Team:TelHai-Migal Israel/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.

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:

After Midi restrictive digestion results:

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:

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:

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:

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:

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:

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:

Nanodrop test:

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:

After preparative gel:

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:

Nanodrop after mini and before freezing:

Maxi prep for 1700-1703 gel results:

Nanodrop after maxi:

Nanodrop for next week experiment:

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:

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:

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	Ex-mKate 1 – TS1
6	1701- BBa_K2946011	Ex-mKate 2 – TS1 – no florescence
7	1702- BBa_K2946013	Ex-mKate 1 – TS2
8	1703- BBa_K2946014	Ex-mKate 2 – TS2
9	1700+1701- BBa_K2946012	TS1
10	1702+1703- BBa_K2946015	TS2

FACS results 1-3 (FL1-H):

FACS results mock,mKate:

FACS mock 1700-1703 (mkate):

FACS results mock, 11, 14 (ECFP):

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 (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

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:

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:

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 –

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

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.