iGEM 2019 — Team TelHai-Migal_Israel

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# Team:TelHai-Migal Israel/Notebook

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# Notebook

## Week 1 – 12/05-18/05

* Transformation of plasmids to Top10 competent bacteria.
* Mini-prep for pLN74, pLN75 and pLN193.
* Re-suspending g-Blocks 1658-1662 (according to IDT protocol)
* Analytic digestion with Asc1 and Xho1 + agarose gel check.

We chose Asc1 and Xho1 and we expected to get the following fragments:

* pLN75 – 1205bp and 3681bp (Tot. of 4886bp) -backbone
* pLN193 – 421bp, 850bp and 3968bp (Tot. of 5239bp) - backbone
* pLN74 – 415bp, 1179bp and 3489bp (Tot. of 5083bp) - positive control

Each plasmid had 1 native DNA tube as control and 3 similar test tube that contains:

### Gel results:

* pLN74 – all 3 clones were positive.
* pLN75 – clones 1 and 3 were positive.
* pLN193 – clones 1 and 3 were positive.

![](https://static.igem.org/mediawiki/2019/3/35/T--TelHai-Migal_Israel--notebook1.png)

## Week 2 – 19/05-25/05

* Midi starter – incubated in the shaker overnight.
* Midi prep (according to the Kit protocol #7326120)
* Re-suspending g-blocks: 1658-1662 (according to IDT protocol)
* Running samples in agarose gel
* Analytic digestion for pLN74, pLN75 and pLN193 after Midi.

Midi starter - 100ml LB + 110µL Ampicillin + frozen Top10 bacteria in an Erlenmeyer. In each Erlenmeyer we add bacteria that contains a different plasmid.

Midi prep -

G-Blocks Gel results were according to known literature, no need to repeat although the marker is defected:

![](https://static.igem.org/mediawiki/2019/3/37/T--TelHai-Migal_Israel--notebook2.png)

After Midi restrictive digestion results:

![](https://static.igem.org/mediawiki/2019/a/a2/T--TelHai-Migal_Israel--notebook3.png)

## Week 3 – 26/05-01/06

* Preparative restrictive digestion
* Preparative gel with crystal violet
* Purify DNA using DNA extraction Kit (real genomics #YDF100)
* Running samples in agarose gel – marker 7.5µL, samples – 5µL DNA +5µL DDW + 2µL loading dye.
* Ligation according to protocol

Preparative digestion gel results:

![](https://static.igem.org/mediawiki/2019/3/37/T--TelHai-Migal_Israel--notebook4.png)

Ligation: in each tube: 1µL buffer, 1µL ligase, 1µL plasmid, 2µL insert, 5µL DDW.

| Plasmid | Gene | After ligation |
| --- | --- | --- |
| pLN193 | 1658 | 1700 |
| pLN75 | 1659 | 1701 |
| pLN193 | 1660 | 1702 |
| pLN75 | 1661 | 1703 |

\*\* We were short with DNA in 1661 and when we ran the samples in agarose gel, we didn’t see any bends for 1661 and 1662. Although 1661 and didn’t appear on the gel we decide to proceed with the ligation.

## Week 4 – 02/06-08/06

* Competent Top10 starter according to protocol.
* Transformation with top10 according to protocol.
* Mini-prep (FB #1071202) for 1700-1703 – 3 clones each.

## Week 5 – 09/06-15/06

* Analytic digestion – 3µL DNA, 1µL cut-smart, 0.2µL Asc1, 0.2µL BamH1, 5.6µL DDW.
* Running samples in agarose gel.
* Freeze bacteria of clones: 1703-2, 1702-1, 1701-3, 1700-3.
* **Next week: repeating the process from preparative digestion for 1703 and 1704.**

After ligation gel results:

![](https://static.igem.org/mediawiki/2019/9/96/T--TelHai-Migal_Israel--notebook5.png)

As we can see during week #3 there was a problem with 1661 that later became 1703. Therefore, next week we'll repeat the experiment from preparative restrictive digestion for these genes.

### Gel results:

![](https://static.igem.org/mediawiki/2019/f/f6/T--TelHai-Migal_Israel--notebook6.png)

## Week 6 – 16/06-22/06

* Repeat preparative digestion for 1661.
* Repeat ligation for 1703 (According to protocol).
* Repeat Competent Top10 starter according to protocol.
* Transformation with top10 according to protocol.

There was some difficulty with the preparative digestion of 1661. Hence, 1703 ligation was defected, and we had to repeat the process from the preparative digestion through ligation until the transformation with Top10 bacteria.

## Week 7 – 23/06-29/06

* Picking colonies of 1703 for mini prep
* Mini-prep (FB #1071202) – 3 colonies each.
* Digestion after Mini.

### Gel results:

![](https://static.igem.org/mediawiki/2019/d/d8/T--TelHai-Migal_Israel--notebook7.png)

**We had another problem with 1703 probably because there wasn’t enough DNA to run. So, we are repeating Mini prep with different colonies we picked. To ensure we have suitable DNA concentration we used nanodrop test:**

![](https://static.igem.org/mediawiki/2019/b/be/T--TelHai-Migal_Israel--notebook8.png)

We had some ongoing problem with 1703 so we ordered new g-block from IDT.

## Week 8 – 29/06 – 06/07

* Midi-prep (Bio Rad #7326120) for pLN75 and 76.
* Nano drop to verify DNA concentration.
* Preparative gel.

### Midi gel results:

![](https://static.igem.org/mediawiki/2019/8/81/T--TelHai-Migal_Israel--notebook9.png)

### Nanodrop test:

![](https://static.igem.org/mediawiki/2019/e/eb/T--TelHai-Migal_Israel--notebook10.png)

### Preparation for preparative gel:

| Insert/plasmid | DNA [µL] | Asc1 [µL] | BamH1 [µL] | Cut smart [µL] | Upw [µL] |
| --- | --- | --- | --- | --- | --- |
| 1659 | 50 | 1.5 | 1.5 | 8 | 19 |
| 1661 | 25 | 1.5 | 1.5 | 8 | 44 |
| pLN75 | 29 | 1.5 | 1.5 | 8 | 44 |

### Running samples before preparative gel:

![](https://static.igem.org/mediawiki/2019/b/be/T--TelHai-Migal_Israel--notebook11.png)

### After preparative gel:

![](https://static.igem.org/mediawiki/2019/c/ce/T--TelHai-Migal_Israel--notebook12.png)

## Week 9 – 07/07 – 13/07

* Repeat ligation for pLN75 with 1659/1661 respectively according to protocol.
* Starter for competent bacteria top10 according to protocol.
* Transformation according to protocol.

### Ligation:

|  | pLN75 [µl] | Insert [µl] | Ligase [µl] | Buffer [µl] | UPW [µl] |
| --- | --- | --- | --- | --- | --- |
| 75 – neg. control | 1 | 0 | 1 | 1 | 7 |
| 75 +1659 | 1 | 2 | 1 | 1 | 5 |
| 75+1661 | 1 | 2 | 1 | 1 | 5 |

## Week 10 – 14/07 – 20/07

* Mini prep (FB #1071202) for samples from 1701/1703.
* Freezing the best samples of each clone (highest concentration) according to nanodrop test.
* Maxi prep for 1700-1703 after nanodrop test for 1700-1703.
* Nanodrop for pLN74-76 and 193 for next week experiment.

### Mini prep gel results:

![](https://static.igem.org/mediawiki/2019/4/44/T--TelHai-Migal_Israel--notebook13.png)

### Nanodrop after mini and before freezing:

![](https://static.igem.org/mediawiki/2019/7/73/T--TelHai-Migal_Israel--notebook14.png)

### Maxi prep for 1700-1703 gel results:

![](https://static.igem.org/mediawiki/2019/3/34/T--TelHai-Migal_Israel--notebook15.png)

### Nanodrop after maxi:

![](https://static.igem.org/mediawiki/2019/d/de/T--TelHai-Migal_Israel--notebook16.png)

### Nanodrop for next week experiment:

![](https://static.igem.org/mediawiki/2019/e/e7/T--TelHai-Migal_Israel--notebook17.png)

## Week 11 – 28/07 – 03/08

Thaw Hek293T cells according to protocol.

**Hek293T defrost:** we had 12.5X106 cells after centrifuge so we plate total of 6 plates with 2X106 cells/plate according to the scheme below:

![](https://static.igem.org/mediawiki/2019/5/5a/T--TelHai-Migal_Israel--notebook18.png)

* July 30th – changing growth medium for cells plated on July 28th.
* July 30th and August 1st Splitting cells we thawed on July 28th according to protocol.

## Week 12 – 04/08 – 10/08

* Splitting cells – August 4th, 6th and 8th according to protocol.

Preparing the cells for the main experiment!

## Week 13 – 11/08 – 17/08

* Preparing Hek293T cells for transfection.
* Splitting cells – August 11th and 13th according to protocol.
* Preparing new DMEM-C.M. according to protocol.
* DNA transfection for our model clones and plasmids.
* FACS
* Sequencing 1700 – 1703 and compering with the inserts.

Replacing growth medium 3h pre-transfection.

### Transfection plan:

![](https://static.igem.org/mediawiki/2019/c/c2/T--TelHai-Migal_Israel--notebook19.png)

### FACS results:

| Num | Clone |  |
| --- | --- | --- |
| 1 | No Transfection | 0% |
| 2 | Mock (1563) | 0% |
| 3 | GFP | Transfection efficacy |
| 4 | pLN74 | Positive control for m-Kate expression |
| 5 | 1700- [BBa\_K2946010](http://parts.igem.org/Part%3ABBa_K2946010) | Ex-mKate 1 – TS1 |
| 6 | 1701- [BBa\_K2946011](http://parts.igem.org/Part%3ABBa_K2946011) | Ex-mKate 2 – TS1 – no florescence |
| 7 | 1702- [BBa\_K2946013](http://parts.igem.org/Part%3ABBa_K2946013) | Ex-mKate 1 – TS2 |
| 8 | 1703- [BBa\_K2946014](http://parts.igem.org/Part%3ABBa_K2946014) | Ex-mKate 2 – TS2 |
| 9 | 1700+1701- [BBa\_K2946012](http://parts.igem.org/Part%3ABBa_K2946012) | TS1 |
| 10 | 1702+1703- [BBa\_K2946015](http://parts.igem.org/Part%3ABBa_K2946015) | TS2 |

### FACS results 1-3 (FL1-H):

![](https://static.igem.org/mediawiki/2019/d/d4/T--TelHai-Migal_Israel--notebook20.png)

### FACS results mock,mKate:

![](https://static.igem.org/mediawiki/2019/b/b9/T--TelHai-Migal_Israel--notebook21.png)

### FACS mock 1700-1703 (mkate):

![](https://static.igem.org/mediawiki/2019/8/89/T--TelHai-Migal_Israel--notebook22.png)
![](https://static.igem.org/mediawiki/2019/7/7c/T--TelHai-Migal_Israel--notebook23.png)

### FACS results mock, 11, 14 (ECFP):

![](https://static.igem.org/mediawiki/2019/b/ba/T--TelHai-Migal_Israel--notebook24.png)

### Sequencing results:

There was no match between 1701 seq and the insert 1660, Therefore we sent it to sequencing and found it to be a 100% match to pLN75. We assumed the insert wasn't ligated or there is some other problem with the gene we received from IDT and we ordered new 1660 and ran the restrictive digestion again. 1700,1702,1703 were correct. **As shown below:**

#### 1700-primer1563 forward

1700

TCGCCACCACTGCTGCCGACCTCGCAACCACTGCTTTGTCTCTGGCGCGCCAGCTCGCCA

1700-SEQ

TCGCCACCACTGCTGCCGACCTCGCAACCACTGCTTTGTCTCTGGCGCGCCAGCTCGCCA

1658

--------------------------------------CTAGAGGCGCGCCAGCTCGCCA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1700

CCATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGG

1700-SEQ

CCATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGG

1658

CCATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1700

AGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACG

1700-SEQ

AGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACG

1658

AGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1700

AGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCG

1700-SEQ

AGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCG

1658

AGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1700

ACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCA

1700-SEQ

ACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCA

1658

ACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1700

TCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGTAAGTGTGCTCGCTTCGGCAGCACATA

1700-SEQ

TCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGTAAGTGTGCTCGCTTCGGCAGCACATA

1658

TCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGTAAGTGTGCTCGCTTCGGCAGCACATA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1700

TACTATGTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACA

1700-SEQ

TACTATGTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACA

1658

TACTATGTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1700

CTTGCTGGGATTACTTCTTCAGGGACTTCTTAACCCAACAGAAGGCTCGAGAAGGTATAT

1700-SEQ

CTTGCTGGGATTACTTCTTCAGGGACTTCTTAACCCAACAGAAGGCTCGAGAAGGTATAT

1658

CTTGCTGGGATTACTTCTTCAGGGACTTCTTAACCCAACAGAAGGCTCGAGAAGGTATAT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1700

TGCTGTTGACAGTGAGCGCCGCTTGAAGTCTTTAATTAAATAGTGAAGCCCATAGGATGG

1700-SEQ

TGCTGTTGACAGTGAGCGCCGCTTGAAGTCTTTAATTAAATAGTGAAGCCCATAGGATGG

1658

TGCTGTTGACAGTGAGCGCCGCTTGAAGTCTTTAATTAAATAGTGAAGCCCATAGGATGG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1700

CAAGATCCTGGTATCGGTCTGCGAGGATCCGTCGAC------------------------

1700-SEQ

CAAGATCCTGGTATCGGTCTGCGAGGATCCGTCGAGCCTCTGGCCACATCGGTTCCTGCG

1658

CAAGATCCTGGTATCGGTCTGCGAGGATCCG-----------------------------

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

#### 1701-primer1561 forward

1701-seq

NNNNTTNCGTTGCCGTTGTCTTTTTTCCTTGAC-TCGGAGGCGCGCCAGCTCGCCACCAT

pln75

GCTTTTCGGTTGCCGTTGTCTTTTTTCCTTGACTCGGAAGGCGCGCCAGCTCGCCACCAT

pln75\_insert

-----------------------------------------CGCGCCAGCTCGCCACCAT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGG

pln75

GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGG

pln75\_insert

GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

CGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGG

pln75

CGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGG

pln75\_insert

CGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

CAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT

pln75

CAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT

pln75\_insert

CAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

CGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCA

pln75

CGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCA

pln75\_insert

CGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

GCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT

pln75

GCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT

pln75\_insert

GCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGT

pln75

CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGT

pln75\_insert

CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

GAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAA

pln75

GAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAA

pln75\_insert

GAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

GCTGGAGTACAACGCCATCAGCGACAACGTCTATATCACCGCCGACAAGCAGAAGAACGG

pln75

GCTGGAGTACAACGCCATCAGCGACAACGTCTATATCACCGCCGACAAGCAGAAGAACGG

pln75\_insert

GCTGGAGTACAACGCCATCAGCGACAACGTCTATATCACCGCCGACAAGCAGAAGAACGG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

CATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA

pln75

CATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA

pln75\_insert

CATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

CCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTA

pln75

CCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTA

pln75\_insert

CCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

CCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCT

pln75

CCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCT

pln75\_insert

CCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1701-seq

GCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAG

pln75

GCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAG

pln75\_insert

GCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

#### 1702-primer1563 forward

1702

TCGCCACCACTGCTGCCGACCTCGCAACCACTGCTTTGTCTCTGGCGCGCCAGCTCGCCA

1702-SEQ

TCGCCACCACTGCTGCCGACCTCGCAACCACTGCTTTGTCTCTGGCGCGCCAGCTCGCCA

1660

---------------------------------------------CGCGCCAGCTCGCCA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1702

CCATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGG

1702-SEQ

CCATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGG

1660

CCATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1702

AGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACG

1702-SEQ

AGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACG

1660

AGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1702

AGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCG

1702-SEQ

AGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCG

1660

AGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1702

ACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCA

1702-SEQ

ACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCA

1660

ACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1702

TCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGTAAGTGTGCTCGCTTCGGCAGCACATA

1702-SEQ

TCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGTAAGTGTGCTCGCTTCGGCAGCACATA

1660

TCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGTAAGTGTGCTCGCTTCGGCAGCACATA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1702

TACTATGTTGAATGAGGCTTCATGGAGAGATTTGGATTTTTTTTAAAAGAAGAGATTTGG

1702-SEQ

TACTATGTTGAATGAGGCTTCATGGAGAGATTTGGATTTTTTTTAAAAGAAGAGATTTGG

1660

TACTATGTTGAATGAGGCTTCATGGAGAGATTTGGATTTTTTTTAAAAGAAGAGATTTGG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1702

AGAAAGGATCAACATAGGATGGCAAGATCCTGGTATCGGTCTGCGACTC-----------

1702-SEQ

AGAAAGGATCAACATAGGATGGCAAGATCCTGGTATCGGTCTGCGAGGATCCGTCGAGCC

1660

AGAAAGGATCAACATAGGATGGCAAGATCCTGGTATCGGTCTGCGAGGATCCG-------

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

#### 1703-primer1561 forward

1703

gcttttcggttgccgttgtcttttttccttgactcggaaggcgcgccgaagcagagttca

1703-SEQ

NNNNNTNCGTTGCCGTTGTCTTTTTTCCTTGAC-TCGGAGGCGCGCCGAAGCAGAGTTCA

1661

-----------------------------------------CGCGCCGAAGCAGAGTTCA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

gccatgaatggacaaccggagagtggattgatcctttctccaaatctcttcttttaaatt

1703-SEQ

GCCATGAATGGACAACCGGANAGTGGATTGATCCTTTCTCCAAATCTCTTCTTTTAAATT

1661

GCCATGAATGGACAACCGGAGAGTGGATTGATCCTTTCTCCAAATCTCTTCTTTTAAATT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\* \*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

aaatccaaatctctccacataagatggcaagatcctggtatcggtctgcgacggggatgg

1703-SEQ

AAATCCAAATCTCTCCACATAAGATGGCAAGATCCTGGTATCGGTCTGCGACGGGGATGG

1661

AAATCCAAATCTCTCCACATAAGATGGCAAGATCCTGGTATCGGTCTGCGACGGGGATGG

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

gggtactaacacgatcgtttttttccctttttttccagggcttcacatgggagagagtca

1703-SEQ

GGGTACTAACACGATCGTTTTTTTCCCTTTTTTTCCAGGGCTTCACATGGGAGAGAGTCA

1661

GGGTACTAACACGATCGTTTTTTTCCCTTTTTTTCCAGGGCTTCACATGGGAGAGAGTCA

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

ccacatacgaagacgggggcgtgctgaccgctacccaggacaccagcctccaggacggct

1703-SEQ

CCACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCT

1661

CCACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

gcctcatctacaacgtcaagatcagaggggtgaacttcccatccaacggccctgtgatgc

1703-SEQ

GCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGC

1661

GCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGC

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

agaagaaaacactcggctgggaggcctccaccgagatgctgtaccccgctgacggcggcc

1703-SEQ

AGAAGAAAACACTCTGCTGGGAGGCCTCCACCGAGATGCTGTACCCCGCTGACGGCGGCC

1661

AGAAGAAAACACTCGGCTGGGAGGCCTCCACCGAGATGCTGTACCCCGCTGACGGCGGCC

\*\*\*\*\*\*\*\*\*\*\*\*\*\* \*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

tggaaggcagaagcgacatggccctgaagctcgtgggcgggggccacctgatctgcaact

1703-SEQ

TGGAAGGCAGAAGCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGATCTGCAACT

1661

TGGAAGGCAGAAGCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGATCTGCAACT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

tgaagaccacatacagatccaagaaacccgctaagaacctcaagatgcccggcgtctact

1703-SEQ

TGAAGACCACATACAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACT

1661

TGAAGACCACATACAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

atgtggacagaagactggaaagaatcaaggaggccgacaaagagacctacgtcgagcagc

1703-SEQ

ATGTGGACAGAAGACTGGAAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGC

1661

ATGTGGACAGAAGACTGGAAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGC

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

acgaggtggctgtggccagatactgcgacctccctagcaaactggggcacaaacttaatt

1703-SEQ

ACGAGGTGGCTGTGGCCAGATACTGCGACCTCCCTAGCAAACTGGGGCACAAACTTAATT

1661

ACGAGGTGGCTGTGGCCAGATACTGCGACCTCCCTAGCAAACTGGGGCACAAACTTAATT

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

1703

gataaaggtcgtcagactgatgggccctattcggatattgagatgaggatccaagcttgt

1703-SEQ

GATAAAGGTCGTCAGACTGATGGGCCCTATTCGGATATTGAGATGAGGATCCAAGCTTGT

1661

GATAAAGGTCGTCAGACTGATGGGCCCTATTCGGATATTGAGATGAG-------------

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

## Week 14 – 18/08 – 24/08

* Restrictive digestion for 1659
* Preparative digestion for 1703, pLN75, PGEM4Z without 1659
* Renew g-Blocks stock – order from IDT.
* Analytic digestion.
* Ligation and Transformation according to protocol.
* Picking colonies for Mini prep next week.
* Sequencing 1701-1/1701-2.
* Preparative gel for pLN75 and PGEM-4Z.
* Golden PART [BBa\_K2946003](http://parts.igem.org/Part%3ABBa_K2946003) (Improved part)– order g-Block from IDT.

**1659 gel result:** we had 1659 that has been digested. We ran the sample to verify that DNA hasn't degraded. We could see a clear band of 700-1000bp. To complete the preparations, we need to digest our 3 plasmids according to the following orders:

|  | DNA [µl] | Asc1 [µl] | BamH1 [µl] | Cutsmart [µl] | UPW[µl] |
| --- | --- | --- | --- | --- | --- |
| pLN75 10µgr | 29 | 2 | 2 | 5 | 12 |
| 1703 1µgr | 1.5 | 0.5 | 0.5 | 1 | 6.5 |
| PGEM4Z 10µgr | 14 | 1.5 | 1.5 | 5 | 28 |

![](https://static.igem.org/mediawiki/2019/0/0d/T--TelHai-Migal_Israel--notebook25.png)

In addition, we are running an analytic gel to check our previous Mini prep of 1701-1 and 1701-5 with MfeI and BamHI.

We hypothesized 1701-2 and 1701-5 were positively transfected because they were cut in the middle of the sequence. Additionally. We're going to run similar digestion for native pLN75 to verify our genes.

## Week 15 – 25/08 – 31/08

* Mini prep for 1701.2, 1701.4-1701.8
* Restrictive digestion with MfeI/BamH1 and analytic gel.
* Maxi starter + Maxi prep for 1701
* **Golden PART arrived – planning 2 paths of experiments.**
* Golden PART – G-Block Kir3DL1 resuspending according to IDT protocol.
* Golden PART – Preparative digestion for 1717/1718 with Xba1/Not1.
* Golden PART – Analytic gel for 1717/1718.
* Golden PART – Competent bacteria starter.
* Golden PART – Ligation of 1717/1718 with PGEM-4Z according to protocol.

**1701.4 went wrong so we tossed it.**

Maxi starter – 300ml LB + 340µl Amp + 1701-2.2. overnight incubated in the shaker.

Maxi prep for 1701 according to QIAGEN Kit + nanodrop check – 1230ng/µl. in addition we ran our sample in analytic gel with 1701 native as control.

### Analytic gel results 1701:

![](https://static.igem.org/mediawiki/2019/c/c3/T--TelHai-Migal_Israel--notebook26.png)

**Golden PART** – 1717 – old part, 1718 – modified part. We'll try to improve it in 2 different ways: #1 integrate the inner unit of Kir3DL1 with a scFv originated from α-HLA, #2 change the sequence with a silent mutation to get a better expression.

### Analytic gel after preparative digestion:

![](https://static.igem.org/mediawiki/2019/8/85/T--TelHai-Migal_Israel--notebook27.png)

**Competent starter:** 10ml LB + Top10 and incubate in the shaker.

**Ligation:** 1717 + PGEM4z 🡪 1721 and 1718 + PGEM4z 🡪 1722:

| [µL] | Ligase | Buffer | PGEM4z | 1717 | 1718 | UPW |
| --- | --- | --- | --- | --- | --- | --- |
| 1721 | 1 | 1 | 1 | 2 | - | 5 |
| 1722 | 1 | 1 | 1 | - | 2 | 5 |
| control | 1 | 1 | 1 | - | - | 7 |

## Week 16 – 01/09 – 07/09

* Transformation for 1721, 1722, (+) control and (-) according to protocol.
* Picking colonies – 4 of each clone.
* Mini prep for 4\*1721 and 4\*1722 – low concentration.
* Prepping competent bacteria.
* Maxi prep for pLN74,75,76 and 193.
* Preparative digestion for pLN75, 76, 193.
* **Golden PART – Repeating** 1721/22 transformation according to protocol.
* Golden PART – **Repeating** Mini starter – picking colonies – 5 of each clone.
* **Splitting K562 cells** according to protocol.
* **Bronze PART – order CD19 g-Block from IDT.**

**Competent bacteria prepping** – adding 100µl TOP10 bacteria from last week starter into 12ml LB and incubate in the shaker for 1.5h.

**Maxi concentration check by nanodrop –**

![](https://static.igem.org/mediawiki/2019/1/10/T--TelHai-Migal_Israel--notebook28.png)

**Preparative digestion:**

|  | DNA [µl] | Asc1[µl] | BamH1[µl] | Sal1[µl] | Cutsmart[µl] | UPW[µl] |
| --- | --- | --- | --- | --- | --- | --- |
| pLN75 | 2 | 0.2 | 0.2 | ------ | 1 | 6.6 |
| 75 Native | 2 | ------ | ------ | ------ | ------ | 8 |
| pLN76 | 2 | 0.2 | ------ | 0.2 | 1 | 6.6 |
| 76 Native | 2 | ------ | ------ | ------ | ------ | 8 |
| pLN193 | 2 | 0.2 | ------ | 0.2 | 1 | 6.6 |
| 193 Native | 2 | ------ | ------ | ------ | ------ | 8 |

**Expected fragments:**

* pLN75 – 739bp + 4147bp
* pLN76 – 1787bp + 3563bp
* pLN193 – 1759bp + 3480bp

![](https://static.igem.org/mediawiki/2019/b/ba/T--TelHai-Migal_Israel--notebook29.png)

## Week 17 – 08/09 – 14/09

* Bronze PART – g-Blocks arrived (1719) and resuspend (IDT protocol).
* Bronze PART – preparative digestion for CD19 (1719).
* Splitting K562 cells according to protocol (Every 2 days).
* Purifying g-Block for PCR according to Kit protocol.
* Ligation for 1719 with PGEM4z 🡪 1723 according to protocol.
* Transformation of 1723 with TOP10 bacteria according to protocol.
* Golden PART – Maxi prep for 1721/1722
* Golden PART – restrictive digestion for 1721/1722.
* Golden PART – linearization of 1721/1722 (SpeI is located on the specific plasmid, though not in our gene.)
* Bronze PART – picking 5 colonies.
* Ethanol precipitation for 1721 according to protocol.
* Mini prep for 1722 (golden part) and 1723 (bronze part)
* Analytic digestion for 1722/1723 and running samples in gel.
* Maxi starter – 250ml LB + 300µl Amp + 100µl bacteria from 1722-5 and 1723-3.
* Maxi prep – 1722-5 and 1723-3
* Digestion to verify clone's wanted length (1118/1343bp)
* Linearization for chosen samples according to protocol.

![](https://static.igem.org/mediawiki/2019/8/8a/T--TelHai-Migal_Israel--notebook30.png)

### analytic gel after Maxi:

1721 – good results, the sequence was digested as expected.

1722 – not as expected. We will pick new colonies and repeat the process.

|  | Marker | 1721-N | 1721-S | 1722-N | 1722-S |
| --- | --- | --- | --- | --- | --- |
| DNA | 7.5 | 2 | 2 | 2 | 2 |
| UPW | - | 8 | 8 | 8 | 8 |
| L.Dye | - | 2 | 2 | 2 | 2 |
| Total | 7.5 µl | 12 µl | 12 µl | 12 µl | 12 µl |

### Preparative digestion 1721/1722:

N-native S-Spe1 digest

![](https://static.igem.org/mediawiki/2019/6/66/T--TelHai-Migal_Israel--notebook31.png)

**Pre-Maxi analytic digestion gel results:**

![](https://static.igem.org/mediawiki/2019/2/26/T--TelHai-Migal_Israel--notebook32.png)

* we chose samples 1723-3 and 1722-5 for our next experiments.

Post-Maxi analytic digestion gel results:

![](https://static.igem.org/mediawiki/2019/0/0c/T--TelHai-Migal_Israel--notebook33.png)

Spe1 Digestion for 1722/1723:

|  | Cutsmart [µl] | Spe1 [µl] | DNA [µl] | UPW [µl] |
| --- | --- | --- | --- | --- |
| 1722 | 50 | 2 | 41 | 407 |
| 1723 | 50 | 2 | 97 | 351 |

**Spe1 gel results:**

![](https://static.igem.org/mediawiki/2019/1/14/T--TelHai-Migal_Israel--notebook34.png)

**Ethanol precipitation nanodrop results:**

![](https://static.igem.org/mediawiki/2019/8/8b/T--TelHai-Migal_Israel--notebook35.png)

## Week 18 – 15/09 – 21/09

* Stage 1 – RNA elicitation for 1721, 1722, 1723, 1024 (control)
* Stage 2 – RNA elicitation for 1721, 1722, 1723, 1024 (control)
* Transfection of 1721/22/23/1024 with electrophoresis according to protocol.
* FACS according to protocol.

Stage 1 RNA nanodrop check:

![](https://static.igem.org/mediawiki/2019/1/1d/T--TelHai-Migal_Israel--notebook36.png)
![](https://static.igem.org/mediawiki/2019/b/b8/T--TelHai-Migal_Israel--notebook37.png)

\*\*\* 1723 went low so we'll take it all to stage 2.

Stage 1 RNA gel results:

![](https://static.igem.org/mediawiki/2019/c/c8/T--TelHai-Migal_Israel--notebook38.png)

Stage 2 RNA gel results:

![](https://static.igem.org/mediawiki/2019/3/3d/T--TelHai-Migal_Israel--notebook39.png)

Transfection plan:

![](https://static.igem.org/mediawiki/2019/a/af/T--TelHai-Migal_Israel--notebook40.png)

Electrophoresis results:

![](https://static.igem.org/mediawiki/2019/e/e0/T--TelHai-Migal_Israel--notebook41.png)

FACS plan:

![](https://static.igem.org/mediawiki/2019/a/a8/T--TelHai-Migal_Israel--notebook42.png)

FACS results:

![](https://static.igem.org/mediawiki/2019/b/b4/T--TelHai-Migal_Israel--notebook43.png)
![](https://static.igem.org/mediawiki/2019/9/98/T--TelHai-Migal_Israel--notebook44.png)
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# Team:TelHai-Migal Israel/Experiments

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# Experiments

For our wet work, we started the process by planning and ordering the parts ([BBa\_K2946010](http://parts.igem.org/Part%3ABBa_K2946010), [BBa\_K2946011](http://parts.igem.org/Part%3ABBa_K2946011), [BBa\_K2946013](http://parts.igem.org/Part%3ABBa_K2946013), [BBa\_K2946014](http://parts.igem.org/Part%3ABBa_K2946014), [BBa\_K2549001](http://parts.igem.org/Part%3ABBa_K2549001) and [BBa\_K321004](http://parts.igem.org/Part%3ABBa_K321004)). BBa\_K321004 was ordered with a ScFv from our lab in order to improve a part ([BBa\_K2946003](http://parts.igem.org/Part%3ABBa_K2946003)).

They were then ordered as gBlocks from IDT. As our backbone vector for the [BBa\_K2946010](http://parts.igem.org/Part%3ABBa_K2946010), [BBa\_K2946011](http://parts.igem.org/Part%3ABBa_K2946011), [BBa\_K2946013](http://parts.igem.org/Part%3ABBa_K2946013), [BBa\_K2946014](http://parts.igem.org/Part%3ABBa_K2946014) parts we used pLN75 & pLN193 (obtained from Nissim et al., Cell 2017).

Once the gBlocks arrived, they were resuspended, and the cloning process began.

## Cloning

Our gBlocks were digested over night with relevant restriction enzymes.
[BBa\_K2946010](http://parts.igem.org/Part%3ABBa_K2946010) and [BBa\_K2946013](http://parts.igem.org/Part%3ABBa_K2946013) were digested with Asc1 and Xho1, while [BBa\_K2946011](http://parts.igem.org/Part%3ABBa_K2946011) and [BBa\_K2946014](http://parts.igem.org/Part%3ABBa_K2946014) were digested with Asc1 and BamHI

### Each tube contains:

50 µL DNA, 1.5 µL from each enzyme, 5 µL cut-smart, 12 µL UPW – total 70 µL

After complete DNA digestion using the relevant restriction enzymes, the DNA segments were run and separated on 1% agarose gel with TAE buffer and crystal violet. The vectors were cut from the gel and cleaned using Wizard SV Gel and PCR Clean-up System from Promega according to their protocol. [(protocol 1)](https://static.igem.org/mediawiki/2019/6/6a/T--TelHai-Migal_Israel--protocol1.pdf)

For extracted the gblocks from the restriction enzymes we used an extraction kit according to Gel/PCR Fragments Extraction Kit. [(protocol 2)](https://static.igem.org/mediawiki/2019/3/33/T--TelHai-Migal_Israel--protocol2.pdf)

## Ligation

**Ligation** of DNA inserts into their vectors was done by using 1 unit of T4 DNA Ligase from NEB at room temperature for 2 hrs.

### Each tube contains:

1µL buffer, 1µL T4 ligase, 1µL vector, 2µL insert and 5µL DDW.

| Plasmid | Gene | Part number |
| --- | --- | --- |
| pLN193 | TS1 | [BBa\_K2946010](http://parts.igem.org/Part%3ABBa_K2946010) |
| pLN75 | TS2 | [BBa\_K2946011](http://parts.igem.org/Part%3ABBa_K2946011) |
| pLN193 | TS11 | [BBa\_K2946013](http://parts.igem.org/Part%3ABBa_K2946013) |
| pLN75 | TS12 | [BBa\_K2946014](http://parts.igem.org/Part%3ABBa_K2946014) |
| PGEM4Z | CD19 | [BBa\_K2549001](http://parts.igem.org/Part%3ABBa_K2549001) |
| PGEM4Z | kir3DL1+ ScFv | [BBa\_K2946003](http://parts.igem.org/Part%3ABBa_K2946003) |

Table 1: Ligations – Each part was ligated into a specific vector. TS1 and TS11 were each inserted to pLN193, TS2 and TS12 were inserted into pLN75. 1721 were insert into pGEM4Z 5'UT-eGFP-3'UT-A64 (PGEM4Z) and so as 1722.

Following ligation DNA was transformed into TOP10 competent cells according to our labs protocol. [(protocol 3)](https://static.igem.org/mediawiki/2019/a/ae/T--TelHai-Migal_Israel--protocol3.pdf) Mini prep was done to 3-5 colonies for each part using Zyppy Plasmid Miniprep kit. [(protocol 4)](https://static.igem.org/mediawiki/2019/c/ca/T--TelHai-Migal_Israel--protocol4.pdf) After determining positive colonies, one was used for maxi prep using Maxi prep endotoxin free- PureYield Plasmid Maxiprep System – Promega. [(protocol 5)](https://static.igem.org/mediawiki/2019/0/0f/T--TelHai-Migal_Israel--protocol5.pdf)

## DNA transfection using Hek293T

HEK293T cells were grown in DMEM complete medium (C.M.) containing 500 ml medium, 50 ml FCS, 5 ml sodium pyruvate and 2 ml Pen-strep.

Table 2 – DNA transfection plan

|  | Plasmid 1 (1µg) | Plasmid 2 (1µg) |
| --- | --- | --- |
| 1 | Positive control | - |
| 2 | GFP (pEGFP-N3) | - |
| 3 | Mock | - |
| 4 | TS1 | - |
| 5 | TS2 | - |
| 6 | TS11 | - |
| 7 | TS12 | - |
| 8 | TS1 | TS2 |
| 9 | TS11 | TS12 |
| 10 | Non-Transfected | - |

Transfections were done in 6 welled plates using transfection reagent FuGENE HD.

FuGENE HD transfection reagent (Promega, Madison, WI; Catalog #E2311) was mixed with OptiMEM medium (Life Technologies Catalog #31985) according to the materials found in Cell 2017 and methods. This was then added to the mixture of two plasmids (1 mg each).

During the 20-minute incubation of FuGENE HD with the DNA complexes at room temperature, HEK293T suspension cells were prepared and diluted to 1.2x106 cells/mL in culture medium. 0.5 mL of diluted cells were added to each tube, mixed well, and 5 minutes later plated into a 6-well plate containing 2 mL cell culture medium and incubation at 37C with 5% CO2.

24 hours post-transfection, the cells growth medium was replaced, and cells were collected 24 hours after that (48 hours post-transfection).

For parts containing mkate2 exons, cells were collected using 0.5ml trypsin + 1ml DMEM-C.M. These cells were then subjected to flow cytometry and measured by FACSCalibur by BD Biosciences or Attune NxT by Thermo Fisher scientific.

**Improved part ([BBa\_K2946003](http://parts.igem.org/Part%3ABBa_K2946003)) and characterization of part ([BBa\_K2549001](http://parts.igem.org/Part%3ABBa_K2549001))**

As our backbone vector for CD19 ([BBa\_K2549001](http://parts.igem.org/Part%3ABBa_K2549001)) and kir3DL1 ([BBa\_K2946003](http://parts.igem.org/Part%3ABBa_K2946003)) parts we used PGEM4Z as our vector. The genes were cloned into the plasmid similarly to the described above.

## RNA transfection using K562 (CML cell line)

In order to examine the expression of CD19 and kir3DL1 parts we used RNA transfection to insert the parts into K562 cells.

mRNA production was done according to T7 mScript Standard mRNA Production System - Cell Script. [(protocol 6)](https://static.igem.org/mediawiki/2019/9/9e/T--TelHai-Migal_Israel--protocol6.pdf)

K562 cells were grown in RPMI complete medium (C.M.) containing 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, Pen-strep solution 1:200.

Transfection into K562 cells was done by Electroporation.
Electroporation was performed with Gene Pulser Xcell (Bio-Rad Laboratories, Hercules, CA) [(protocol 7)](https://static.igem.org/mediawiki/2019/a/ae/T--TelHai-Migal_Israel--protocol7.pdf) according to the following conditions:

| Cell type | Amount of cells | Amount of mRNA | Pulse type | Volts | Ω | µF | Pulse duration | Cuvette width |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| K562 | 3x106 | 10µg | Exponential | 350V | - | 150µF | - | 4mm |

Table 3 - electroporation conditions

After the transfection we incubated the cells with antibodies for the FACS measurement.
10 µl Anti-Human c-Myc- APC (R&D SYSTEMS, Cat: IC3696A).
5 µl Anti Ha- Alexa Fluor 647 (R&D SYSTEMS, Cat: IC6875R).

## Preparing for FACS

Cells were harvested, washed once with cold FACS buffer (PBS with 0.5M EDTA, 0.01% sodium azide and 1% BSA) and incubated for 45 minutes at 4֯C in the dark with the respective Ab-conjugate at concentration recommended by the manufacturer. Cells were washed again with 3 ml FACS buffer, resuspended in 0.3 ml PBS and subjected to flow cytometry using FACSCalibur (Becton Dickinson, San Jose, CA). Data were analyzed by FCSexpress (DeNovo Software, Los Angeles, CA).

## Reference

1. Nissim, L. et al. Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy. Cell 171, 1138-1150.e15 (2017).

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# Team:TelHai-Migal Israel/Results

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* [Home](https://2019.igem.org/Team%3ATelHai-Migal_Israel)
* Project
  + [Description](https://2019.igem.org/Team%3ATelHai-Migal_Israel/Description)
  + [Design](https://2019.igem.org/Team%3ATelHai-Migal_Israel/Design)
  + [Experiments](https://2019.igem.org/Team%3ATelHai-Migal_Israel/Experiments)
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* [Parts](https://2019.igem.org/Team%3ATelHai-Migal_Israel/Parts)
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* Human Practices
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# Results

Here we show that unlike the original Cell modules, LoGENEgate totally precludes expression of a model gene in off-target cells. Our findings pave the way for numerous therapeutic applications of LoGENEgate.

## Overview

* We design 2 circuits and constructed 3 Parts for each circuit.
* Assembled and demonstrated a functional AND gate gene circuit in Hek293T cells
* Compared the results to find which trans-splicing sites are more effective.
* Compared our system to literature, ours is safer!

## Results

* Design and Construction of Circuits

In order to achieve our goal, we first designed and constructed the Trans Splicing (TS)-based AND gate circuits, LoGENEgate.

We have assembled and analyzed two different circuits, each comprising two modules (see below for details), with different intronic sequences designated to induce TS. The first circuit, comprised of TS1 + TS2, include a 'basic' TS site design based on the original intron separating the two mKate2 exons used in cell 20171. The second circuit, TS11 + TS12, contain a TS site design based on a thorough analysis to maximize the efficacy. Each of the modules is regulated by a separate promoter (Figure 2). Module 1 consists of P1 (SSX1p), which regulates the expression of exon 1 of mKate2 as well half of the intron that include a strong donor splice site and linker. Module 2 consists of P2 (H2A1p), which regulates a TS guiding sequence, the acceptor splice site and mKate2 exon 2 (Figure 1).

### First circuit:

[![](https://static.igem.org/mediawiki/2019/d/da/T--TelHai-Migal_Israel--results1.png)](https://static.igem.org/mediawiki/2019/d/da/T--TelHai-Migal_Israel--results1.png)
 [![](https://static.igem.org/mediawiki/2019/5/59/T--TelHai-Migal_Israel--circle555small350.png)](https://static.igem.org/mediawiki/2019/5/59/T--TelHai-Migal_Israel--circle555small350.png)

### Second circuit:

[![](https://static.igem.org/mediawiki/2019/b/ba/T--TelHai-Migal_Israel--results3.png)](https://static.igem.org/mediawiki/2019/b/ba/T--TelHai-Migal_Israel--results3.png)
 [![](https://static.igem.org/mediawiki/2019/f/f3/T--TelHai-Migal_Israel--results4.png)](https://static.igem.org/mediawiki/2019/f/f3/T--TelHai-Migal_Israel--results4.png)

Following trans-splicing, the exons are assembled into a mature output mRNA.

In this system, the output protein is expressed only when the promoters regulating both modules are mutually active. However, when only one of the promoters is active or when none of the promoters are active, they cannot produce any functional protein. thus, standing alone, is permanently in state ‘0’.

We defined two different states for this circuit: in state [1,0], module 1 is active, while module 2 is inactive (off); and in state [1,1], both module 1 and module 2 are active (on).

![](https://static.igem.org/mediawiki/2019/0/03/T--TelHai-Migal_Israel--results5.png)

* Expression in HEK293T cells

The data below (Figures 3+4) display the transient transfection of HEK293T cells with plasmids containing LoGENEgate by flow cytometry. Any expression below the negative control (<3.09%) is irrelevant, hence amounts to 0% expression of GOI.

As expected in [1,0] states there is no product, 0% expression, while in [1,1] stats the mean of expression is 24.02%.

For further validation, the [experiment](https://2019.igem.org/Team%3ATelHai-Migal_Israel/Experiments)was done twice each on 105 cells, with similar results.

As expected the second 'optimized' circuit, TS11+12, yielded higher levels of expression [31.55% (Figure 4) compare to 17.5% (Figure 3)] then the first 'basic' circuit. However, we note that further optimization is needed.

![](https://static.igem.org/mediawiki/2019/5/59/T--TelHai-Migal_Israel--results6.png)
![](https://static.igem.org/mediawiki/2019/a/a8/T--TelHai-Migal_Israel--results7.png)

* Comparing to cell 2017

We next compared the results of LoGENEgate to the results from cell 2017 1 (Figure 5). Although the sponge system is more efficient, with a higher level of expression in state [1,1], LoGENEgate is much safer, with 0% levels of expression compare to 15% in state [1,0].

![](https://static.igem.org/mediawiki/2019/a/a7/T--TelHai-Migal_Israel--results8.png)

## Conclusion and Future plans

We presented proof-of-concept [demonstration](https://2019.igem.org/Team%3ATelHai-Migal_Israel/Demonstrate) that LoGENEgate works!

The protein product of the two-module circuit is only translated when both modules are co-expressed and TS occurs, but is absolutely absent when each of the two modules is expressed alone. Even so our experiments didn't test the promotors themselves. Accordingly, the next step will be to find promotors that are specific to a type of cancer and test the expression of the GOI in healthy cells compare to cancer cells.

Additionally, there is no doubt that several rounds of optimization steps could substantially improve the circuit efficacy, while not impairing its safety at all.

We have patented LoGENEgate and our lab is committed to continue this research and get it to clinical phases.

## Reference

1. Nissim, L., M.-R. Wu, E. Pery, A. Binder-Nissim, H. I. Suzuki, D. Stupp, C. Wehrspaun, Y. Tabach, P. A. Sharp, and T. K. Lu. 2017. Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy. Cell 171: 1138-1150.

2. Xie, M., and M. Fussenegger. 2018. Designing cell function: assembly of synthetic gene circuits for cell biology applications. Nat. Rev. Mol. Cell Biol. 19: 507–525.

3. Yang, Y., and C. E. Walsh. 2005. Spliceosome-Mediated RNA Trans-splicing. Mol. Ther. 12: 1006–1012.

4. Puttaraju, M., S. F. Jamison, S. G. Mansfield, M. A. Garcia-Blanco, and L. G. Mitchell. 1999. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy. Nat. Biotechnol. 17: 246–252.

5. Puttaraju, M., J. DiPasquale, C. C. Baker, L. G. Mitchell, and M. A. Garcia-Blanco. 2001. Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing. Mol. Ther. 4: 105–14.

6. Suñé-Pou, M., S. Prieto-Sánchez, S. Boyero-Corral, C. Moreno-Castro, Y. El Yousfi, J. Suñé-Negre, C. Hernández-Munain, and C. Suñé. 2017. Targeting Splicing in the Treatment of Human Disease. Genes (Basel). 8: 87.

7. Schlesinger, J., D. Arama, H. Noy, M. Dagash, P. Belinky, and G. Gross. 2003. In-cell generation of antibody single-chain Fv transcripts by targeted RNA trans-splicing. J Immunol Methods 282: 175–186.

8. Poddar, S., P. S. Loh, Z. H. Ooi, F. Osman, J. Eul, and V. Patzel. 2018. RNA Structure Design Improves Activity and Specificity of trans -Splicing-Triggered Cell Death in a Suicide Gene Therapy Approach. Mol. Ther. - Nucleic Acids 11: 41–56.

9. Sullenger, B. A., and T. R. Cech. 1994. Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature 371: 619–622.

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