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Abstract Construct Lab note [ June / July / August ] Result

Lab note

To make main construct, we made several assays.

Detail protocols about this is:

June.

July.

August.

Main construct

Main construct

If you want to know detail explanation, please read Construct section.

Assay

Terminator leak switch

Terminator leak switch assay

To realize 4C3 leak switch, we should choose proper terminator which terminates transcription when connected two or more but leak when single.

We made "A-M-RO/Char" assay (see Fig, named from the famous animation, "MOBILE SUIT GUNDAM") to select proper terminator:

AOI

no terminator

-> cre protein express rapidly

-> lox site is removed rapidly

-> gfp may expression rapidly

MURASAKI

one terminator

-> cre protein express slowly

-> lox site is removed slowly

-> gfp may expression slowly

ROKUJYO

two terminator

-> cre protein can't express

-> lox site remain

-> gfp expression may not express

First we use single terminator BBa_B1006. Then other terminators are tested: 80%-terminate terminator and 99%-terminate terminator to determine the best threshold to realize terminator leak switch.

Location sequence

Parts making

How to make location parts

Ligate small parts - recognation site, location site and rbs.

Expression check

Location sequence

location sequence を用いた、mRNA の相補鎖による翻訳の抑制を調べるassay。アンチセンス鎖 として働くL1'-L4' をc-pro 􀀀を用いて転写。c-pro 􀀀は恒常的に作動するプロモーター。iGEM パー ツに存在するconstitutive promoter で最も強いものを使用。pBAD(rev) はアラビノースを投入する ことで作動するプロモーター。アラビノースの濃度と転写量の関係は不明。そこで、まずは各プロ モーターの転写を開始させる強さを調べるアッセイを行う。それぞれのプロモーター(pBAD は正方 向のものを用いる) の下流にgfp を発現するparts をつなげることで、gfp をレポートタンパク質と して用いて、転写の強さを調べる。 対照実験として、ポジティブコントロールを作成。アンチセンス鎖(L1'-L4') が存在しないなかで、 L1-L4&loading sequence の配列を通過してtet 耐性が見られるかどうかを確認。 として、現在設計されているlocation sequence に開始コドンに対応する塩基配列が含まれ ているものを発見(L1,L4)。rbs の後ろに配列があるため、gfp に余分なアミノ酸が結合するか、も しくは、完全に配列が変化するかして、いい実験結果が得られない可能性が高いため、assay 系は、 L2,L4 のみを用いて行う。

MS2 virus

Parts making

We get MS2 gene RT-PCR product. This original product include a lot of restricted enzyme site: two EcoRI site, two XbaI site and one VspI site.

To run our project, we don’t have to remove EcoRI site in the region. So we modified XbaI site by PCR as the following method:

How to make MS2 parts

1. We divided RT-PCR product into two parts by XP enzyme digestion: E-X-E-E-V-X -> “Goten” X-S-P -> “Tranks” (named from the famous animation, "DRAGON BALL")

2. Insert is ligated with the vector, chloramphenicol-tolerance: Goten -> EX vector Tranks -> XP vector

3. Tranks -> PCR adding V-X region

4. Each part -> VP enzyme digestion, ligation each other

Expression check

Virus expression check assay

We use MS2 phage to transmit information of location and number. The expression of the phage should start after 4C3 leak switch turns on. MS2 phage transport RNA which has loding sequence, so we knock out self assembly and made our E.coli translate loading seaquence at another point. To check whether transportion go correctly, we made assay.

flpe

Parts making

How to make flpe parts

The original flpe include two restriction enzyme sites(EcoRI, SpeI).

We modified this flpe by PCR:

1st PCR : cloning

-> ligation with vector

-> 2nd PCR : modified SpeI site

-> 3rd PCR : modified EcoRI site

We use this part as the form of reverse, so we made this part reverse by PCR.

Expression check

flpe expression check assay

To check whether flpe works correctly, we made assay shown in Fig.

The top construct is a nagative control. GFP can't be expressed because of the double terminators.

The bottom construct express GFP when flpe works correctly. When flpe is expressed correctly, flpe recognaize the frt site and double terminator which terminates the expression of gfp is removed, so GFP may be expressed.

Hin

Parts making (reverse)

How to make Hin parts

We get Hin parts from HQ (BBa_J31000).

We use this part as the form of reverse, so we made this part reverse by PCR.

To check PCR is done correctly, we did VspI digestion.

Expression check

Hin expression check assay

To check whether flpe works correctly, we made assay shown in Fig, similar to the assay of flpe check.

The top construct is a nagative control. GFP can't be expressed because of the double terminators.

The bottom construct express GFP when Hin works correctly. When Hin is expressed correctly, Hin recognaize the hix site and double terminator which terminates the expression of gfp is removed, so GFP may be expressed.

Parts list