iGEM 2016 — Team Technion_Israel

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# Team:Technion Israel/Notebook

S.tar, by iGEM Technion 2016

[![](https://static.igem.org/mediawiki/2016/2/2d/T--Technion_Israel--logo.png)](https://2016.igem.org/Team%3ATechnion_Israel)

* [Project](https://2016.igem.org/Team%3ATechnion_Israel/Description)
* [S.Tar](https://2016.igem.org/Team%3ATechnion_Israel/S.Tar_intro)
  + [Overview](https://2016.igem.org/Team%3ATechnion_Israel/S.Tar_intro)
  + [Tar Improvements and Characterization](https://2016.igem.org/Team%3ATechnion_Israel/Tar_improvements)
  + [Computational Design](https://2016.igem.org/Team%3ATechnion_Israel/Modifications/Rosetta)
  + [PctA-Tar Chimera](https://2016.igem.org/Team%3ATechnion_Israel/Proof)
  + [NarX-Tar Chimera](https://2016.igem.org/Team%3ATechnion_Israel/Modifications/narx)
  + [hERα-Tar Chimera](https://2016.igem.org/Team%3ATechnion_Israel/Modifications/EstoTar)
  + [Intein](https://2016.igem.org/Team%3ATechnion_Israel/Modifications/Intein)
* [FlashLab](https://2016.igem.org/Team%3ATechnion_Israel/Design)
  + [Overview](https://2016.igem.org/Team%3ATechnion_Israel/Design)
  + [Modeling](https://2016.igem.org/Team%3ATechnion_Israel/Model)
  + [Hardware](https://2016.igem.org/Team%3ATechnion_Israel/Hardware)
  + [Chromoprotein](https://2016.igem.org/Team%3ATechnion_Israel/Color)
* Wet Lab
  + [Highlight Results](https://2016.igem.org/Team%3ATechnion_Israel/Results)
  + [Experiments](https://2016.igem.org/Team%3ATechnion_Israel/Experiments)
  + [Protocols](https://2016.igem.org/Team%3ATechnion_Israel/Protocols)
  + [Notebook](https://2016.igem.org/Team%3ATechnion_Israel/Notebook)
* Judging
  + [Proof of concept](https://2016.igem.org/Team%3ATechnion_Israel/Proof)
  + [Biobricks](https://2016.igem.org/Team%3ATechnion_Israel/Parts)
  + [Software](https://2016.igem.org/Team%3ATechnion_Israel/Software)
  + [Measurments](https://2016.igem.org/Team%3ATechnion_Israel/Measurement)
  + [Safety](https://2016.igem.org/Team%3ATechnion_Israel/Safety)
  + [Medals](https://2016.igem.org/Team%3ATechnion_Israel/Medals)
* People
  + [Human Practice](https://2016.igem.org/Team%3ATechnion_Israel/Human_Practices)
  + [Team Members](https://2016.igem.org/Team%3ATechnion_Israel/Team)
  + [Attributions](https://2016.igem.org/Team%3ATechnion_Israel/Attributions)
  + [Collaborations](https://2016.igem.org/Team%3ATechnion_Israel/Collaborations)

S.tar, by iGEM Technion 2016

![](https://static.igem.org/mediawiki/2016/2/2b/T--Technion_Israel--notebookcov.jpg)

* [April](#tab1)
* [May](#tab2)
* [June](#tab3)
* [July](#tab4)
* [August](#tab5)
* [September](#tab6)
* [October](#tab7)

The following calendar presents the work we have done in each day.
For more information on a specific entry, press the colored buttons. Press again to close the explanation.

S.Tar
 FlashLab
 HP & Collaboration
 Other

| Sunday | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | 1 | 2 |
| --- | --- | --- | --- | --- | --- | --- |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |

**12.4.16**
 Shirane Tsedef & Nofar Shasha's birthday!

**18.4.16**
Received Strain UU1250.
O\N Starter to UU1250.

**18.4.16**
Meeting with Tel-Hai iGEM group.

**19.4.16**
Glycerol stock for UU1250.

**22.4.16**
 Shani Weiner's birthday!

**24.4.16**
 Lab preparations.

The following calendar presents the work we have done in each day.
For more information on a specific entry, press the colored buttons. Press again to close the explanation.

S.Tar
 FlashLab
 HP & Collaboration
 Other

| Sunday | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| --- | --- | --- | --- | --- | --- | --- |
| 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| 15 | 16 | 17 | 18 | 19 | 20 | 21 |
| 22 | 23 | 24 | 25 | 26 | 27 | 28 |
| 29 | 30 | 31 |  |  |  |  |

**03.5.16**
 Meeting with Ran Levi.

**05.5.16**
Meeting with Prof. Esti Segal.

**08.5.16**
 "Science On the Bar" event - Prof. David (Dedi) Meiri.

Project presentation at a special convention of Technion ATF (French Technion Society).

**09.5.16**
Meeting with Prof. Gilad Yossifon.

**10.5.16**
 Chemical competent cells preparation for strains Top 10 & UU1250.

**15.5.16**
Meeting with PhD student Jonathan Avesar and Prof. Shulamit Levenberg for consultation regarding the model.

**16.5.16**
Tar & Color:
DNA extraction from 2016 distribution kit parts: K1357008, K1357009 , K1357010 and J23100.
Transformation to Top10 by heat shock to K1357008 , K1357009 , K1357010 and J23100.

**18.5.16**
 "Science On the Bar" event - Prof. Avraham Haim.

**18.5.16**
Tar & Color:
O\N starter to: Top 10 with K1357008, Top 10 with K1357009 & Top 10 with K1357010

**19.5.16**
Tar & Color:
Glycerol stock for Top10+K1357008, Top10+K1357009 & Top10+K1357010.
Miniprep for K1357008 ,K1357009 & K1357010.
Transformation of J23100 plasmid to Top10 by heat shock.

**19.5.16**
Meeting with PhD student Jonathan Avesar.

**21.5.16**
Tal Fried's birthday!

**22.5.16**
 O\N starter to Top10 with J23100

**23.5.16**
Tar & Color:
Miniprep for J23100.

**23.5.16**
 "Science On the Bar" event-Prof. Meytal Landau.

**24.5.16**
Tar & Color:
Restriction of J23100 with the enzymes: SpeI & Pst.
DNA extraction from 2016 distribution kit: k777001 & k777000.
Transformation to Top10 by heat shock to:
1. k777001
2.k777000.
O\N Starters for: MG1655, UU1250,K1357008, K1357009 & J23100.

**25.5.16**
Tar & Color:
Glycerol stock for Top10 with J23100.
Miniprep for K1357008, K1357009 & J23100.
Restriction of K1357008 & K1357009 with the enzymes: PstI & XbaI
Gel electrophoresis, cutting and cleaning K1357008 & K1357009

Chemotaxis:
Chemical in plug assay for MG1655 & UU1250 strains.

**25.5.16**
Introductory meeting for iGEM 2017 team - explanation about synthetic biology and the project.

**26.05.16**
Chemotaxis:
The Chemical in plug assay for MG1655 & UU1250 strains did not work as expected.
After talking with Oshri Afanzar
we understood the requirement for improving the separation of the bactria from the liquid.

**29.5.16**
Meeting with Prof. Moran Berkovichi.

**30.5.16**
Tar:
O\N starters of Top10 with k777001 & Top10 with k777000.

**31.5.16**
Tar & Color:
Glycerol stock for Top10 with k777001 & Top10 with k777000.
Miniprep for k777001 & k777000.

Restriction of k777001 with the enzymes: pstI & speI.
Gel electrophoresis, cutting and Cleaning k777001 plasmid.
Ligation of:
1. k777001 with purple chromogenic protein.
2. k777001 with blue chromogenic protein.
3.J23100 with K1357008.
4.J23100 with K1357009.

The following calendar presents the work we have done in each day.
For more information on a specific entry, press the colored buttons. Press again to close the explanation.

S.Tar
 FlashLab
 HP & Collaboration
 Other

| Sunday | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- | --- | --- |
| 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| 12 | 13 | 14 | 15 | 16 | 17 | 18 |
| 19 | 20 | 21 | 22 | 23 | 24 | 25 |
| 26 | 27 | 28 | 29 | 30 |  |  |

**02.6.16**
Tar & Color:
Transformation to Top10 by heat shock of:
1.Tar+blue chromogenic protein
2.Tar+purple chromogenic protein
3.Purple chromogenic protein
4.Blue chromogenic protein

**03.6.16**
Tar & Color:
Color colonies for all 4 strains:
Top 10 with Tar+blue chromogenic protein, Top 10 with Tar+purple chromogenic protein,
Top 10 with Purple chromogenic protein & Top 10 with Blue chromogenic protein.

**05.6.16**
Tar & Color:

O\N Starters, (LB,37C) of:
1.Top 10 with Tar+blue chromogenic protein.
2.Top 10 with Tar+purple chromogenic protein.
3.Top 10 with purple chromogenic protein.
4.Top 10 with blue chromogenic protein.

**06.6.16**
Tar & Color:
No visible color in the Tar+color strain starter.
All strains showed color after centrifuge.
Glycerol stock for:
1.Top 10 with Tar+blue chromogenic protein.
2.Top 10 with Tar+purple chromogenic protein
3.Top 10 with purple chromogenic protein
4.Top 10 with blue chromogenic protein
Miniprep to Tar+blue chromogenic protein, Tar+purple chromogenic protein, purple chromogenic protein & blue chromogenic protein.

**07.6.16**
Chemotaxis:
O\N starters of UU1250 & MG1655 for Swarming assay.

**07.6.16**
"Science On the Bar" event-Prof. David (Dedi) Meiri.

**08.6.16**
Chemotaxis:
Swarming assay to UU1250 and MG1655.

**09.6.16**
Chemotaxis:
Swarming assay conclusion:
MG1655 is a suitable strain for positive control and UU1250 for negative control.
The assay is suitable only for ligands that are metabolized.

**12.6.16**
Rosetta
First run of the "Rosetta" software with TSR, an  *E.coli*  receptor for Serine, using Oxytocin as the new ligand.
TSR was used for technical reasons.

**12.6.16**
Rosetta
Conclusion: for optimal results from the software we need more computing power than a normal home PC.
We began searching for a suitable computer.

**13.6.16**
Chemotaxis:
O\N starter to MG1655.

**13.06.16**
Chemotaxis experiments on paper chip.

**14.6.16**
Tar & Color:
Transformation to UU1250 by heat shock of:
1.Tar+purple chromogenic protein.
2.Tar+blue chromogenic protein.
3.Purple chromogenic protein.
4.Blue chromogenic protein.
Preparation of MG1655 electro competent cells.
Transformation to MG1655 by electroporation of:
1.Purple chromogenic protein.
2.Blue chromogenic protein.

Rosetta
Meeting with David Cohen who is in charge of the Atlas computer cluster in the Technion.

**14.06.16**
Chemotaxis experiments on paper chip.

**15.6.16**
Rosetta
Rosetta installed on Atlas!

Tar & Color:
Strains of Tar and color presented visible color but no color was visible in MG1655 or UU1250.
O\N Starters to:
1.UU1250 with Tar+blue chromogenic protein
2.UU1250 with Tar+purple chromogenic protein.
3.MG1655 with purple chromogenic protein.
4.UU1250.
5.UU1250 with AMP resistance.
6.MG1655.
7.MG1655 with AMP resistance.

**15.06.16**
Chemotaxis experiments on paper chip.

**16.6.16**
Tar & Color:
Glycerol stocks for:
1.UU1250 with Tar+blue chromogenic protein.
2.UU1250 with Tar+purple chromogenic protein.
Restriction to Tar+purple chromogenic with the enzymes: Xbal, Kpnl, Xbal+Kpnl.

Chemotaxis:
Swarming assay and Chemical in Plug assay to:
1.UU1250 with AMP resistance.
2.MG1655 with AMP resistance.
3.UU1250 with Tar+purple chromogenic protein
4.UU1250 with Tar+blue chromogenic protein.
Two swarming assays were tested: original swarming assay with TB as the chemoattractant and swarming assay with motility buffer as control.

**16.06.16**
Chemotaxis experiments on paper chip.
working on PDMS chip with PhD student Jonathan Avesar.

**17.06.16**
Chemotaxis:
No swarming was visible in the motility buffer swarming assays meaning the experiment had failed.
In the original swarming assay (with TB):
Both of the cloned strains failed to express color nor swarm as expected.
But UU1250 with Tar+blue did show better swarming abilities compared to the UU1250 strain.

**19.6.16**
Chemotaxis:
Meeting with Oshri Afanzar in Weismann institute.
The purpose of our meeting was to learn more abut chemotaxis Assays:
Chemical in Plug assay, CHIP (BRET),Under the microscope chemotaxis and Optimal concentrations.

**19.06.16**
Chemotaxis experiments on paper chip.
By comparing to drop test we could see effect of the diffusion on the chip.

**20.06.16**
Chemotaxis experiments on paper chip.
Sodium benzoate was used in too high concentration that killed the bacteria before it had a chance to flee.

**21.06.16**
Chemotaxis experiments on paper chip.
We found the temperature and time needed to grow the bacteria for our assay.

**22.6.16**
Sharbel Zaharan's birthday!

**22.06.16**
Chemotaxis experiments on paper chip.
We were able to see motile bacteria in the chip.

**29.6.16**
Chemotaxis:
O/N starters to strains from Weismann institute:
1.B275 ΔFliM- ΔF - negative control for chemotaxis assay.
2.B275 Δzras-ΔZ - positive control for chemotaxis assay.

**30.6.16**
Chemotaxis:

Glycerol stock for:
1.B275 ΔFliM- ΔF
2.B275 Δzras-ΔZ

The following calendar presents the work we have done in each day.
For more information on a specific entry, press the colored buttons. Press again to close the explanation.

S.Tar
 FlashLab
 HP & Collaboration
 Other

| Sunday | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | 1 | 2 |
| --- | --- | --- | --- | --- | --- | --- |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| 31 |  |  |  |  |  |  |

**04.7.16**
Meeting with Prof. Dan Shechtman regarding possible educational collaboration.

**08.7.16**
Shilo Ohayon's birthday!

**11.7.16**
Color:
O/N Starters in order to test color formation to:
1.Top10 with RFP.
2.UU1250 with Tar+purple chromogenic protein.
3.UU1250 with Tar+blue chromogenic protein.
4.Top10 with purple chromogenic protein.
5.Top10 with Blue chromogenic protein.
6.Top10 with Tar+purple chromogenic protein.
7.Top10 with Tar+Blue chromogenic protein.
Color formation check at LB and TB in both 37C and 30C.

**12.7.16**
Color:
Checking results.
Got some beautiful colored bacteria but still not the right conditions for chemotaxise assays.
Isolation plating form TB O/N starters.

**17.7.16**
Color:
O\N starters to Top 10 with purple chromogenic protein & Top 10 with blue chromogenic protein.

**18.7.16**
Color:
Checking color results in Top 10 with purple chromogenic protein and Top 10 with blue starters.
Transformation to UU1250 by heat shock to:
1.Purple chromogenic protein.
2.Blue chromogenic protein.
3.RFP.

**20.7.16**
Color:
Color formation test in TB and LB in both 30C and 37C to:
1.UU1250 with Tar+purple chromogenic protein.
2.UU1250 with Tar+blue chromogenic protein.

**21.7.16**
Color:

Checking results of Color formation test.

**24.7.16**
Tar:
PCR to:
1.promotor + purple chromogenic protein.
2.Tar - adding overlap for gibson assambly.
Cleaning from PCR and checking concentrations.
O\N starters to:
1.UU1250 with blue chromogenic protein.
2.UU1250 with RFP chromogenic protein.
3.UU1250 with purple chromogenic protein.
4.MG1655 with AMP resistance.
5.MG1655.

**25.7.16**
Tar:
Glycerol stock to UU1250 blue chromogenic protein and UU1250 RFP.
PCR to promoter+blue chromogenic protein and promoter+purple chromogenic protein .
Gel electrophoresis.
Unsuccessful purification due to an error in using undiluted wash buffer.

Chemotaxis:
Starters to:
1.UU1250.
2.MG1655 with AMP resistance.

**25.7.16**
 Chemotaxis experiments on commercial chip.

**26.7.16**
Chemotaxis:
Chemotaxis test in 30C in both LB and TB for:
1.UU1250 with purple chromogenic protein.
2.UU1250 with blue chromogenic protein.
3.UU1250 with RFP.
4.UU1250 with Tar + purple chromogenic protein.
5.UU1250 with Tar + blue chromogenic protein.
Microscope assay - we weren't able to see the bacteria under the microscope.

Tar:
PCR to promoter+purple chromogenic protein again, Gel electrophoresis and DNA purification.
Gibson assembly to Tar+overlap with promoter+purple chromogenic protein Open plasmid.
Transformation to Top 10 by heat shock to Tar+promoter+ purple chromogenic protein .

**26.7.16**
 Chemotaxis experiments on commercial chip.

**26.7.16**
 Our special video is online! Preparing for the Giant Jamboree like true Olympics athletes.

**27.7.16**
Tar:
Colony PCR to 5 colonies from Gibson assembly - all colonies were negative.

Chemotaxis:
O/N Starters for:
1.UU1250.
2.MG1655 with AMP resistance.

**28.7.16**
Tar:
Re-doing Colony PCR to 5 new colonies.
No positive colonies.
Place bacteria in 30 degrees for 8 hours.

**29.7.16**
Tar:
Re-donig all cloning from start:
PCR to:
1.Tar-adding overlap for gibson assambly.
2.promoter+ blue chromogenic protein .
Purification of PCR products and Concentration check.
Gibson assembly of the products.
Transformation to Top 10 by heat shock to Gibson assembly products.

**31.7.16**
Chemotaxis:
Chemotaxis test in 32C and 35C in both LB and TB to:
1.UU1250 with purple chromogenic protein.
2.UU1250 with Blue chromogenic protein.
3.UU1250 with RFP.
4.UU1250 with Tar + purple chromogenic protein.
5.UU1250 with Tar + blue chromogenic protein.
Tar:
Colony PCR to 5 white colonies.
The positive result in the negative control might be due to the fact the the Top10 bacteria has TAR in its genome.
We should do restriction protocol to verify Tar in the plasmid.????
O/N Starters for the 5 colonies.
Test contamination in PCR ingredients, conclusion: Primers are contaminated with the TAR.

Intein:
Resived 12 primers of intein - Preparing diluted stock 1:10.

The following calendar presents our work in each day.
For more information, press on the colorful buttons. Press again to close.

S.Tar
 FlashLab
 HP & Collaboration
 Other

| Sunday | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
| --- | --- | --- | --- | --- | --- | --- |
|  | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 |  |  |  |

**01.8.16**
Tar:
Colonies verification via restriction.
Glycerol stock and miniprep to positive colonies.
Tar 1 is ready!!
PCR to clone nRBS from miniprep of Tar 1 to get Tar 2 - faild.
O/N Starters to:
1.Tar 1.
2.UU1250.
3.MG1655 with AMP resistance.

Rosetta
finish first Rosetta run with native ligand - Aspartate (calibration of the system).

**02.8.16**
Tar & color:
New PCR to clone nRBS from miniprep of Tar 1 to get Tar 2.
O/N starters to:
1.UU1250 with purple chromogenic protein.
2.UU1250 with Blue chromogenic protein.
3.UU1250 with RFP.
4.UU1250 with Tar + purple chromogenic protein.
5.UU1250 with Tar + blue chromogenic protein.
6.UU1250.
7.MG1655 with AMP resistance.

Chemotaxis:
The microscope malfunctioned. The experiment was canceled.

**03.8.16**
Tar & color:
For color visibility's test - move the following strains:
1.UU1250 with purple chromogenic protein.
2.UU1250 with Blue chromogenic protein.
3.UU1250 with RFP.
4.UU1250 with Tar + purple chromogenic protein.
5.UU1250 with Tar + blue chromogenic protein.
From 37 to 32C and 35C dilution (1:60).

DpnI - for Tar1 +nRBS.
Cleaning from enzymatic reaction.
Phosphorylation.
Ligation of Tar 1 with nRBS.

DNA extraction from 2016 distribution kit: double terminator - TT.
Transformation to Top 10 by heat shock to TT.

**03.8.16**
Transfered the bacteria to a fresh medium and let them grew to about OD=0.9.
Finding the right stimulus concentrations to work with.
Attempted to detected chemotaxis on the chip.
Statistical observation.

**04.8.16**
Tar & color:
Transformation to Top 10 by heat shock to ligation product - Tar 2.
PCR to Tar 1 backbone - open for intein.
Cleaning from PCR.
Checking color visibility's test results - some color was visibal in 35C.

**04.8.16**
Finding the right stimulus concentrations to work with.
Attempted to detected chemotaxis on the chips.
Statistical observation.

**04.8.16**
Collaborate with MadaTech - activity with Mexican high school students.

**07.8.16**
Tar:
Glycerol stock and miniprep to B0015.
Restriction to B0015 with the enzymes: EcoRI & XbaI.

**08.8.16**
Tar:
Restriction for Verification of Tar 2 with the enzyms: Ecor1 & Xba1.
PCR to open Tar2 in 3 different places (as we did in Tar1).

Glycerol stock and miniprep to B0015.
Restriction to B0015 with the enzymes: EcoRI & XbaI.

Intein:
PCR – to amplify ESTR\_LBD from intein gBlock.
Purification of PCR product - we got low concentration.
Gibson assembly to:
1.Intein gBlock with Tar1 in place 1.
2.Intein gBlock with Tar1 in place 2.
3.ESTR\_LBD with Tar1 (only tail).
Transformation to Top 10 by heat shock to all Gibson products.
\*We used the rong consentration.
NarX/PctA:
Gibson assembly on 3 parts: Terminator, 2 parts of PctA gBlocks.
Gibson assembly on 3 parts: Terminator, 2 parts of NarX gBlocks.
Transformation to Top 10 by heat shock to the Gibson assembly products.

**08.8.16**
Meeting with Prof. Moran Berkovichi.

**09.8.16**
Intein:
Gibson assembly de-novo with the correct intein gBlock concentration.
Colony PCR for the Gibson from 8.8.16.
All colonies were negative.

**10.8.16**
NarX/PctA:
Restriction for Verification to:
1.PctA with the enzymes: PstI-HF & SalI-HF.
2.NarX with the enzymes: PstI-HF & ClaI.

**10.8.16**
Skype meeting with Aachen iGEM group.

**11.8.16**
Chemotaxis:
Meeting with Einat Tamar - Help with Chemotaxis.

Tar:
Miniprep to Tar 2.

Intein
O/D Starter to: Top 10 with Estr+Tar1.
PCR to Estr\_LBD from diluted gBlock.
Purification to PCR products.
Gibson assambly to:
1.Tar1 with intein1.
2.Tar1 with intein2.
3.Tar1 –only tail with ESTR\_LBD.
Transformation to Top10 by heat shock to all gibson product.
Miniprep to Estr+Tar1.

NarX/PctA:
Colony PCR to PctA and NarX.
Gibson assambly to:
1.PctA + B0015.
2.NarX + B0015.
Transformation to Top 10 by heat shock to Gibson product.
Starters to PctA and NarX.

Rosetta
Success to filter Rosetta output for the first time with the correct filters.

**11.8.16**
Meeting with Mrs. Shifra Antebi from the Department of Education in Haifa - build an educational program.
Skype meeting with Freiburg.

**12.8.16**
NarX/PctA:
Glycerol stock and miniprep to:
1.PctA.
2.NarX.

**13.8.16**
Tar:
Verification to Tar2 via PCR reaction.

NarX/PctA:
Verification with restriction to:
1.PctA with the enzymes: SalI-HF & PstI-HF
2.NarX with the enzymes: PstI-HF & claI.
Good results.
Starters to: UU1250 with NarX-Tar.

Intein:
Estr+Tar1 verification by restriction enzymes: NcoI-HF & kpnI-HF.
Starter to Top10 Estr+Tar1.
Colony PCR to 4 colonies from 1st Gibson transformation.
No result.

**14.8.16**
NarX/PctA:
Miniprep to NarX.
Transformation to UU1250 by heat shock to:
1.NarX.
2.PctA.

Tar:
PCR to Tar1.

Intein:
Miniprep to Tar1+Est.
PCR to intein gBlock.
Transformation to UU1250 by heat shock to Tar1+Estr.
PCR to intein gBlock again.

**14.8.16**
Meeting with Prof. Moran Berkovichi.

**15.8.16**
NarX/PctA:
PctA + NarX sending to sequence.
Starter to:
1.UU1250 with NarX.
2.UU1250 with PctA.

Tar:
Starters of 2 colonies of Tar 1.

Intein:
Estr\_LBD - sending to sequence.
Gibson assambly to:
1.Intein gBlock+ Tar in place 1.
2.Intein gBlock+ Tar in place 2.
Using PCR product for intein gBlock.
Transformation to Top 10 by heat shock to Gibson intein products.

**15.8.16**
Tzila Davidov birthday!

**16.8.16**
Intein:
Colony PCR to Tar 1 intein 1 and Tar1 intein 2.
Bad results.

Tar:
Glycerol stock to Tar 1.
Miniprep to Tar1.
Verification of Tar 1 with PCR reaction.
Starters to Tar 1.

**17.8.16**
Intein
Starters from colony backup Tar1-intein1 and Tar1-intein2.
Miniprep to starters.

Tar:
Miniprep to Tar1.

NarX/PctA:
Glycerol stock to:
1.UU1250 with PctA.
2.UU1250 with NarX.

**17.8.16**
Skype meeting with Peshawar iGEM group - introduction.
Skype meeting with Aachen iGEM group.

**18.8.16**
Intein:
Miniprep to Estr+Tar1.
Glycerol stock to UU1250 with Estr+Tar1.
Verification to Tar1-intein1 and Tar1-intein2 with Restriction Enzymse: NcoI-HF & XbaI.
Not so clear results.
Starters from colony backup Tar1-intein1 and Tar1-intein2.

Color:
Starters to:
Top 10 with NarX, Top 10 with PctA, Top 10 with Tar1 & Top 10 with Estr-Tar 1.

**19.8.16**
Intein:
Miniprep to: Tar1-intein1 and Tar1-intein2
Verification to:
Tar1-intein1 and Tar1-intein2.
With the enzymes Enzymes: PstI-HF & XbaI.

Color:
Miniprep to:
NarX, PctA, Tar1 & Estr-Tar 1.

**20.8.16**
Chemotaxis:
Starters O/N in TB 30 for:
UU1250, ΔZ, ΔF,UU1250 with Tar 1,UU1250 with PctA & UU1250 with NarX.

**21.8.16**
Chemotaxis:
Swarming assay on 6X2 plates (duplicat) to:
UU1250, ΔZ, ΔF, Tar 1, PctA & NarX.
O/N Starters in TB, 30C to:
ΔZ ,ΔF,UU1250,UU1250 with ESTR & MG1655.

Intein:
Transformation to UU1250 by heat shock to:
1.Tar 1 intein 1.
2.Tar1 intein 2.
O/N Starters to:
 Top 10 with Tar1 intein 1 and Top 10 with Tar1 intein 2.
PCR to Tar 1 backbone to:
1.Open for intein in place 1.
2.Open for intein in place 2.
Tar:
PCR to:
1.rhlR.
2.Tar 1 plasmids.
3.Tar 2 plasmids.
starter to Tar2

**22.8.16**
Chemotaxis:
Chemical in Plug assay to: ΔZ ,ΔF,UU1250 and MG1655.
O/N Starters in TB to:
ΔZ , ΔF & UU1250 with Tar 1+ NarX.

GFP:
DNA extraction from 2016 distribution kit: part E0040.
Transformation to Top 10 by heat shock to part E0040.

intein:
Starters to:
UU1250 with Tar1 intein1 ,UU1250 with Tar1 intein2 & Top10 with ESTR-Tar1
Glycerol stock to UU1250 with tar1 intein1 & UU1250 with tar1 intein 2.
Miniprep to ESTR-Tar1.

Color:
Transformation to Top 10 by heat shock to p15A.

Tar:
Cleaning PCR products.
Glycerol stock and miniprep for Tar 2.
Restriction to:
Tar1, Tar2 & rhlR.
With the enzymes: XbaI & BglII.
Cleaning from gel.

**22.8.16**
Meeting with Prof. Meytal Landau - help with visualization software for collaboration with Aachen iGEM group.

**23.8.16**
Chemotaxis:
Chemical in Plug assay to: ΔZ & ΔF.
O/N Starters in TB, 30C to:
UU1250 with Tar1+PctA,UU1250 Tar1+NarX,UU1250 with Estr-Tar1, ΔF and ΔZ.

GFP:
Starter to GFP.

Color:
PCR to:
PctA backbone, NarX backbone, Tar & Tar 1.
Cleaning from PCR.
PCR to Tar from Gibson - no results.
Gibson Assembly with the PCR product without purification - Tar1 & NarX.
Transformation to Top 10 by heat shock to Gibson product.
Gibson Assembly from purified PCR products to:
1. Tar1 with NarX.
2. Tar1 wuth PctA.
Transformation to Top 10 by heat shock to Gibson product.
O/N Starter to p15A Kan antibiotic.
Tar:
O/N Starters to UU1250 with Tar2.
Ligation of:
1.rhlR with Tar 1.
2.rhlR with Tar 2.
Transformation to Top 10 by heat shock to Ligation product.
Sending Tar1 & Tar2 for sequencing.

**23.8.16**
Skype meeting with Aachen iGEM group.

**24.8.16**
GFP:
PCR to GFP part.
Cleaning from PCR.
PCR to Tar in two stages.

Color:
Colony PCR to rhlR - Tar.
Starters to positive colony.

Tar:
Colony PCR to rhlR - Tar.
Starters to all 3 colonies.

Chemotaxis:
swarming assay after O/N in 37C or 30C to:
ΔF, ΔZ,UU1250 & PctA.
we could see results in two Temperatures.
Swarming assay to Tar2
Chemical in Plug assay to:
ΔF, ΔZ & UU1250 with Tar+ESTR.

Rosetta
Create automation script to run the whole protocol.

**24.8.16**
Skype meeting with Peshawar iGEM group - Help with Mathematical Model.
Skype meeting with Aachen iGEM group.

**25.8.16**
GFP:
PCR of TAR again but with one stag.

Intein:
Sending ESTR-Tar1-1 for sequencing again.

Tar:
Glycerol stock and Miniprerp to all 3 colonies.
Restriction to Tar with the enzymes: NcoI & KpnI .

**25.8.16**
Skype meeting with Peshawar iGEM group - Help with Mathematical Model.

**26.8.16**
Chemotaxis:
O/N Starters at 30C for:
ΔZ ( LB and TB) ,ΔF ( LB and TB) ,Tar1+PctA ( LB and TB),Tar1+intein 1 (TB) & Tar 1+intein 2 (TB).

GFP:
Gibson assembly of Tar and GFP.

Tar:
PCR to Tar2/3/4 on pSB1C3.
Cleaning from PCR.
Restriction to TAR 3/4(CM resistance) with the enzymes: XbaI & Bgl II.

**27.8.16**
Tar:
Cleaning from restriction.
Ligation of Tar 3 and Tar 4.
Transformation to Top 10 by heat shock to:
1.Tar-GFP(AMP).
2.Tar 2(CM).
3.Tar 3 (CM).
4.Tar 4 (CM.
Transformation to UU1250 by heat shock to Tar 1+ promoter+ blue chromogenic protein (CM).

GFP:
Transformation to Top 10 by heat shock to Gibson product.

Chemotaxis:
Starters to ΔZ, ΔF, UU1250,UU1250 with Tar 1+Narx,UU1250 with Tar+Estr, K12+GFP

**28.8.16**
Tar:
Transformation to UU1250 by heat shock to Tar 4 (AMP).

Intein:
Sending Tar1 intein 1 and Tar1 intein 2 for sequencing.

GFP:
Colony PCR to tow colonies.
Starters to two colonies.

Chemotaxis:
Chemical in plug assay for: ΔZ ,ΔF and UU1250 with ESTR-Tar1.

**28.8.16**
Meeting with Eng. Kfir Cohen.
Meeting with PhD student Jonathan Avesar

**28.8.16**
Meeting with Hagit - CEO of eco99 radio station and marketing expert.
New ideas about marketing out product to the public.

**29.8.16**
Tar:
Colony PCR to Tar 3 and Tar 4.
Miniprep to Tar 2 (CM) ,Tar 3 (CM) and Tar-GFP (CM).
Restriction to:
1.Tar 3 with the enzymes: NcoI-HF & KpnI-HF.
2.Tar-GFP with the enzymes: NcoI-HF & XbaI.
PCR to open rhlR.
Transformation to Top 10 by heat shock to:
1. promoter+ blue chromogenic protein .
2.promoter+ purple chromogenic protein .
Transformation to UU1250 by heat shock to:
1.Tar1 (CM).
2.Tar2 (CM).
PCR to open Tar 1 (CM) and Tar 2 (CM) for rhlR.
Cleaning from PCR.

Intein:
O/N Starters to:
UU1250, UU1250 with Tar1, UU1250 with Tar1+ intein1 & UU1250 with Tar1+intein 2.

**30.8.16**
Tar:
Cleaning Tar 1 from PCR.
Glycerol stock to UU+Tar 4 (AMP).
Restriction to:
1.Tar 1 opened.
2.opened rhlR.
With the enzymes: Xbal & Bgl II.
Cleaning from restriction.
Transformation to UU1250 by heat shock to p15A and Tar 1 (CM).
Starters to:
UU1250 with Tar 1, UU1250 with Tar 2,UU1250 with Tar 1 +promoter+ blue chromogenic protein (CM+AMP), Top 10 with Tar 1, Top 10 with Tar 2,Top 10 with promoter+ purple chromogenic protein ,Top 10 with promoter+ blue chromogenic protein (AMP).

Intein:
PCR to Tar 2 backbone - open for intein in place 1 and place.
Intein incubation protocol.

Chemotaxis:
O/N Starters in TB to: UU1250 with narX, UU1250 with Tar1,UU1250 with ESTR, ΔF, ΔZ and UU1250.

**30.8.16**
Meeting with Liat Reter, administration manager in the faculty of Biotechnology and Food Engineering - about the national Israeli Mini Jamboree.

**31.8.16**
Tar:
Miniprep to Tar 1 and Tar 2.
Glycerol stock to UU1250 with Tar 1 (CM) and UU1250 with Tar 2 (CM).
Ligation of rhlR with Tar3 (CM).
Transformation to Top 10 by heat shock to:
1.Tar 3.
2.Tar 3 - new rhlR.

Chemotaxis:
Test of new small plats for farther use.
Chemical in Plug assay to: UU1250 with narX, UU1250 with Tar1,UU1250 with ESTR, ΔF, ΔZ and UU1250.

**31.8.16**
Skype meeting with Peshawar iGEM group - Help with Human Practices.

The following calendar presents our work in each day.
For more information, press on the colorful buttons. Press again to close.

S.Tar
 FlashLab
 HP & Collaboration
 Other

| Sunday | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | 1 | 2 | 3 |
| --- | --- | --- | --- | --- | --- | --- |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| 25 | 26 | 27 | 28 | 29 | 30 |  |

**01.9.16**
Tar:
Colony PCR to rhlR+Tar.
Starters to:
UU1250 with Tar 1+ promoter+ blue chromogenic protein (CM+AMP),UU1250 with Tar 1 (CM),UU1250 with Tar 2 (CM), ΔZ, ΔF, Top 10 with promoter+ purple chromogenic protein & Top 10 with promoter+ blue chromogenic protein (AMP).

**01.9.16**
First 3D printed mold for the chip.

**01.9.16**
Meeing with Dr. Fabian Glaser - Homology modelling for Aachen.
Skype meeting with Freiburg-mathematical model.
Meeting with Orit the responsible of education in the scientific kindergarten.

**02.9.16**
Tar:
Miniprep for Tar 3 (CM).
New glycerol stock to Top 10 with promoter+ blue chromogenic protein & Top 10 with promoter+ purple.
Swarming assay to UU1250 with Tar 1+promoter+ blue chromogenic protein (CM+AMP)[the starter is blue!!], UU1250 with Tar 1 (CM), UU1250 with Tar 2 (CM), ΔZ & ΔF.
Restriction of Tar 3 (CM) with the enzymes: XbaI & Spel.

**03.9.16**
Tar:
Transformation to UU1250 by heat shock to Tar 3.

**04.9.16**
Tar:
Glycerol stock for UU1250 with Tar 3 (CM).
PCR to open Tar 2 (CM) for Tar 4.

Intein:
PCR to amplify intein gBlock 12 cycles.
Cleaning from gel.
PCR to open Tar 1 and Tar 2 for intein 1, intein 2 and Tar 2 for ESTR.

Chemotaxis:
O/N Starters to: UU1250 with Tar 3 (CM),UU1250 with Tar 1 (CM), UU1250 with Tar 2 (CM), UU1250 with Tar4 (Amp),ΔZ and ΔF.

**05.9.16**
Intein:
Concentration check to PCR purification product.
PCR to open Tar 1 and Tar 2 tail for ESTR.
Cleaning PCR product.
PCR to amplify intein gBlock 25 cycles.
Cleaning from gel.

GFP:
PCR to open Tar 1 (CM) and Tar 2 (CM) for GFP.
Cleaning Tar1 from PCR.
PCR for GFP.
PCR to open Tar 1 (CM) and Tar 2 (CM) for GFP again.

Chemotaxis:
1:50 dilution of starters to a final volume of 5ml (Tar4-Amp didn’t grow).
3 hour incubation in 30C with inducer (0µM ,1µM and 50µM).
Swarming assay performance - experiment didn’t work!
microscope testing for ΔZ, ΔF & UU1250

Rosetta
Run Rosetta design against new ligands: Histamine, Glocuse, Lactose, Rohypnol, Ampicilin.

**06.9.16**
Tar:
PCR to amplify Tar 3.

Intein:
Gibson assembly to:
1.Tar 1 in place 1+ intein gBlock.
2.Tar 1 in place 2+intein gBlock.
3.Tar 2 in place 1+ intein gBlock.
4.Tar 2 in place 2+ intein gBlock.
5.Tar 2+ ESTR.

GFP:
PCR to open Tar 1 (CM) and Tar2 (CM) for GFP again.
Cleaning from PCR using another kit.
Gibson assembly to:
1.Tar 1 with GFP.
2.Tar 2 with GFP.
Transformation to UU1250 by heat shock to all gibson products.

Chemotaxis:
O/N Starters for: ΔZ, UU1250 with PctA & UU1250.
Microscope testing for ΔZ, UU1250, UU1250 with Tar1 & UU1250 with NarX.

**06.9.16**
Skype meeting with Aachen iGEM group.

**07.9.16**
Intein:
Colony PCR to UU1250 with Tar 1 intein 1, UU1250 with Tar 1 intein 2, UU1250 with Tar 2 intein 1 & UU1250 with Tar 1 intein 2 (for 48 colonies!!).
Chemotaxis:
Swarming assay to:
 ΔZ ,UU1250 with PctA & UU1250.

Rosetta - Histamine:
Design primers for cloning Histamine variants.

**08.9.16**
Intein:
Colony PCR to promising looking colonies with higher annealing temperature - bed results.

GFP:
Colony PCR for Gibson products (4 colonies from each kind).

Chemotaxis:
Microscope testing for UU1250 ,UU1250 with Tar1 and UU1250 with Hisamine variants: 4,6,7,8,9 & 10.

**11.9.16**
Intein:
O/D Starters to the promising colonies & Top10 with Tar2+ESTR.
Miniprep to the starters.

GFP:
O/N Starter for the posetive colonies.

**12.9.16**
Intein:
Restriction verification for:
1. Tar 2 + ESTR with the enzymes: HindIII-HF.
2. Tar + intein (in place 1 and 2) with the enzymes: KpnI-HF.
PCR to open Tar 1 and Tar 2 in place 1 & 2.

GFP:
Dilution of starters 1:100 to TB at 30C for 5 hour.
MAX for all semples in order to check fluorescence.
Microscope testing for Top 10 with Tar-GFP.

**12.9.16**
Skype meeting with Aachen iGEM group.

**12.9.16**
Educational activity at Scientific kindergarten.

**13.9.16**
Intein:
Sending Tar 2 +ESTR for sequencing.

Chemotaxis:
Swarming assay on BA to: UU1250, ΔZ,UU1250 with Tar1 and UU1250 with PctA.

**13.9.16**
Meeting with Prof. Moran Berkovichi.

**13.9.16**
Educational activity at Scientific kindergarten.

**14.9.16**
Intein:
Gibson assembly from new gBlock ,intein divided to 2 parts, to:
1.Tar 1 in place 1+ intein gBlocks.
2.Tar 1 in place 2+intein gBlocks.
3.Tar 2 in place 1+ intein gBlocks.
4.Tar 2 in place 2+ intein gBlocks.
Transformation to Top 10 by heat shock to gibson products.

Rosetta - Histamine:
Primers arrived!! Dilution and preparation.
First PCR to His variant: 1, 2, 3, 4, 11.

**14.9.16**
Educational activity at Scientific kindergarten.

**15.9.16**
Intein:
Verification with colony PCR for 4 colonies from each type.

Rosetta - Histamine:
Gel electrophoresis - variant 1, 2 & 3 were good!
Purification for His variant 1, 2 & 3.
DpnI to His variant 1, 2 & 3.
Purification for His variant 1, 2 & 3.
First PCR to His variant 4, 5, 6, 7, 8, 9, 10 & 11.
DpnI for His variant 4,5,6,7,8,9,10 & 11.
Purification for His variant 4,5,6,7,8,9,10 & 11.
Phosphorylation for His variant 1-11.
Ligation.
Transformation to Top 10 by heat shock to His Variant 1-11.

Chemotaxis:
Swarming assay on BA to:
UU1250, ΔZ, UU1250 with Tar1 & UU1250 with PctA.
Swarming assay to:
UU1250 with Tar2+GFP, UU1250 with Tar1+GFP, ΔZ & UU1250.

**15.9.16**
Experiment with Trap & Track assay at esti segal lab on: ΔZ

**16.9.16**
Rosetta - Histamine:
Starters for His variant 1A,1B,2A,2B,3A,3B,6A,6B,8A,9A,11A,11B.

**17.9.16**
Rosetta - Histamine:
Miniprep for His variant 1A,1B,2A,2B,3A,3B,6A,6B,8A,9A,11A,11B.
Concentration check.

**18.9.16**
Intein:
Verification with Colony PCR for 4 other colonies from each type - negative results.
Gibson assembly from new gBlock second chance (intein divided to 2 parts):
1.Tar 1 in place 1+ intein gBlocks.
2.Tar 1 in place 2+intein gBlocks.
3.Tar 2 in place 1+ intein gBlocks.
4.Tar 2 in place 2+ intein gBlocks.
Transformation to Top 10 by heat shock to gibson product.
O/N Starters to 2 colonies.

Rosetta - Histamine:
Send for sequencing His variant: 1A, 2B, 3B, 6B, 9A, 11B.

Chemotaxis:
Swarming assay to : ΔZ, UU1250, UU1250 with Tar1+GFP, UU1250 with Tar2+GFP, UU1250 with Tar+YFP, UU1250 with Tar1+CheZ & UU1250 with Tar1+promoter+ blue chromogenic protein .
Microscope testing - UU1250, UU1250 with Estr-tar & UU1250 with Tar1.

**18.9.16**
 Testing color on commercial Ibidi chip

**19.9.16**
Intein:
Miniprep to the starters of 2 colonies.
verification via restriction enzyme with: KpnI-Hf.
Colony PCR to the last colony back up with different primers.
Agine negative results.
PCR to intein original gBlock.

Chemotaxis:
Microscope testing for: UU1250 with Tar1, UU1250 with Tar2, ΔZ & UU1250

**19.9.16**
Educational activity at Scientific kindergarten.

**20.9.16**
Intein:
PCR to amplify old gBlock.
Cleaning from gel.

GFP:
PCR to open PctA for GFP.
Purification from PCR.
Microscope testing for Top 10 with Tar-GFP.

Rosetta - Histamine:
Sequencing is back!! His variant 9 and 6 were as expected.
After PCR1, 5+6 are duplicates so we can use 6 as a template for 5 seem go's to 8+9 so we can use 9 as a template for 8.

Chemotaxis:
Swarming assay for Tar 1 (CM) and Tar 2 (CM).
Microscope testing for: UU1250 with Tar1 & UU1250 with Tar2.

**20.9.16**
Graphic work for the iGEM Technion Mini Jamboree.
Educational activity at Scientific kindergarten.

**21.9.16**
GFP:
PCR to open NarX and ESTR.
Purification from PCR.

Rosetta - Histamine:
First PCR to His variant 1-4,7, 10, 11.
Second PCR to His variant 5, 6, 8 and 9.
Purification for all 11 variants.
DpnI for all 11 variants.
Purification for all 11 variants.
Phosphorylation for all 11 variants.
Ligation.
Transformation to Top 10 by heat shock to all 11 variants.
Grow well: His\_1,2,3,4,6,7,8,10,11.
O/N Starters for His 11 for sequencing.

**21.9.16**
Trap & Track assay at esti segal lab on:
ΔZ & UU1250 with Tar1.

**21.9.16**
Shopping for the iGEM Technion Mini Jamboree.
Educational activity at Scientific kindergarten.

**21.9.16**
Asif Gil birthday!

**22.9.16**
GFP:
Gibson assembly to:
1.Pct A with GFP.
2.NarX with GFP.
3.ESTR with GFP.

Rosetta - Histamine:
Miniprep to His 11.
Concentration check.
Send to sequencing His 11 (triplicate).

Chemotaxis:
Microscope testing for: UU1250 with NarX, UU1250, UU1250 with PctA & UU1250 with Tar-Estr.
Meeting with Einat Tamar - Swarming assays troubleshooting.

**22.9.16**
 Meeting with PhD student Einat Tamar.

**22.9.16**
 Israeli Researchers Night 2016 in the MadaTech & in Tel-Hai college.

**23.9.16**
Chemotaxis:
Microscope testing for: UU1250 with PctA, UU1250 with Tar1, UU1250 with Tar2 & UU1250.

**24.9.16**
Rosetta - Histamine:
Miniprep for PCR 1 His variant: 1-4, 7,10 & 11.
Mini prep for PCR 2 His variant: 6 & 8.
Send to sequencing His variant: 1-4,6-8 & 10.

Chemotaxis:
Microscope testing for: ΔZ, UU1250 with PctA & UU1250 with Tar1 + purple chromogenic protein

**25.9.16**
Intein:
PCR to open Tar 1 and Tar 2 for blunt ligation with intein in 2 places.
Cleaning from PCR.

Rosetta - Histamine:
Transformation to Top 10 by heat shock to His variant: 5 & 9 (PCR 2).
Transformation to UU1250 by heat shock to His variant: 6 & 8 (PCR 2).

Chemotaxis:
Documentation of swarming assays at Prof. Roy Kishony’s lab

**25.9.16**
Working in Prof. Moran Berkovichi’s lab-printing chips.
Trap & Track assay at esti segal lab on: ΔZ

**25.9.16**
Skype meeting with Peshawar iGEM group - Help with Cloning.
Sincopa pub quiz.

**26.9.16**
Intein:
Phosphorylation to whole intein gBlock.
Ligation with Tar 1 or Tar 2.

GFP:
O/N Starter to NarX.

Rosetta - Histamine:
Sequencing for 11 is back! we succeed to insert the desirable mutation.

**26.9.16**
 Working in Prof. Moran Berkovichi’s lab-printing chips.

**26.9.16**
Educational activity at Scientific kindergarten.

**27.9.16**
Intein:
Transformation to Top 10 by heat shock to ligation product.
GFP:
Miniprep for NarX.
PCR to NarX.

Rosetta - Histamine:
PCR 2 for His variants: 1-5, 7 & 9-11.
Only His\_11 worked.
Cleaning and DpnI for His 11.
PCR 2 again for His variant:1-5,7,9 & 10.Same conditions as first time

Chemotaxis:
Microscope testing for Histamine variant 6 & histamine variant 8.

**27.9.16**
Educational activity at Scientific kindergarten.

**28.9.16**
Intein:
Colony PCR to blunt ligation.
 Only one colony looked promising.
Second try to find positive colonies with colony PCR.
Two more colonies looked promising.
Starters for 3 colonies to check orientation.

GFP:
Colony PCR to 4 colonies.

Rosetta - Histamine:
PCR 2 for His variant: 1 & 2, with grad of temperature for each.
PCR 2 for His variant: 3,4,5,7,9 & 10.
Purification & DpnI for His variant: 1-5,7,9 & 10.

**28.9.16**
Trap & Track assay at esti segal lab on: ΔZ & UU1250

**28.9.16**
Preparations to the 2nd Annual iGEM Technion Mini Jamboree.
Educational activity at Scientific kindergarten.
Skype meeting with Aachen iGEM group.

**29.9.16**
GFP:
Glycerol stock to NarX-GFP

Rosetta - Histamine:
Purification for all 9 His variant: 1-5,7 & 9-11.
Phosphorylation.
Ligation.
Transformation to UU1250 by heat shock to His variants:1-7 & 9-11.
His variant 5,7,11 didn't grow at all. The rest grew vey well!

**29.9.16**
Trap & Track assay at esti segal lab on: ΔZ.
Chip test for UU1250 with Tar1 + purple chromogenic protein.

**29.9.16**
The 2nd Annual iGEM Technion Mini Jamboree.

**30.9.16**
Rosetta - Histamine:
O/N Starter for His variants: 1-4,7,9,10 in duplicate.
Transformation to UU1250 by heat shock for His variant: 5,7 & 11 (again).
Variants 7 & 11 grow. Variant 5 didn't grow.

The following calendar presents our work in each day.
For more information, press on the colorful buttons. Press again to close.

S.Tar
 FlashLab
 HP & Collaboration
 Other

| Sunday | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  | 1 |
| --- | --- | --- | --- | --- | --- | --- |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| 23 | 24 | 25 | 26 | 27 | 28 | 29 |
| 30 | 31 |  |  |  |  |  |

**01.10.16**
Intein
Restriction to verifiad the orientation of the insert to 3 colonies, with the enzymes: KpnI-HF.
One "positive" result to Tar 1 intein in place 2.
Transformation to UU1250 by heat shock to Tar 1 intein 2.

Rosetta - Histamine:
Miniprep for His variants:1-4 & 9-10.
O/N Starters His variants: 7 & 11.

**01.10.16**
Diffusion Experiments-testing the model.
Trap & Track assay at esti segal lab on: ΔZ

**02.10.16**
Rosetta - Histamine:
Miniprep for His variants: 7 & 11.
PCR2 for His variant 5 with grad.
Purification & DpnI for His variant 5 61.1°C.
Purification & Phosphorylation for His variant 5.
Ligation for His variant 5.

**03.10.16**
Intein
O/N Starter to: Top 10 with Tar 1 intein 2 & UU1250 with Tar 1 intein 2.

Rosetta - Histamine:
Transformation to UU1250 by heat shock to His variant 5.
Didn't grow.

**03.10.16**
Trap & Track assay at esti segal lab on: ΔZ

**04.10.16**
Intein
Miniprep to Tar 1 intein 2.

Intein incubation protocol.

Rosetta - Histamine:
Send to sequencing His variant 1,2,3,4,7,9,10 & 11.

**05.10.16**
Intein
Send Tar 1 intein 2 to sequencing.

Rosetta - Glucose
PCR for all 8 Glu variants.
Only Glu 7 worked! Repeat PCR for rest of variants.
PCR for Glu variants: 1-6 & 8.
Nothing worked...
PCR for Glu variants again: 1-6 & 8.
This time we used GC buffer and checked the TM in Neb calculator. We decided to use 72°C for the annealing step in all variants.

Chemotaxis:
Documentation of swarming assays at Prof. Roy Kishony’s lab.

**06.10.16**
Intein
once again only part of the intein got in.

Rosetta - Histamine:
PCR3 for His variant: 2 & 3.

Rosetta - Glucose
2-stage PCR for Glu variants: 1,2,3,4,5,6,7,8.
This time we used 3 different concentration of DMSO(3%, 5%, 10%) and a lower concentration of the primers (0.25µM). We also used Q5 polymerase.
Purification, DpnI, Purification, Phosphorylation & Ligation for Glu variants: 1,2,3,4,5,6,7 & 8.
Transformation to Top 10 by heat shock of Glu variants: 1-8.
Only Glu\_7 growth.

**06.10.16**
Chip test for UU1250 with Tar1 + purple chromogenic protein.

**07.10.16**
Chemotaxis:
Microscope testing for: UU1250 with Tar1, UU1250 with Tar2, UU1250 & ΔZ

**07.10.16**
Testing the quantitive system for bacteria concentration.
Trap & Track assay at esti segal lab on: ΔZ

**08.10.16**
Rosetta - Glucose
Repeat phosphorylation & ligation for Glu variants: 1,2,3,4,5,6 & 8.
O/N Starter for Glu variant 7 in duplicat.

**08.10.16**
Testing the quantitive system for bacteria concentration.

**09.10.16**
Rosetta - Histamine:
Send for sequencing His variants: 1 ,3 & 11 in duplicat and 10.

Rosetta - Glucose
Miniprep for Glu variant 7 in duplicat.
Send to sequencinc Glu variant 7 in duplicat.
Transformation to UU1250 by heat shock to Glu variants: 1,2,3,4,5,6,8

Part submission:
Resived primers for Basic part submission.
PCR to parts:
PctA, PctA-GFP, Tar-GFP & Histamine 9.
The wrong bufer was used. No results.

**10.10.16**
Part submission:
PCR to parts:
PctA, PctA-GFP, Tar-GFP & Histamine 9.
We got our Basic parts.
Cleaning from PCR.
Restriction to Backbone with the enzymes: EcorI & PstI.
Ligation to each part with Backbone.

Gibson assambly to each part with Backbone.

Transformation to Top 10 by heat shock to Gibson assambly and Ligation products.

**10.10.16**
Meeting with Prof. Roee Kishony.
Trap & Trak to: UU1250 with Tar 1

**11.10.16**
Part submission:
Colony PCR to: PctA, Histamine 9 & Tar-GFP.

**12.10.16**
Part submission:
O/N Starters to positive colonnies of: PctA, Histamine 9 & Tar-GFP.

**13.10.16**
GFP - Histamine:
PCR to open Histamine variant 9.

Chemotaxis:
Microscope testing for: UU1250 with Histamine 9 & UU1250 with Tar 1.
Swarming assay to Glucose variants: 1-6 & 7.
 Experiment fail

Part submission:
Miniprep to: PctA, Histamine 9 & Tar-GFP Basic parts
O/N starter to composite parts: PctA & Tar 1.

**13.10.16**
Trap & Trak to: UU1250.

**14.10.16**
GFP - Histamine:
Cleaning from PCR.
Gibson assambly to Histamine 9 with GFP.
Transformation to Top 10 by heat shock to Gibson assambly product.
Colony PCR to His\_9+GFP
O/N starters in LB and TB for positive colonies.
Chemotaxis:
Microscope testing for: UU1250 with Histamine 9.

**15.10.16**
GFP - Histamine:
Colony PCR to His\_9+GFP
O/N starters in LB and TB for positive colonies.

**16.10.16**
GFP - Histamine:
Miniprep for His\_9+GFP.

**17.10.16**
Part submission:
Part Shipment to iGEM HQ.

**17.10.16**
Naor Garnik birthday!

**19.10.16**
Wiki freeze!!!!

**25.10.16**
Shiran Sarig birthday!

**27.10.16**
iGEM 2016 Giant Jamboree!

**28.10.16**
iGEM 2016 Giant Jamboree!
Our presentation:
Room 306, 14:00.

**29.10.16**
iGEM 2016 Giant Jamboree!

**30.10.16**
iGEM 2016 Giant Jamboree!

**27.10.16**
iGEM 2016 Giant Jamboree!

...

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