iGEM 2016 — Team Tsinghua

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# Team:Tsinghua/Notebook

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[Description](https://2016.igem.org/Team%3ATsinghua/Description)

[Design](https://2016.igem.org/Team%3ATsinghua/Experiments)

[Results](https://2016.igem.org/Team%3ATsinghua/Results)

[Proof of concepts](https://2016.igem.org/Team%3ATsinghua/Proof)

[Notebook](https://2016.igem.org/Team%3ATsinghua/Notebook)

[Modeling](https://2016.igem.org/Team%3ATsinghua/Model)

[Safety](https://2016.igem.org/Team%3ATsinghua/Safety)

# notebook

## Jun 2016

**01/07/16**

JQA

Preparation of dCas9 plasmid: hym12, hym5, jqa2.

**03/07/16**

JQA

Transformation of Recombinant of sgRNA, sgNC(negative control). In total 6 plates

**06/07/16**

JZR

Identification of Positive Recombinant of sgRNA, sgNC. And inculated 2 single colony of transformed bacteria
into LB medium. Shaking overnight at 37℃. Transformation of Recombinant of sgActin.

**07/07/16**

JZR

Identification of Positive Recombinant of sgActin. And inculated 2 single colony of transformed bacteria into
LB medium. Shaking overnight at 37℃.

**08/07/16**

JZR

Prepare plasmids of sgActin and sgNC with the Kittool. Sent to sequencing.

**09/07/16**

JZR

PCR for sgActin with KOD system.

PCR system:

* Template 1µl* Primer F & R 1.5 \* 2 = 3µl* Buffer 5 µl* dNTP 5 µl* Mg2+ 4 µl* KOD+ enzyme 1 µl* ddH2O 31 µl

total 50 µl

The result of agarose gel electrophoresis is smeared.

**10/07/16**

GCH,LMJ

In vitro transcription of sgRNA using the promega kittool.The result is negative.

Isolation of total RNA and cDNA synthesis. KOD PCR for linear DNA with T7 promoter.

PCR system:

* Template 1µl* Primer F & R 1.5 \* 2 = 3µl* Buffer 5 µl* dNTP 5 µl* Mg2+ 4 µl* KOD+ enzyme 1 µl* ddH2O 31 µl

total 50 µl

**11/07/16**

LMJ, GCH

*In vitro* transcription of sgRNA using the promega kittool. RNA purification.

Reaction system:

* 0.1 Volume of NaOAC* 1 Volume phenol-chloroform-isopropanol

Mix(invert 6 times)

Transfer to phase lock gel(PLG)

15000 rpm, 10min, 4℃

supernatant + same volume of chloroform

Mix

PLG

Transfer to phase lock gel(PLG)

15000 rpm, 10min, 4℃

Add 1.5 volume of isopropanol

-20$#8451 for 1 hour

18000rpm, 4℃

200 µl 75% EtOH wash

5 µl ddH2O resolve

![](https://static.igem.org/mediawiki/2016/7/75/T--Tsinghua--notebook1.png)

**18/07/16**

LY

KOD PCR for sgActin & yActin linear DNA with T7 promoter

PCR system:

* Template 1µl* Primer F & R 1.5 \* 2 = 3µl* Buffer 5 µl* dNTP 5 µl* Mg2+ 4 µl* KOD+ enzyme 1 µl* ddH2O 31 µl* total 50 µl

PCR procedure:

* 94℃ 3’* 94$℃ 30’’* 60$℃ 30’’ \* 25 cycles* 72$℃ 30’’ \* 25 cycles* 72$℃ 7’* 16$℃ ∞

PCR result: sgActin failed. yActin succeeded.

**19/07/16**

LY

sgActin1-4 were sent to sequencing.(M13F)

PCR of sgActin, procedure is the same as that in 7.18

sgActin PCR purification using TIANGEN Kit.

The result of agarose gel ep is negative. – Nothing!

**28/07/16**

JQA

dCas9 protein

A280 test suggests a 0.62 ng/ml

**28/07/16**

LMJ

dCas9 +RT+sgRNA transformation into yeast

dCas9 +PAMMer+sgRNA transformation into yeast

using leu2 auxotroph yeast to select positive strains.

**29/07/16**

JQA

Bridge PCR of Actin with 3 parallels.

Colony PCR of RT with KOD(20 µl system)

![](https://static.igem.org/mediawiki/2016/b/b4/T--Tsinghua--notebook2.png)

Reaction buffer:

* 20 mM Tris-HCL pH7.5* 75mM KCl* 5mM MgCl2* 1mM DTT* 5% glycerol

## Aug 2016

**01/08/16**

GCH, LMJ

POT assembly system:

* 10X T4 ligase buffer 1µl* 100X BSA 0.1µl* BsmBI 0.5 µl* T4 ligase 0.2µl* Parts: P,O,T 2µl* POT vectors

*In vitro* transcription with promega Kit (20 µl system):

* Buffer 4µl* ATP, CTP, GTP, UTP: 1.5µl\*4=6µl* Enzyme mix(T7) 2µl* Template: 1-2 µg* Nuclease free water 7µl

37$#8451 incubation for 2hrs

DNAase overnight

PCI extraction

EtOH precipitation

Free water resolve

Agarose gel to confirm

**02/08/16**

LMJ

SgRNA extraction

SgRNA template PCR purification

![](https://static.igem.org/mediawiki/2016/9/95/T--Tsinghua--Notebook3.png)

**03/08/16**

JQA

POT assembly of RT, yTK

Couldn't find 555,556, so did plasmid preparation again

**20/08/16**

JQA

C2C2 assembly:

PCR system:

* C2C2 + primer* dCas9 POT + BD primer/AD primer* Template 5µl* Primer F & R 2.5 \* 2 = 3µl* Buffer 5 µl* dNTP 5 µl* Mg2+ 4 µl* KOD+ enzyme 1 µl* ddH2O 25 µl

total 50 µl

PCR procedure:

* 94℃ 3’* 94℃ 30’’* 55℃ 30’’ \* 25 cycles* 68℃ 4’ \* 25 cycles* 68℃ 10’* 16℃ ∞

**21/08/16**

JQA

BD/AD PCR failed

Using PID 13, 14 to restart the experiment again.

PCR system:

* Template 5µl* Primer F & R 2.5 \* 2 = 5µl* Buffer 5 µl* dNTP 5 µl* Mg2+ 4 µl* KOD+ enzyme 1 µl* ddH2O 31 µl

total 50 µl

PCR procedure:

* 94℃ 3’* 94℃ 30’’* 55℃ 30’’ \* 35 cycles* 68℃ 345’ \* 35 cycles* 68℃ 10’* 16℃ ∞

**23/08/16**

JQA

One pot digestion & ligation reaction to construct dC2C2-BD-AD on HCKanO

KOD PCR to change vector for dCas9

**24/08/16**

LY

Construct PQlink-His-dCas9

PQ-H double digestion:

* 10X NEB buffer 3.1 2µl* EagI 0.5µl* SalI-HF 0.5µl* ddH2O 13µl

Total 20µl

37℃ 2hrs and 65℃ 20’

**27/08/16**

LMJ

PCR + Histag to dCas9

PCR system:

* Template 5µl* Primer F & R 2 \* 2 = 4µl* Buffer 5 µl* dNTP 5 µl* Mg2+ 4 µl* KOD+ enzyme 1 µl* ddH2O 25 µl

total 50 µl

PCR procedure:

* 94℃ 3’* 94℃ 30’’* 63℃ 30’’ \* 25 cycles* 68℃ 6’ \* 25 cycles* 68℃ 10’* 16 ∞

Ligation

Transformation into *E.coli*

Shaking overnight

**30/08/16**

JQA

C2C2 assembly:

Result is negative again!

![](https://static.igem.org/mediawiki/2016/d/d1/T--Tsinghua--Notebook4.png)

**31/08/16**

JZR

Using fluorescence to locate dCas9, via electroporation

Sg+ group: sg+ Cas9+PAMer

Sg- group: Cas9+PAMer

Most signal is located in the nucleus

## Sep 2016

**04/09/16**

LMJ

Transformation of pGEX-suvCas9 plasmid

**05/09/16**

LY

C2C2 assembly again

BD, AD, C1C2 gel clean up digestion

* DNA 4µl* Custmart 1µl* BsaI 0.2µl* ddH2O 4.8µl

**06/09/16**

LY

C1-C2, BD-AD PCR

* Q5 buffer 5µl* dNTP 4µl* Primer 2.5\* 2µl* Q5 0.5µl* Ligation product 30.5µl

**18/09/16**

LY

Transform BL21 with pGEX-suvCas9

Inculate in LB, 37℃ for 8hrs

**23/09/16**

JZR

Insert sequencing

DNA purification in 15µl system

Nanodrop to measure the concentration:

* Insert5-1: 3.6λ* Insert12-1: 8.7λ

**24/09/16**

JZR

Transfection with yeast using 2 system:

system1 :insert5-1

system2 :insert12-1

[>>Read More<<](https://static.igem.org/mediawiki/2016/9/93/T--Tsinghua--notebook.pdf)

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