,a3)
> abline(0,1/1.343758,col="red")
> lm(a3~a1)

Call:
lm(formula = a3 ~ a1)

Coefficients:
(Intercept) a1
-0.00182 0.66564

> plot(a3,a1)
> abline(0,1/0.66564,col="red")

#logでもハマるか一応
> plot(log(a1),log(a2))
> e<-exp(1)
> e
[1] 2.718282
> abline(-log(1.0643829),1,col="red")
> plot(log(a2),log(a3))
> abline(-log(1.343758),1,col="red")
> abline(0,1,col="blue")
> plot(log(a3),log(a1))
> abline(0,1,col="blue")
> abline(-log(0.66564),1,col="red")

e0160319_116455.png


赤線が回帰の線
青線は1:1の線

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# by koretoki | 2014-01-07 01:18