iGEM 2015 — Team BGU_Israel

# Team:BGU Israel/Notebook

# Team:BGU Israel

|  |  |  |
| --- | --- | --- |
| ![](https://static.igem.org/mediawiki/2015/d/df/Bgu_banner4.png) | | |

* [Home](https://2015.igem.org/Team%3ABGU_Israel)
* Project
  + [Description](https://2015.igem.org/Team%3ABGU_Israel/Description)
  + [Design](https://2015.igem.org/Team%3ABGU_Israel/Design)
  + [Results](https://2015.igem.org/Team%3ABGU_Israel/Results)
  + [Protocols](https://2015.igem.org/Team%3ABGU_Israel/Experiments)
  + [Collaborations](https://2015.igem.org/Team%3ABGU_Israel/Collaborations)
  + [Achievements](https://2015.igem.org/Team%3ABGU_Israel/Achievements)
* [Team](https://2015.igem.org/Team%3ABGU_Israel/Team)
  + [Team](https://2015.igem.org/Team%3ABGU_Israel/Team)
  + [Attributions](https://2015.igem.org/Team%3ABGU_Israel/Attributions)
* Practices
  + [Practices and Polices](https://2015.igem.org/Team%3ABGU_Israel/Practices)
  + [Entrepreneurship](https://2015.igem.org/Team%3ABGU_Israel/Entrepreneurship)
  + [Ethics](https://2015.igem.org/Team%3ABGU_Israel/Ethics)* Parts
    + [Team Parts](https://2015.igem.org/Team%3ABGU_Israel/Parts)
    + [Basic Parts](https://2015.igem.org/Team%3ABGU_Israel/Basic_Part)
    + [Part Improvement](https://2015.igem.org/Team%3ABGU_Israel/Part_improvement)
    + [Part Collection](https://2015.igem.org/Team%3ABGU_Israel/Part_Collection)
  * [Notebook](https://2015.igem.org/Team%3ABGU_Israel/Notebook)
  * [Safety](https://2015.igem.org/Team%3ABGU_Israel/Safety)

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | June | | | | | | | | --- | --- | --- | --- | --- | --- | --- | |  | 1 | 2 | 3 | 4 | 5 | 6 | | 7 | 8 | 9 | 10 | 11 | 12 | 13 | | 14 | 15 | 16 | 17 | 18 | 19 | 20 | | [21](#2106) | [22](#2206) | 23 | 24 | 25 | 26 | 27 | | [28](#2806) | [29](#2906) | [30](#3006) |  |  |  |  | | | July | | | | | | | | --- | --- | --- | --- | --- | --- | --- | |  |  |  | [1](#0107) | 2 | 3 | 4 | | 5 | [6](#0607) | [7](#0707) | [8](#0807) | 9 | 10 | 11 | | [12](#1207) | [13](#1307) | [14](#1407) | [15](#1507) | [16](#1607) | [17](#1707) | [18](#1807) | | [19](#1907) | [20](#2007) | [21](#2107) | [22](#2207) | [23](#2307) | [24](#2407) | 25 | | [26](#2607) | [27](#2707) | [28](#2807) | [29](#2907) | 30 | 31 |  | | | August | | | | | | | | --- | --- | --- | --- | --- | --- | --- | |  |  |  |  |  |  | 1 | | [2](#0208) | [3](#0308) | [4](#0408) | [5](#0508) | [6](#0608) | 7 | 8 | | [9](#0908) | [10](#1008) | [11](#1108) | [12](#1208) | [13](#1308) | 14 | [15](#1508) | | [16](#1608) | [17](#1708) | [18](#1808) | [19](#1908) | [20](#2008) | [21](#2108) | [22](#2208) | | [23](#2308) | [24](#2408) | [25](#2508) | [26](#2608) | [27](#2708) | [28](#2808) | [29](#2908) | | [30](#3008) | [31](#3108) |  |  |  |  |  | | | September | | | | | | | | --- | --- | --- | --- | --- | --- | --- | |  |  | [1](#0109) | [2](#0209) | [3](#0309) | [4](#0409) | 5 | | [6](#0609) | [7](#0709) | [8](#0809) | 9 | [10](#1009) | 11 | 12 | | 13 | 14 | 15 | 16 | 17 | 18 | 19 | | 20 | 21 | 22 | 23 | 24 | 25 | 26 | | 27 | 28 | 29 | 30 |  |  |  | |

**All protocols of our work can be found on our
[Protocols page](https://2015.igem.org/Team%3ABGU_Israel/Experiments)
.**

**21.06.15**

**In the lab today: Shai and Shoham**

* We received from Addgene 2 plasmids (in bacterial stab): **dCas9-vp64** (#47107) and **SaCas9** (#61591). they were spread on LB agar plates
  with ampicillin ON at 37°C.
* We transformed **pSB1C3** backbone into DH5α E. coli and seeded it on LB agar plates with Chloramphenicol.

**22.06.15**

**In the lab today: Shai and Shoham**

* 10ml of LB (+ampicillin) was inoculated with one colony from **dCas9-vp64** LB agar plate.
* The same was done for **SaCas9**.

**28.06.15**

**In the lab today: Shai and Shoham**

* 10ml of LB (+ampicillin) was inoculated with one colony from **dCas9-vp64** LB agar plate.
* The same was done for **SaCas9**.

**29.06.15**

**In the lab today: Shai**

* 10ml of LB (+Chloramphenicol) was inoculated with one colony from **pSB1C3** LB agar plate.
* We performed miniprep for **dCas9-vp64** and **SaCas9**.

**30.06.15**

**In the lab today: Shai and Shoham**

* **SaCas9**
  (DH5α) glycerol stock was prepared.
* **dCas9-vp64**
  (DH5α) glycerol stock was prepared
* We performed miniprep for **pSB1C3**.

**01.07.15**

**In the lab today: Shai and Shoham**

* Restriction of **pSB1C3** backbone with EcoRI and PstI. Alkaline phosphatase was added. Incubation for 30 minutes at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol. Nanodrop concentration results were 60 ng/μl.

* Received **U6-gMLP,gMLP,U6-gUBB,gUBB** from synthesis in a form of DNA fragments.

Restriction of **U6-gMLP,gMLP,U6-gUBB,gUBB** with EcoRI and PstI. Incubation for 45 minutes at 37°C.

The restriction products were purified using PCR purification kit. Nanodrop concentration results were 3ng/μl.

* Ligation of **pSB1C3** (vector) with **U6-gMLP,gMLP,U6-gUBB,gUBB** (insert). (each time with different insert).
* 2X10ml of LB (+Chloramphenicol) was inoculated with one colony from **pSB1C3** LB agar plate.

**06.07.15**

**In the lab today: Shoham**

* Ligation products from 01.07 (**U6-gMLP-pSB1C3,U6-gUBB-pSB1C3,gMLP-pSB1C3,gUBB-pSB1C3**) were transformed into DH5α and seeded on LB agar plates
  with Chloramphenicol ON.
* Received virus kit from Agilent (cat. No. 240071) (the kit includes helper plasmids, RC plasmids, AAV-MCS plasmids, HEK cells for virus production).
* We transformed **pAAV-MCS** into DH5α and seeded it on LB agar plates with ampicillin.

**07.07.15**

**In the lab today: Shoham**

* No colonies found on any of the plates of 01.07 ligations.
* Colonies found on **pAAV-MCS** plates from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony.

**08.07.15**

**In the lab today: Shoham**

* We performed miniprep for **pAAV-MCS**. Nanodrop concentration results were 110ng/μl and 117ng/μl (2 Eppendorfs).
* PCR
  was carried out on **gMLP**, **gUBB,** **U6-gMLP**, **U6-gUBB** using default PCR program.

The PCR products were purified using PCR purification kit.

Nanodrop concentration results:

**gMLP**
: 96.6ng/μl, **gUBB**: 40.5ng/μl, **U6-gMLP**: 38.0ng/μl, **U6-gUBB**: 26.0ng/μl.

We prepared 1% Agarose gel, and run the PCR products to check whether the PCR worked as expected. Gel results were good (band in expected size).

Restriction of PCR products with EcoRI and PstI. Incubation for 35 minutes at 37°C.

The restriction products were purified using PCR purification kit. Nanodrop concentration results:

**gMLP**
: 18.8ng/μl, **gUBB**: 6.1ng/μl, **U6-gMLP**: 6.7ng/μl, **U6-gUBB**: 9.4ng/μl.

**12.07.15**

**In the lab today: Shai and Shoham**

* Restriction of **pSB1C3** backbone with EcoRI and PstI. Alkaline phosphatase was added. Incubation for 30 minutes at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol.

* Ligation of **pSB1C3** (vector) with **U6-gMLP** from 08.07(insert).
* Ligation of **pSB1C3** (vector) with **U6-gUBB** from 08.07 (insert).
* Ligation products (**U6-gMLP-pSB1C3,** **U6-gUBB-pSB1C3**) were transformed into DH5α and seeded on LB agar plates
  with Chloramphenicol ON.
* We received from synthesis by Syntezza Bioscience Ltd. **hTERT** promoter (in pU257 plasmid). It was spread on LB agar plates with ampicillin ON
  at 37°C.

**13.07.15**

**In the lab today: Shai and Shoham**

* PCR
  was carried out again for **gMLP**, **gUBB,** **U6-gMLP**, **U6-gUBB** using default PCR program.

The PCR products were purified using PCR purification kit.

Nanodrop concentration results:

**gMLP**
: 106.7ng/μl, **gUBB**: 249.8ng/μl, **U6-gMLP**: 110.1ng/μl, **U6-gUBB**: 57.6ng/μl.

Since concentrations of **gMLP**, **U6-gMLP**, **U6-gUBB** are low we tried to run them in purification columns again with 10μl of elution buffer.

New nanodrop concentration results were:

**gMLP**
: 97.2ng/μl, **U6-gMLP**: 47.4ng/μl, **U6-gUBB**: 73.1ng/μl.

* 10ml of LB (+ampicillin) was inoculated with one colony of **pAAV-MCS** plate from 07.07.

**Cell studies**:

* seeding the following cell lines in 24-well plate (50,000 cells per well
  in triplicates):

+ **HepG2** (Hepatocellular carcinoma, ATCC)
+ **A549** (Lung carcinoma, ATCC)
+ **HT1080** (Fibrosarcoma, Agilent)
+ **MDA-MB 231** (Breast cancer cells, ATCC)
+ Human dermal fibroblasts (**HF**)- normal fibroblasts (control)

**14.07.15**

**In the lab today: Shai, Shoham and Vlad**

* We prepared 1% Agarose gel, and run the PCR products from yesterday to check whether the PCR worked as expected. Gel results indicate PCR was fine.

[![](https://static.igem.org/mediawiki/2015/6/60/Bgu_israel_gel_14.07.15_01.jpg)](https://static.igem.org/mediawiki/2015/6/60/Bgu_israel_gel_14.07.15_01.jpg)

* We performed miniprep for **pAAV-MCS** from yesterday. Nanodrop concentration results were 169.2ng/μl.
* Colonies found on **phTERT** plates from 12.07. 10ml of LB (+ampicillin) was inoculated with one colony.

**Cell studies**:

* qPCR for gene expression of **hTERT** and **Survivin (BIRC5)**- total RNA
  was isolated using the EZ-RNA RNA purification kit (Biological Industries), and
  500 ng of RNA from each sample was reverse transcribed into cDNA using the High
  Capacity cDNA Reverse Transcription Kit (Life Technologies). For gene expression
  analysis, mRNA levels were determined by real-time PCR using StepOnePlus™
  Applied detection system (Life Technologies) according to the manufacturer's instructions.
  Gene expression analyses of **hTERT** and **Survivin (BIRC5)** were performed
  using TaqMan gene expression assays (Life Technologies). Data was analyzed by the
  delta delta Ct method using ACTB as the house-keeping gene.

**15.07.15**

**In the lab today: Shai, Shoham, Vlad and Bar**

* Restriction of PCR products from 13.07 (**gMLP**, **gUBB,** **U6-gMLP**, **U6-gUBB**) with EcoRI and PstI. Incubation for 35 minutes at
  37°C.

We prepared 1% Agarose gel, and run the restriction products of **gUBB** and **U6-gUBB**.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol. Because of human error, **U6-gUBB** extraction failed.
Nanodrop concentration of **gUBB**: 26.1ng/μl (1.83/2.38).

**gMLP**
and **U6-gMLP** restriction products were purified using PCR purification kit (so we can compare if one of the purification methods gives better results
than the other). Nanodrop concentration results:

**gMLP**
: 31.7ng/μl (1.75/1.55), **U6-gMLP**: 12.5ng/μl (2.24/1.01).

* We performed miniprep for **phTERT** from yesterday. Then restrictions withEcoRI and PstI. Restrictions done in incubation for 30 minutes at
  37°C.

We run restriction products on the gel prepared earlier today, extracted the appropriate DNA band (according to size) and performed gel extraction
protocol.

**16.07.15**

**In the lab today: Shai, Shoham**

* Restriction of **pSB1C3** backbone with EcoRI and PstI. Alkaline phosphatase was added. Incubation for 30 minutes at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol.

* Ligation of **pSB1C3** (vector) with **phTERT** from yesterday (insert).

Ligation products (**phTERT-pSB1C3**) were transformed into DH5α and seeded on LB agar plates with Chloramphenicol ON.

* Ligation of **pSB1C3** (vector) with **gMLP** from yesterday (insert).
* Ligation of **pSB1C3** (vector) with **U6-gMLP** from yesterday (insert).
* PCR was carried out onligation products (**gMLP-pSB1C3,** **U6-gMLP-pSB1C3**) to check whether the ligation worked as
  expected (using F primer of pSB1C3 and R primer of insert). We prepared 1% Agarose gel, and run the PCR products. Gel results were good (band in expected
  size) so ligation products were transformed into DH5α and seeded on LB agar plates with Chloramphenicol ON.

[![](https://static.igem.org/mediawiki/2015/b/b9/Bgu_israel_gel_16.07.15_01.jpg)](https://static.igem.org/mediawiki/2015/b/b9/Bgu_israel_gel_16.07.15_01.jpg)

**17.07.15**

**In the lab today: Shoham**

* No colonies found on any of the plates from yesterday.

**18.07.15**

**In the lab today: Shai**

* 10ml of LB (+Chloramphenicol) was inoculated with one colony from **pSB1C3** LB agar plate.

**19.07.15**

**In the lab today: Shai, Shoham**

* Ligation products of **gMLP-pSB1C3,** **U6-gMLP-pSB1C3** from 16.07 were transformed again into DH5 α and seeded on LB agar plates with Chloramphenicol ON.
* We performed miniprep for **pSB1C3** from yesterday. Nanodrop concentration results were 85.2ng/μl (2.04,1.84).
* PCR
  was carried out on **gMLP**, **gUBB,** **U6-gMLP**, **U6-gUBB** using default PCR program (except Tm was changed form 60°C to 65°C).

The PCR products were purified using PCR purification kit.

Nanodrop concentration results:

**gMLP**
: 100.1ng/μl (1.79,0.72), **U6-gMLP**: 139.8ng/μl (1.81,1.78),

**gUBB**
: 81.1ng/μl (1.80,1.08), **U6-gUBB**: 78.9ng/μl (1.83,0.60).

**20.07.15**

**In the lab today: Shai, Shoham, Bar, Vlad**

* Colonies found on **gMLP-pSB1C3,** **U6-gMLP-pSB1C3** plates from yesterday.

Colony PCR was carried out on2 colonies of**gMLP-pSB1C3** and 3 colonies of **U6-gMLP-pSB1C3** to check whether the
ligation worked as expected (using F primer of pSB1C3 and R primer of insert). We prepared 1% Agarose gel, and run the PCR products. Gel results were good
(band in expected size).

3X10ml of LB (+Chloramphenicol) was inoculated with 1 colony each of **U6-gMLP-pSB1C3** and the same was done for the two colonies of **gMLP-pSB1C3**.

* Restriction of **phTERT** withEcoRI and PstI. Restriction done in incubation for 30 minutes at 37°C. Restriction products ran on gel
  made earlier today.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol. Nanodrop concentration results were 6.9ng/μl.

[![](https://static.igem.org/mediawiki/2015/c/c1/Bgu_israel_gel_20.07.15_01.jpg)](https://static.igem.org/mediawiki/2015/c/c1/Bgu_israel_gel_20.07.15_01.jpg)

**21.07.15**

**In the lab today: Shai, Shoham, Vlad, Shalev**

* We performed miniprep for **gMLP-pSB1C3,** **U6-gMLP-pSB1C3** from yesterday. Nanodrop concentration results were:

**gMLP-pSB1C3** (colony no. 1) 123.4ng/μl.

**gMLP-pSB1C3** (colony no. 2) 117.0ng/μl.

**U6-gMLP-pSB1C3** (colony no. 1) 103.0ng/μl.

**U6-gMLP-pSB1C3** (colony no. 2) 95.0ng/μl.

**U6-gMLP-pSB1C3** (colony no. 3) 110.0ng/μl.

* Restrictions of **gMLP-pSB1C3** and **U6-gMLP-pSB1C3** withXhoI (to check ligation). All restrictions done in incubation
  for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of **gMLP-pSB1C3** and **U6-gMLP-pSB1C3** succeeded - Send for sequencing.

[![](https://static.igem.org/mediawiki/2015/2/25/Bgu_israel_gel_21.07.15_01.jpg)](https://static.igem.org/mediawiki/2015/2/25/Bgu_israel_gel_21.07.15_01.jpg)

* 10ml of LB (+Chloramphenicol) was inoculated with one colony from **pSB1C3** LB agar plate.
* 10ml of LB (+ampicillin) was inoculated with one colony from **phTERT** LB agar plate.

**22.07.15**

**In the lab today: Shai, Shoham, Vlad, Shalev**

* Restrictions of **pSB1C3** and **phTERT** from yesterday with EcoRI and PstI. Restrictions done in incubation for 30 minutes at 37°C.
  Alkaline phosphatase was added to**pSB1C3**.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol.

Ligation of **pSB1C3** (vector) with **phTERT** from (insert).

* Ligation of **pSB1C3** (vector) with **U6-gUBB** from 19.07(insert).
* Ligation of **pSB1C3** (vector) with **gUBB** from 19.07 (insert).
* Ligation products (**phTERT-pSB1C3**,**U6-gUBB-pSB1C3,** **gUBB-pSB1C3**) were transformed into DH5 α and seeded on LB agar plates with Chloramphenicol ON.

**23.07.15**

**In the lab today: Shai and Shoham**

* Colonies found on all plates from yesterday (**phTERT-pSB1C3**,**U6-gUBB-pSB1C3,** **gUBB-pSB1C3**). 4 colonies were taken
  from each plate and 12X10ml of LB (+Chloramphenicol) was inoculated with one colony each.

**24.07.15**

**In the lab today: Shai and Shoham**

* We performed miniprep for all **phTERT-pSB1C3**,**U6-gUBB-pSB1C3,** **gUBB-pSB1C3** from yesterday. Nanodrop concentration
  checked for all:

|  |  |  |  |
| --- | --- | --- | --- |
|  | **phTERT-pSB1C3** (ng/μl) | **U6-gUBB-pSB1C3** (ng/μl) | **gUBB-pSB1C3** (ng/μl) |
| LB no. 1 | 233 | - | 231 |
| LB no. 2 | 309 | 249 | 154 |
| LB no. 3 | 199 | 227 | 123 |
| LB no. 4 | 248 | 246 | 187 |

* Restrictions of miniprep productswithEcoRI and PstI (to check ligation). All restrictions done in incubation for 30 minutes at
  37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of **phTERT-pSB1C3**,**U6-gUBB-pSB1C3,** **gUBB-pSB1C3** succeeded - Send for sequencing of 2
colonies from each.

[![](https://static.igem.org/mediawiki/2015/1/14/Bgu_israel_gel_24.07.15_01.jpg)](https://static.igem.org/mediawiki/2015/1/14/Bgu_israel_gel_24.07.15_01.jpg)

**26.07.15**

**In the lab today: Shoham**

* 10ml of LB (+ampicillin) was inoculated with one colony from **dCas9-vp64** LB agar plate.
* The same was done for **SaCas9**.

**27.07.15**

**In the lab today: Shai and Shoham**

* We performed miniprep for **SaCas9**, **dCas9-vp64** from yesterday (2 Eppendorfs each). Nanodrop concentration results were:

|  |  |  |
| --- | --- | --- |
|  | **SaCas9** | **dCas9-vp64** |
| Eppendorf no. 1 | 277ng/μl (2.01/2.06) | 593ng/μl (1.85/2.09) |
| Eppendorf no. 2 | 215ng/μl (1.98/2.09) | 522ng/μl (1.89/2.30) |

* We received **pSurvivin-mCherry** from outsourcing synthesis by Syntezza Bioscience Ltd.
* We transformed plasmids from virus kit(**pAAV-MCS**, **pRC**, **pHelper**) and **pSurvivin-mCherry** into DH5α and seeded
  them on LB agar plates with ampicillin.

**28.07.15**

**In the lab today: Shai and Shoham**

* Colonies found on allplates from yesterday. 2 colonies were taken from each plate and 8X10ml of LB (+ampicillin) was inoculated with one
  colony each.

**29.07.15**

**In the lab today: Vlad and Shalev**

* We performed miniprep for **pAAV-MCS**, **pRC**, **pHelper, pSurvivin-mCherry** from yesterday (2 Eppendorfs each). Nanodrop concentration
  results were:

|  |  |  |
| --- | --- | --- |
|  | colony no. 1 | colony no. 2 |
| **pHelper** | 602ng/μl | 236ng/μl |
| **pRC** | 520ng/μl | 399ng/μl |
| **pAAV-MCS** | 186ng/μl | 137ng/μl |
| **pSurvivin-mCherry** | 305ng/μl | 357ng/μl |

**02.08.15**

**In the lab today: Shai and Shoham**

* We transformed **gMLP-pSB1C3** (from colony no. 2 at 21.07) into DH5α E. coli and inserted into 10ml of LB (+Chloramphenicol) ON.
* We transformed **U6-gMLP-pSB1C3** (from colony no. 3 at 21.07) into DH5α E. coli and seeded it on LB agar plates with Chloramphenicol.
* 10ml of LB (+Chloramphenicol) was inoculated with colony no. 3 of **phTERT-pSB1C3** from 23.07.
* 10ml of LB (+Chloramphenicol) was inoculated with colony no. 2 of **U6-gUBB-pSB1C3** from 23.07.
* We transformed **pGFPN1** (GFP plasmid found on our lab) into DH5α and seeded it on LB agar plates with ampicillin.

**03.08.15**

**In the lab today: Shai and Shoham**

* Nothing grew in LB of **gMLP-pSB1C3** and **U6-gMLP-pSB1C3** from yesterday, so we did transformation again, this time with heat
  shock:

**gMLP-pSB1C3** (colony no. 2 from 21.07), **U6-gMLP-pSB1C3** (colony no. 2 from 21.07) and **U6-gMLP-pSB1C3** (colony no. 3 from 21.07) all
were inserted into 10ml of LB (+Chloramphenicol) ON.

* Colonies found on **pGFPN1** plate from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony.

**04.08.15**

**In the lab today: Vlad**

* We performed miniprep for: **gMLP-pSB1C3** (colony no. 2), **U6-gMLP-pSB1C3** (colony no. 2), **U6-gMLP-pSB1C3** (colony no.
  3), **pGFPN1** (all from yesterday) and **phTERT-pSB1C3** from 02.08. Nanodrop concentration results were:

**gMLP-pSB1C3**(2):245ng/μl, **U6-gMLP-pSB1C3**(2):221ng/μl,

**U6-gMLP-pSB1C3**(3):256ng/μl, **phTERT-pSB1C3**(3): 58.6ng/μl, **pGFPN1**: 2.4ng/μl.

* Restriction of **pAAV-MCS** and **pGFPN1** with EcoRI and XbaI. Alkaline phosphatase was added to **pAAV-MCS**. Incubation for 30 minutes
  at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

Gel results indicate that restriction of **pGFPN1** failed.

**05.08.15**

**In the lab today: Shai**

* We performed miniprep for: **U6-gUBB-pSB1C3** (colony no. 2)from 02.08. Nanodrop concentration results were 160ng/μl.
* Send for sequencing miniprep results of yesterday and today (**phTERT-pSB1C3**(3), **gMLP-pSB1C3**(2), **U6-gMLP-pSB1C3**
  (2)**,** **U6-gMLP-pSB1C3**(3)**, U6-gUBB-pSB1C3**(2)).

**06.08.15**

**In the lab today: Shai, Vlad**

* We decided to try another GFP plasmid (instead of **pGFPN1**). New plasmid: **pAM-EGFP**.
* Restriction of **pAAV-MCS** and **pAM-EGFP** with HindIII and BamHI. Alkaline phosphatase was added to **pAAV**. Incubation for 30 minutes
  at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA bands (according to size) and performed gel extraction protocol. Nanodrop concentration results were

**GFP**
: 19.6ng/μl and **pAAV**: 9.7ng/μl.

[![](https://static.igem.org/mediawiki/2015/1/1b/Bgu_israel_gel_06.08.15_01.jpg)](https://static.igem.org/mediawiki/2015/1/1b/Bgu_israel_gel_06.08.15_01.jpg)

* Ligation of **pAAV** (vector) with **GFP** (insert).

Ligation products (**GFP-pAAV**) were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

**09.08.15**

**In the lab today: Shai**

* No colonies found on **GFP-pAAV** plates from yesterday, so we did restriction again.

Restriction of **pAAV-MCS** and **pAM-EGFP** with HindIII and BamHI. Alkaline phosphatase was added to **pAAV**. Incubation for 30 minutes at
37°C.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA bands (according to size) and performed gel extraction protocol.

* Ligation of **pAAV** (vector) with **GFP** (insert).

Ligation products (**GFP-pAAV**) were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

* 2X100ml of LB (+ampicillin) were made for midiprep tomorrow. One was inoculated with a colony of **pHelper** and the other with **pRC**.

**10.08.15**

**In the lab today: Shai**
, **Shoham**

* 2 colonies found on **GFP-pAAV** plates from yesterday. 2X10ml of LB (+ampicillin) was inoculated with one colony each.
* We performed midiprep for **pHelper**, **pRC, pAAV-MCS**. Nanodrop concentration results were:

|  |  |  |
| --- | --- | --- |
|  | Vessel no. 1 | Vessel no. 2 |
| **pRC** | 600ng/μl (1.82/2.22) | 646ng/μl (1.84/2.13) |
| **pHelper** | 658ng/μl (1.87/2.24) | 581ng/μl |
| **pAAV-MCS** | 217ng/μl (1.80/1.53) | 277ng/μl (1.79/1.56) |

**11.08.15**

**In the lab today: Vlad**
, **Shalev**

* We received phTERT-eGFP-pAAV plasmid(**master-AAV**) from IDT**.** It was transformed into DH5α and inserted into 10ml of LB (+ampicillin) ON.
* We performed miniprep for **GFP-pAAV** from yesterday.

Nanodrop concentration results were:

colony no. 1: 189.6ng/μl (1.96,2.05), colony no. 2: 182.6ng/μl (1.94,2.03).

* Restriction of **GFP-pAAV** with HindIII and BamHI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C.
  Restriction products ran on 1% Agarose gel.

Results indicate ligation of **GFP-pAAV** in colony no. 2 succeeded.

**12.08.15**

**In the lab today: Shoham, Vlad and Shalev**

* **master-AAV**
  (DH5α) glycerol stock was prepared
* **GFP-AAV**
  (DH5α) glycerol stock was prepared
* PCR
  was carried out on **U6-gMLP**, **U6-gUBB**, **Affibody** using default PCR program.

The PCR products were purified using PCR purification kit.

Nanodrop concentration results of **U6-gMLP** were 160.2ng/μl (1.84/1.92).

Nanodrop concentration results of **U6-gUBB** were 219.8ng/μl (1.85/1.08).

Nanodrop concentration results of **Affibody** were 109.0ng/μl (1.84/2.11).

We prepared 1% Agarose gel, and run the PCR products.

[![](https://static.igem.org/mediawiki/2015/4/46/Bgu_israel_gel_12.08.15_01.jpg)](https://static.igem.org/mediawiki/2015/4/46/Bgu_israel_gel_12.08.15_01.jpg)

* We performed midiprep for **master-AAV**. Nanodrop concentration results were 286.8ng/μl (1.87/2.28).
* Restriction
  of **U6-gMLP, U6-gUBB** with SalI and PacI. Incubation for 30 minutes at 37°C.

**13.08.15**

**In the lab today: Shoham, Vlad, Bar and Shalev**

* Restriction of DNA
  . All restrictions done in incubation for 30 minutes at 37°C. Then we run restriction products on 1% Agarose gel, extracted the appropriate DNA
  band (according to size) and performed gel extraction protocol. Nanodrop concentration checked.

|  |  |  |  |
| --- | --- | --- | --- |
|  | Restriction enzymes | Alkaline phosphatase | Nanodrop concentration (ng/μl) |
| **pAAV\_MCS** | EcoRI, XhoI | Added | 50 |
| **master-AAV** (1) | SalI, PacI | Added | 56 |
| **master-AAV** (2) | SalI, XbaI | Added | 74 |
| **master-AAV** (3) | XhoI, BamHI | Added | 64 |
| **pSurvivin-mCherry** | SalI, PacI |  | 13.2 |
| **dCas9-vp64** | SalI, XbaI |  | 43 |
| **SaCas9** | XhoI, BamHI |  | 29 |
| **Affibody** \* | EcoRI, XhoI |  | 50 |

\***Affibody** was purified using PCR purification kit (not gel extraction).

* Ligations
  :
* Ligation of **p****AAV** (vector) with **Affibody** (insert).
* Ligation of **master-AAV** (vector) with **U6-gMLP** (insert).
* Ligation of **master-AAV** (vector) with **U6-gUBB** (insert)
* Ligation of **master-AAV** (vector) with **dCas9-vp64** (insert).
* Ligation of **master-AAV** (vector) with **SaCas9** (insert).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

**15.08.15**

**In the lab today: Shoham**

* No colonies found on any of the plates from yesterday. So we do all restrictions and ligation again.
* **Restriction of DNA.**
  All restrictions done in incubation for 30 minutes at 37°C. Then we run restriction products on 1% Agarose gel, extracted the appropriate DNA
  band (according to size) and performed gel extraction protocol. Since it is Saturday nanodrop room is closed so we didn't check the concentration and
  didn't continue to ligation (nanodrop results added later).

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | Restriction enzymes | Alkaline phosphatase | Nanodrop results - eppendorf 1 | Nanodrop results - eppendorf 2 |
| **pAAV\_MCS** (from stock) | EcoRI, XhoI | Added | - | - |
| **master-AAV** (1) | SalI, PacI | Added | 78.7 (2.47/0.55) | 119.0(1.87/0.69) |
| **master-AAV** (2) | SalI, XbaI | Added | 74.5 (2.51/0.51) | 68.0 (1.85/1.43) |
| **master-AAV** (3) | XhoI, BamHI | Added | 72.9 (2.19/0.29) | 56.9 (1.94/1.04) |
| **pSurvivin-mCherry** | SalI, PacI |  | 34.2 (2.37/0.06) | 11.1 (1.79/0.13) |
| **dCas9-vp64** | SalI, XbaI |  | 43.3 (2.78/0.18) | 30.3 (2.06/1.41) |
| **SaCas9** (from stock) | XhoI, BamHI |  | 30.0 | 20.0 |
| **U6-gUBB** | SalI, PacI |  | 25.6 (5.26/0.21) | 14.0 (1.67/0.30) |
| **U6-gMLP** | SalI, PacI |  | 21.7 (4.59/0.06) | 12.8 (1.71/0.08) |
| **Affibody** \* | EcoRI, XhoI |  | 17.0 | - |

\***Affibody** was purified using PCR purification kit (not gel extraction).

* Since we can't do ligation with current products of restriction, we tried ligation of yesterday's product again:
* Ligation of **p****AAV** (vector) with **Affibody** (insert).
* Ligation of **master-AAV** (vector) with **U6-gMLP** (insert).
* Ligation of **master-AAV** (vector) with **U6-gUBB** (insert)
* Ligation of **master-AAV** (vector) with **dCas9-vp64** (insert).
* Ligation of **master-AAV** (vector) with **SaCas9** (insert).
* Ligation of **master-AAV** (vector) with **pSurvivin-mCherry** (insert).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

**16.08.15**

**In the lab today: Shoham, Vlad**

* Colonies found on **pSurvivin-mCherry-pAAV** plates from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony.
* Colonies found on **CMV-dCas9-VP64-pAAV** plates from yesterday. 2X10ml of LB (+ampicillin) was inoculated with one colony each.
* Colonies found on **Affibody-AAV** plates from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony.
* No colonies found on the other plates from yesterday. So we do ligation with yesterday's restriction products:
* Ligation of **master-AAV** (vector) with **U6-gMLP** (insert).
* Ligation of **master-AAV** (vector) with **U6-gUBB** (insert)
* Ligation of **master-AAV** (vector) with **SaCas9** (insert).

**17.08.15**

**In the lab today: Vlad, Shalev**

* Nothing grew in LB of **pSurvivin-mCherry-pAAV** from yesterday.
* LB of **CMV-dCas9-VP64-pAAV** and LB of **Affibody-AAV** are OK. Miniprep was performed on both. Nanodrop concentration results
  were 193.5ng/μl (1.95/20.9) for **CMV-dCas9-VP64-pAAV** and 209.7ng/μl (1.97/2.09) for **p****Affibody-AAV**.

We prepared 1% Agarose gel, and run Miniprep products.

[![](https://static.igem.org/mediawiki/2015/3/32/Bgu_israel_gel_17.08.15_01.jpg)](https://static.igem.org/mediawiki/2015/3/32/Bgu_israel_gel_17.08.15_01.jpg)

Gel results indicate that ligation of **CMV-dCas9-VP64-pAAV** failed.

Ligation of **p****Affibody-AAV** succeeded**.**

**Affibody-AAV** (DH5α) glycerol stock was prepared.

* Yesterday's ligation products (**U6-gMLP-pAAV**, **U6-gUBB-pAAV**, **CMV-SaCas9-pAAV**) were transformed into DH5α and seeded on LB agar plates with ampicillin ON.
* Ligation products of **CMV-dCas9-VP64-pAAV** (from 15.08) were transformed again into DH5α and seeded on LB agar plates
  with ampicillin ON.

**18.08.15**

**In the lab today: Shoham, Vlad, Shalev**

* Restrictions again of **master-AAV, pSurvivin-mCherry, U6-gUBB, U6-gMLP** withSalI, PacI. All restrictions done in incubation for 30 minutes at
  37°C. Alkaline phosphatase was added to **master-AAV**.
* Restriction products of **master-AAV, pSurvivin-mCherry** ran on 1% Agarose gel.

[![](https://static.igem.org/mediawiki/2015/c/c6/Bgu_israel_gel_18.08.15_01.jpg)](https://static.igem.org/mediawiki/2015/c/c6/Bgu_israel_gel_18.08.15_01.jpg)

Gel results indicate about a problem. Tomorrow we will use new **master-AAV** from midiprep and new **pSurvivin-mCherry** from glycerol stock (10ml of LB
(+ampicillin) was inoculated).

* The restriction products of **U6-gUBB, U6-gMLP** were purified using PCR purification kit. Nanodrop concentration results were 21.1ng/μl for **U6-gUBB** and 35.9ng/μl for **U6-gMLP**.
* We inoculated 10ml of LB (+ampicillin) with one colony from **CMV-SaCas9-pAAV** from yesterday.
* We inoculated 10ml of LB (+ampicillin) with one colony from **pSurvivin-mCherry-pAAV** from 16.08.

**19.08.15**

**In the lab today: Vlad, Shalev**

* Nothing grew in LB of **pSurvivin-mCherry-pAAV** from yesterday.
* LB of **CMV-SaCas9-pAAV** is OK. Miniprep was performed.

We prepared 1% Agarose gel, and ran Miniprep products.

Gel results indicate that ligation of **CMV-SaCas9-pAAV** failed.

* We performed midiprep for **master-AAV** and miniprep for **pSurvivin-mCherry**. Then restrictions of both withSalI, PacI. All restrictions
  done in incubation for 30 minutes at 37°C. Alkaline phosphatase was added to **master-AAV**.

We run restriction products on the gel prepared earlier today, extracted the appropriate DNA band (according to size) and performed gel extraction
protocol.

[![](https://static.igem.org/mediawiki/2015/0/0c/Bgu_israel_gel_19.08.15_01.jpg)](https://static.igem.org/mediawiki/2015/0/0c/Bgu_israel_gel_19.08.15_01.jpg)

* Ligations
  :
* Ligation of **master-AAV** (vector) with **pSurvivin-mCherry** (insert). (**Survivin** from today).

Ligation of **master-AAV** (vector) with **pSurvivin-mCherry** (insert). (**Survivin** from 16.08).

Ligation of **master-AAV** (vector) with **pSurvivin-mCherry** (insert). (**Survivin** from 13.08).

Ligation of **master-AAV** (vector) with **U6-gMLP** (insert). (**U6-gMLP** from 18.08).

Ligation of **master-AAV** (vector) with **U6-gUBB** (insert). (**U6-gUBB** from 18.08).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

**20.08.15**

**In the lab today: Vlad, Shalev**

* No colonies found on any of the plates from yesterday.
* We think maybe there is a problem with the **master-AAV**. So we decided to try **master-AAV** from 2 dates: 19.08 and 16.08 for today's ligations:

Ligation of **master-AAV** (vector) with **pSurvivin-mCherry** (insert). (**master-AAV** from 19.08).

Ligation of **master-AAV** (vector) with **pSurvivin-mCherry** (insert). (**master-AAV** from 16.08).

Ligation of **master-AAV** (vector) with **U6-gUBB** (insert). (**master-AAV** from 19.08).

Ligation of **master-AAV** (vector) with **U6-gMLP** (insert). (**master-AAV** from 16.08).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

**21.08.15**

**In the lab today: Shoham, Vlad, Shalev**

* No colonies found on any of the plates from yesterday.
* We start working on **pRC** and **pHelper** of AAV kit.

Both were transformed into DH5α and inserted into 10ml of LB (+ampicillin) ON.

**22.08.15**

**In the lab today: Shoham**

* We performed midiprep for **pRC**. Nanodrop concentration results were 3745ng/μl (1.81/2.24).
* We performed midiprep for **pHelper**. Nanodrop concentration results were 4072ng/μl (1.79/2.17).

**23.08.15**

**In the lab today: Vlad, Shalev**

* Since no colonies found on any of the plates in 21.08, we do DNA restrictions again.

Restriction
of **master-AAV**, **pSurvivin-mCherry** with SalI and PacI. Incubation for 30 minutes at 37°C. Alkaline phosphatase was added to **master-AAV**.

We ran restriction products on 1% Agarose gel.

[![](https://static.igem.org/mediawiki/2015/1/16/Bgu_israel_gel_23.08.15_02.jpg)](https://static.igem.org/mediawiki/2015/1/16/Bgu_israel_gel_23.08.15_02.jpg)

* Gel result indicates that **master-AAV** restriction didn't work as expected. Maybe **master-AAV** eppendorf is contaminated. So **master-AAV** from
  original stock was transformed into DH5α and inserted into 10ml of LB (+ampicillin) ON.
* **pSurvivin-mCherry**
  gel results are OK.
* Meanwhile, we decided to try ligation with current **master-AAV**. So both **master-AAV** and **pSurvivin-mCherry** extracted from gel.
* Since it didn't work so far, we tried to improvise with the ligation protocol. Instead of 50ng of vector, we took 100ng. Instead of 150ng of insert,
  we took 400ng. So the vector/insert ratio was changed from 1:3 to 1:4.

Ligation of **master-AAV** (vector) with **dCas9-vp64** (insert).

Ligation of **master-AAV** (vector) with **SaCas9** (insert).

* After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON. We also made a change in transformation
  protocol - instead of 10 minutes in ice, we left the DH5α for 40 minutes in the ice.

**Cell studies**:

* Seeding six 15 cm tissue culture plates of **AAV-293** cells (Agilent) for viral
  stocks production (2 plates per each vector).
* Seeding 6-well plates of **HT1080** and **HF** cells (200,000 cell/ well)
  for CaP transfection.

**24.08.15**

**In the lab today: Shoham, Vlad, Shalev**

* We performed miniprep for **master-AAV** from yesterday (from stock). Nanodrop concentration result was 233ng/μl.

Restriction of **master-AAV** was made withSalI, PacI. Restriction done in incubation for 35 minutes at 37°C. Alkaline phosphatase was added.

Restriction products ran on 1% Agarose gel. **master-AAV** gel results are good.

[![](https://static.igem.org/mediawiki/2015/3/39/Bgu_israel_gel_24.08.15_01.jpg)](https://static.igem.org/mediawiki/2015/3/39/Bgu_israel_gel_24.08.15_01.jpg)

* Colonies found on **CMV-SaCas9-pAAV** plate from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony ON
* Ligations:

2X Ligation of today's **master-AAV** (vector) with yesterday's **pSurvivin-mCherry** (insert).

Ligation of yesterday's **master-AAV** (vector) with **U6-gMLP** (insert).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON. Like yesterday, for one of **pSurvivin-mCherry-pAAV** ligations we changed the protocol - instead of 10 minutes in ice, we left the DH5α for 40 minutes in the ice.

* AAV plasmids arrived from cloning: **phTERT-SaCas9-pAAV, phTERT-dCas9-VP64-pAAV, pMLPm-eGFP-pAAV, pSurvivin-gMLP-pAAV, pSurvivin-gUBB-pAAV**.

Plasmids were transformed into DH5α and inserted into 100ml of LB (+ampicillin) ON, for midiprep tomorrow.

**Cell studies**:

* Three transfections were performed in **AAV-293** cells according to AAV production
  protocol.

The transfections were with the following Plasmids:

+ **GFP-pAAV**
+ **master-AAV**
+ **Affibody-AAV**

* In addition two transfections were performed according to CaP transfection protocol
  in **HT1080** and **HF**.

+ **GFP-pAAV**
+ **master-AAV**

**25.08.15**

**In the lab today: Vlad, Shalev**

* We performed midiprep for **pSurvivin-gMLP-pAAV, pSurvivin-gUBB-pAAV, phTERT-dCas9-VP64-pAAV** . Nanodrop concentration results were:

**pSurvivin-gMLP-pAAV**
: 1379ng/μl (1.85/2.25) - concentration is good.

**pSurvivin-gUBB-pAAV**
: 270ng/μl (1.86/2.24) - concentration is too low.

**phTERT-dCas9-VP64-pAAV:**
concentration is too low.

* Restrictions of **CMV-SaCas9-pAAV** with NotI, AgeI (to check ligation). All restrictions done in incubation for 30 minutes at
  37°C. Restriction products ran on 1% Agarose gel. Gel results indicate ligation of **CMV-SaCas9-pAAV** failed.

[![](https://static.igem.org/mediawiki/2015/2/25/Bgu_israel_gel_25.08.15_01.jpg)](https://static.igem.org/mediawiki/2015/2/25/Bgu_israel_gel_25.08.15_01.jpg)

* Colonies found on all plates from yesterday (2X**pSurvivin-mCherry-pAAV,** 1X**U6-gMLP-pAAV**). 10ml of LB (+ampicillin) was
  inoculated with 2 colonies each.
* Colonies found on **CMV-SaCas9-pAAV** plates from 23.08. 10ml of LB (+ampicillin) was inoculated with one colony.

**Cell studies**:

* Medium change in all plates.

**26.08.15**

**In the lab today: Vlad, Shalev**

* Restrictions of **pSurvivin-mCherry-pAAV** withSalI, AgeI (to check ligation). All restrictions done in incubation for 30
  minutes at 37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of **pSurvivin-mCherry-pAAV** succeeded.

* Restrictions of **U6-gMLP-pAAV** withSalI, PstI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C.
  Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of **U6-gMLP-pAAV** succeeded.

[![](https://static.igem.org/mediawiki/2015/0/00/Bgu_israel_gel_26.08.15_01.jpg)](https://static.igem.org/mediawiki/2015/0/00/Bgu_israel_gel_26.08.15_01.jpg)

* Plasmids (**pSurvivin-mCherry-pAAV** and **U6-gMLP-pAAV**) were transformed into DH5α and inserted into 100ml of LB
  (+ampicillin) ON, for midiprep tomorrow.
* AAV Plasmids (**phTERT-SaCas9-pAAV, phTERT-dCas9-VP64-pAAV, pMLPm-eGFP-pAAV, pSurvivin-gUBB-pAAV**) were transformed into DH5α and inserted into 100ml of LB
  (+ampicillin) ON, for midiprep tomorrow.
* Ligation of **master-AAV** from 23.08 (vector) with **U6-gUBB** (insert).

After ligation, products were transformed into DH5α and seeded on 2 LB agar plates with ampicillin ON.

**Cell studies**:

* Observation: CaP transfection in **HT1080** and **HF** at 48h, we looked at
  cells under inverted fluorescence microscope (IX70, Olympus) connected to an Olympus
  (DD71) digital capture system.
* Seeding eight 15 cm tissue culture plates of **AAV-293** cells (Agilent) for
  viral stocks production.

**27.08.15**

**In the lab today: Vlad, Shalev**

* We performed midiprep. Nanodrop concentration checked.

|  |  |
| --- | --- |
|  | Nanodrop results |
| **phTERT-SaCas9-pAAV** | 893ng/μl (1.86/2.29) |
| **phTERT-dCas9-VP64-pAAV** | 688ng/μl (1.84/2.35) |
| **pMLPm-eGFP-pAAV** | 956ng/μl (1.86/2.28) |
| **pSurvivin-gUBB-pAAV** | 349ng/μl (1.84/2.24) |
| **pSurvivin-mCherry-pAAV** | 450ng/μl (1.84/2.25) |

* Colonies found on both plates of **U6-gUBB-pAAV** from yesterday. 5X10ml of LB (+ampicillin) was inoculated with one colony each ON (3
  colonies from plate 1, and 2 colonies from plate 2).

**Cell studies**:

* Preparing viral stocks according to AAV production protocol for:

+ **GFP-pAAV**
+ **master-AAV**
+ **Affibody-AAV**

* Four transfections were performed in **AAV-293** cells according to AAV production
  protocol.

The transfections were with the following Plasmids:

+ **pSurvivin-mCherry-pAAV**
+ **phTERT-dCas9-VP64-pAAV**
+ **pSurvivin-gMLP-pAAV**
+ **pMLPm-eGFP-pAAV**

**28.08.15**

**In the lab today: Vlad, Shalev**

* We performed miniprep for **U6-gUBB-pAAV** from yesterday

Then we perform Restrictions of withSalI, PstI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C. Restriction
products ran on 1% Agarose gel.

Gel results indicate that in the second colony of the first plate ligation of **U6-gUBB-pAAV** succeeded.

[![](https://static.igem.org/mediawiki/2015/2/2a/Bgu_israel_gel_28.08.15_01.jpg)](https://static.igem.org/mediawiki/2015/2/2a/Bgu_israel_gel_28.08.15_01.jpg)

* Restriction of DNA
  . All restrictions done in incubation for 30 minutes at 37°C. Then we run restriction products on 1% Agarose gel, extracted the appropriate DNA
  band (according to size) and performed gel extraction protocol.

|  |  |  |
| --- | --- | --- |
|  | Restriction enzymes | Alkaline phosphatase |
| **master-AAV** (1) | XhoI, BamHI | Added |
| **master-AAV** (2) | SalI, XbaI | Added |
| **dCas9-vp64** | SalI, XbaI |  |
| **SaCas9** | XhoI, BamHI |  |

[![](https://static.igem.org/mediawiki/2015/1/15/Bgu_israel_gel_28.08.15_02.jpg)](https://static.igem.org/mediawiki/2015/1/15/Bgu_israel_gel_28.08.15_02.jpg)

[![](https://static.igem.org/mediawiki/2015/e/ec/Bgu_israel_gel_28.08.15_03.jpg)](https://static.igem.org/mediawiki/2015/e/ec/Bgu_israel_gel_28.08.15_03.jpg)

**Cell studies**:

* Change medium in **AAV-293** cell culture plates

**29.08.15**

**In the lab today: Vlad, Shalev**

* Ligations of restrictions from yesterday:

Ligation of **master-AAV** (vector) with **dCas9-vp64** (insert).

Ligation of **master-AAV** (vector) with **SaCas9** (insert).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

* **U6-gUBB-pAAV**
  (DH5α) glycerol stock was prepared.

**30.08.15**

**In the lab today: Vlad, Shalev**

* Colonies found on both plates (**CMV-dCas9-VP64-pAAV** and **CMV-SaCas9-pAAV**) from yesterday. 10ml of LB (+ampicillin) was
  inoculated with one colony from each ON.
* We still had some restriction products of **master-AAV** and **dCas9-vp64** from 28.08, so we did Ligation of **master-AAV** (vector) with **dCas9-vp64** (insert) again. (in case ligation failed in current colonies). After ligation, products were transformed into DH5α and seeded on LB
  agar plates with ampicillin ON.

**Cell studies**:

* Preparing viral stocks of according to AAV production protocol

+ **pSurvivin-mCherry-pAAV**
+ **phTERT-dCas9-VP64-pAAV**
+ **pSurvivin-gMLP-pAAV**
+ **pMLPm-eGFP-pAAV**

* Seeding **HT1080** and **HF** cells in 24-well plates (50,000 cells per well),
  and in 6- well plates (200,000 cells per well)

**31.08.15**

**In the lab today: Vlad, Shalev**

* Colonies found on **CMV-dCas9-VP64-pAAV** plate from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony ON.
* **pSurvivin-mCherry-pAAV**
  (DH5α) glycerol stock was prepared.
* Restrictions of **CMV-dCas9-VP64-pAAV** withSpeI, PacI (to check ligation). All restrictions done in incubation for 30 minutes at
  37°C. Restriction products ran on 1% Agarose gel. Gel results indicate about a problem. Maybe PacI enzyme is not working as expected. We will try
  tomorrow other restriction enzymes set.
* Restrictions of **CMV-SaCas9-pAAV** withAgeI, NotI (to check ligation). All restrictions done in incubation for 30 minutes at
  37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of **CMV-SaCas9-pAAV** succeeded.

[![](https://static.igem.org/mediawiki/2015/2/2b/Bgu_israel_gel_31.08.15_01.jpg)](https://static.igem.org/mediawiki/2015/2/2b/Bgu_israel_gel_31.08.15_01.jpg)

* **CMV-SaCas9-pAAV** (DH5α) glycerol stock was prepared.

**Cell studies**:

* **Functional experiment, activation platform:** transduction of **HT1080**
  cells and **HF** cells (24- well plates) with 40 µl from AAV stocks of:

+ Three components of activation system: **phTERT-dCas9-VP64-pAAV, pSurvivin-gMLP-pAAV, pMLPm-eGFP-pAAV**
+ Control for transduction efficiency: **GFP-pAAV**
+ Controls for promoters activation: **pSurvivin-mCherry-pAAV, phTERT-eGFP-pAAV**
+ Same groups of AAV vectors were tested in **HT1080** and **HF** (6- well plates)
  using CaP transfection protocol. In this experiment we included also a group of
  **pMLPm-eGFP-pAAV** alone to exclude false positive GFP expression results by
  activation of the minimal promoter.

**01.09.15**

**In the lab today: Vlad, Shalev**

* Restrictions of **CMV-dCas9-VP64-pAAV** from 29.08 and 30.08 (to check ligation). We decided to use different restriction enzymes because
  maybe PacI is problematic. So new enzymes are: SalI,XBaI. All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1%
  Agarose gel.

Gel results indicate ligation of **CMV-dCas9-VP64-pAAV** from 29.08 succeeded.

[![](https://static.igem.org/mediawiki/2015/d/dd/Bgu_israel_gel_01.09.15_01.jpg)](https://static.igem.org/mediawiki/2015/d/dd/Bgu_israel_gel_01.09.15_01.jpg)

* **CMV-dCas9-VP64-pAAV** (DH5α) glycerol stock was prepared.
* 100ml of LB (+ampicillin) where made for midiprep. Each contained one of the following: **CMV-dCas9-VP64-pAAV, CMV-SaCas9-pAAV, U6-gMLP-pAAV, U6-gUBB-pAAV**.

**Cell studies**:

* Change medium to **HT1080** and **HF** cells in 6-well plates

**02.09.15**

**In the lab today: Shoham, Shalev, Vlad**

**Cell studies**:

* **Functional experiment, activation platform** - Observation: CaP transfection and AAV transduction in **HT1080** and **HF**
  at 48h, we looked at cells under inverted fluorescence microscope (IX70, Olympus)
  connected to an Olympus (DD71) digital capture system. [See results here](https://2015.igem.org/Team%3ABGU_Israel/Results#prototype)
* Seeding 15 cm tissue culture plates of **AAV-293** cells (Agilent) for viral
  stocks production.

**03.09.15**

**In the lab today: Vlad, Shalev**

* We performed midiprep. Nanodrop concentration checked.

|  |  |
| --- | --- |
|  | Nanodrop results |
| **CMV-dCas9-VP64-pAAV** | 1467ng/μl |
| **CMV-SaCas9-pAAV** | 697ng/μl |
| **U6-gMLP-pAAV** | 1685ng/μl |
| **U6-gUBB-pAAV** | 1209ng/μl |

**Cell studies**:

* Six transfections were performed in **AAV-293** cells according to AAV production
  protocol.

The transfections were with the following Plasmids:

+ **CMV-dCas9-VP64-pAAV**
+ **CMV-SaCas9-pAAV**
+ **U6-gMLP-pAAV**
+ **U6-gUBB-pAAV**
+ **phTERT-SaCas9-pAAV**
+ **pSurvivin-gUBB-pAAV**

**04.09.15**

**In the lab today: Shoham, Shalev, Vlad**

**Cell studies**:

* Change medium in **AAV-293** cell culture plates

**06.09.15**

**In the lab today: Shoham, Shalev, Vlad**

**Cell studies**:

* Seeding **HT1080** and **HF** cells in 24-well plates (50,000 cells per well),
  and in 6- well plates (200,000 cells per well).
* For affibody exp- seeding **HT1080** cells on chamber slides (10,000 cells per
  well)

**07.09.15**

**In the lab today: Shoham, Shalev, Shai**

**Cell studies**:

* **Functional experiment, knockout platform:** transduction of **HT1080** cells
  and **HF** cells (24- well plates) with 40 µl from AAV stocks of:

+ Two components of knockout system, specific promoters:

**phTERT-SaCas9-pAAV, pSurvivin-gUBB-pAAV**

+ Two components of knockout system, constitutive promoters:

**CMV-SaCas9-pAAV, U6-gUBB-pAAV**

+ Controls: **phTERT-eGFP-pAAV, pSurvivin-mCherry-pAAV**
+ Same groups of AAV vectors were tested in **HT1080** and **HF** (6- well plates)
  using CaP transfection protocol.

* Transduction of **affibody-AAV** was performed in **HT1080** (chamber slides)

**08.09.15**

**In the lab today: Shoham, Shalev, Shai**

**Cell studies**:

* Change medium to **HT1080** and **HF** cells in 6-well plates

**10.09.15**

**In the lab today: Shoham, Shalev, Shai**

**Cell studies**:

* Observation: we looked at cells under inverted fluorescence microscope (IX70, Olympus)
  connected to an Olympus (DD71) digital capture system.
* Immunostaining for **Affibody expression** according to Immunofluorescent staining protocol:
  cells in chamber slides were stained with primary Ab for FLAG peptide (F1804, Sigma,
  1:500 dilution). Protocols with and w/o permeabilization step were used, to distinguish
  between external and intracellular expression. Alexa-488-conjugated Ab (A21200,
  Life Technologies, 1:250 dilution) was used for visualization. Negative control
  included samples w/o primary Ab. The cells were observed under laser scanning confocal
  microscope (C1si, Nikon). [See results here](https://2015.igem.org/Team%3ABGU_Israel/Collaborations#Stockholm)

## Follow us:

## Address:

|  |
| --- |
| Ben-Gurion University of the Negev   Ben Gurion 1, Beer Sheva 8410501, Israel  Mail: igembgu2015@gmail.com |

[![Attribution 3.0 Unported](http://i.creativecommons.org/l/by/3.0/88x31.png)](http://creativecommons.org/licenses/by/3.0/)