iGEM 2022 — Team technion-israel

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{% block title %}Notebook{% endblock %}
{% block lead %}This is our notebook of the whole project, in it you can find the progress we made each week from the initial brainstorming sessions, and up to the last stages. {% endblock %}
{% block page\_content %}

## January

### [Week 1 - 2/1](#collapse1)

First meeting - kickoff and introduction to iGEM.

### [Week 2 - 9/1](#collapse11)

Introducing iGEM – starting to read about old projects.s

Dividing into groups and each group presented an old winning iGEM project.

### [Week 3 - 16/1](#collapse21)

Exploring the structure of iGEM competition.

### [Week 4 - 23/1](#collapse31)

Exploring the structure of iGEM competition.

### [Week 5 - 31/1](#collapse32)

Exam period – a break from iGEM 🙁

## February

### [Week 6 - 6/2](#collapse2)

### General

Brain storming of 6 ideas to the project and writing 1 paper on each one.

### [Week 7 - 13/2](#collapse12)

### General

Brain storming of 6 ideas to the project and writing 1 paper on each one.

### [Week 8 - 20/2](#collapse22)

### General

Brain storming of 6 ideas to the project and writing 1 paper on each one.

### [Week 9 - 27/2](#collapse33)

### General

Brain storming of 6 ideas to the project and writing 1 paper on each one.

## March

### [Week 10 - 6/3](#collapse3)

Brainstorming.

### [Week 11 - 13/3](#collapse13)

Brainstorming.

### [Week 12 - 20/3](#collapse23)

### General

3 ideas presentation – and writing 5 page paper on each one and first presentation.

### [Week 13 - 27/3](#collapse34)

### General

Second presenting of the 3 projects.

## April

### [Week 14 - 3/4](#collapse35)

Deciding on an idea.

### [Week 15 - 10/4](#collapse14)

Deciding on an idea.

### [Week 16 - 17/4](#collapse24)

### General

iGEM crash course on synthetic biology.

### [Week 17 - 24/4](#collapse36)

### General

Final presentation and idea decision.

## May

### [Week 18 - 1/5](#collapse5)

Deciding on groups.

### [Week 19 - 8/5](#collapse15)

Planning future work.

### [Week 20 - 15/5](#collapse25)

Planning future work.

### [Week 21 - 22/5](#collapse37)

Planning future work.

### [Week 22 - 29/5](#collapse38)

### Model

• We chose relevant equations for the kinetics model.

## June

### [Week 23 - 5/6](#collapse6)

### Model

• We found relevant constants for the kinetics model.

### [Week 24 - 12/6](#collapse16)

### Model

• finish the basic kinetic model.

### Human Practices

• 15.6 - hair donation event.

### [Week 25 - 19/6](#collapse26)

### Model

• Meeting with dry lab team of Athens group.

### Wet lab

##### Oracell

• Consulting Amir Argoetti regarding measurement of decursin (Luciferase assay).

### [Week 26 - 26/6](#collapse39)

Exam period – a break from iGEM 🙁

## July

### [Week 27 - 3/7](#collapse7)

Exam period – a break from iGEM 🙁

### [Week 28 - 10/7](#collapse17)

Exam period – a break from iGEM 🙁

### [Week 29 - 17/7](#collapse27)

Exam period – a break from iGEM 🙁

### [Week 30 - 24/7](#collapse40)

Exam period – a break from iGEM 🙁

### [Week 31 - 31/7](#collapse41)

Starting the iGEM summer!

### General

• Started working on the wiki.

### Human Practices

• Staring to work on human practice

### Wet lab

• Ordering the parts and plasmid.

## August

### [Week 32 - 7/8](#collapse8)

### General

• Working on the mini jamboree.

• Interview with Dr. Eid, a former social worker Rambam hospital

• Meeting with Assistant Prof. Omer Yehezkeli

• Meeting with Prof. Ayelet Fishman

### Human Practices

• Interview with Dr. Eid, a former social worker Rambam hospital.

### Model

• Expansion of the basic model so that it will be more accurate.

### Wet lab

##### POC

• Meeting with Prof. Ayelet Fishman discussing HPLC of metabolites

• Meeting with Omer Yehezkeli, Ph.D. discussing NMR of metabolites

##### Cloning

• Multiple Starters of A133 and pTRE.

• Transformation of Pet9d to top10 cc (we got an Eppendorf of this plasmid from a lab in the faculty).

• Glycerol stocks of all plasmids.

• Miniprep (plasmid extraction) of A133, Pet9d and pTRE plasmid from starters.

Nanodrop results:

|  | A133 | | | Ptre | Pet9d | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Conc. [ng/μl] | 247.1 | 80 | 486 | 448.9 | 208.1 | 225 |
| 260/280 | 1.87 | 1.87 | 1.97 | 1.87 | 1.88 | 2.09 |
| 260/230 | 2.04 | 1.63 | 2.12 | 2 | 1.95 | 1.96 |

Some of the minipreps were done using multiple starters on one minicolumn in order to increase the final concentrations.
The reason behind the multiple minipreps of each plasmid is to make sure we have enough to send for sequencing, and continue on working with them according to our designs.

• Sent plasmids for sequencing:

A133, Pet9d and pTre match their maps.

• Digestion of A133 plasmid by the restriction enzymes NdeI, AgeI to get it ready for P2A ligation.

• Preparation of low temp 1.5% gel and loading of digested A133 – unsuccessful run because the gel dissolved, so we repeated the digestion of A133, and run in low temp 1.5% gel after cooling it.

The wanted fragment is the backbone (3974bp), we cut it from the gel.

![Digested A133 at Low temp.](https://static.igem.wiki/teams/4179/wiki/notebook/fig-1.png)

![Digested A133 at Low temp. after cut](https://static.igem.wiki/teams/4179/wiki/notebook/fig-2.jpg)

Figure 1: Gel electrophoresis in low temp gel of A133 before and after cutting 11.08.22

Cleaned using Wizard kit.

Nanodrop results:

|  | Digested A133 |
| --- | --- |
| Conc. [ng/μl] | 47 |
| 260/280 | 1.93 |
| 260/230 | 0.09 |

### [Week 33 - 14/8](#collapse18)

### General

• Photo shoot day.

• Launch our Tik-Tok education page.

• Meeting with Prof.Roee Amit.

• Meeting with Shir Shaked (Graphic Design).

### Wet lab

• Digestion of 5 Gblocks of prenyltransferases: PsPT1, FcPT1, UDT, Chimera1, Chimera2, using the restriction enzymes NdeI, NheI.
We accidentally switched between the binding and washing solution.
We had to repeat this step in the upcoming weeks.

• Additional starter and miniprep pf Pet9d.

Nanodrop results:

|  | Pet9d |
| --- | --- |
| Conc. [ng/μl] | 62.7 |
| 260/280 | 1.97 |
| 260/230 | 1.67 |

• Starters of CMV and K133 plasmids, and miniprep.

Nanodrop results:

|  | CMV | K133 |
| --- | --- | --- |
| Conc. [ng/μl] | 311.5 | 122.5 |
| 260/280 | 1.89 | 1.95 |
| 260/230 | 2.07 | 1.78 |

### [Week 34 - 21/8](#collapse28)

### Human Practices

• Pharmaseed tour.

### Model

• Interpretation of the model results & diffusion model.

• Meeting with Athens about modeling.

### Wet Lab

• Isolation plating of petduet and Luciferase plasmids from agar stock.

• Preparation of starter of petduet and Luciferase plasmids.

• Glycerol stock of petduet and Luciferase plasmids.

• Miniprep of petduet and Luciferase plasmids.

Nanodrop results:

|  | Petduet | Luciferase |
| --- | --- | --- |
| Conc. [ng/μl] | 350.7 | 786 |
| 260/280 | 2.08 | 1.89 |
| 260/230 | 2.28 | 2.31 |

• Sent petduet, K133 and Luciferase plasmids for sequencing, had in issue during the process, we repeat this step in following weeks.

• Did reverse PCR of petduet to remove ximE– due to miscalculation of elongation time, we got unsuccessful gel run result (desired product + additional byproducts):

![](https://static.igem.wiki/teams/4179/wiki/notebook/igem-no-xime-bad-first-result-24-08-22-1-45min-elongation.jpg)
Figure 2: Gel electrophoresis of reverse PCR to remove ximE from petduet 24.08.22

• Another reverse PCR to remove ximE with corrected elongation time (4 minutes), with successful results, no\_ximE\_petduet, (1kb ladder neb):

![](https://static.igem.wiki/teams/4179/wiki/notebook/petduet-no-xime-250822.jpg)
Figure 3: Gel electrophoresis of reverse PCR to remove ximE from petduet 25.08.22

Desired length: 7990 bp.

### [Week 35 - 28/8](#collapse42)

### Human Practices

• Volunteering with the oncology department for children.

• Meeting with Team Latvia

### Model

• We started working on the Mutations model

### Wet Lab

##### POC

• First HPLC test run – running one sample of DMS, umbelliferone and decursinol and finalizing the program.

##### Cloning

• Preparation of additional starters for K133, petduet and Luciferase plasmids, as well as BFP plasmid.

• Glycerol stock and miniprep of said plasmids.

Nanodrop results:

|  | K133 | Petduet | Luciferase | BFP |
| --- | --- | --- | --- | --- |
| Conc. [ng/μl] | 212.1 | 83 | 920.4 | 207 |
| 260/280 | 2.05 | 1.96 | 1.86 | 1.9 |
| 260/230 | 2.16 | 2.1 | 2.25 | 2.29 |

• Restriction of no\_ximE\_petduet with same restriction enzyme for self ligation.

• Transformation of self-ligated no\_ximE\_petduet.

![](https://static.igem.wiki/teams/4179/wiki/notebook/photo-2022-08-26-09-38-29.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/photo-2022-08-26-09-38-29-2.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/photo-2022-08-26-09-38-28.jpg)

Figure 4: Plates with petduet\_no\_ximE: regular + rest + control 26.08.22

• Colony PCR to ensure desired self-ligated product.

Gel results (1kb ladder neb):

![](https://static.igem.wiki/teams/4179/wiki/notebook/colony-no-xim-e-28-8-22.jpg)
Figure 5: Colony PCR for E. coli Top10 colonies suspected no\_ximE\_petduet 28.8.22

Desired length: 237 bp.

Smaller fragments (lower band) indicate desired colonies.

• Starters from colonies 1 and 10 of no\_ximE\_petduet.

• Miniprep of colonies 1 and 10.

Nanodrop results:

|  | Colony 1 | Colony 10 |
| --- | --- | --- |
| Conc. [ng/μl] | 75 | 77.5 |
| 260/280 | 1.9 | 1.9 |
| 260/230 | - | - |

• Digestion of K133, and run in low temp gel to separate desired backbone.

• Cut K133 from gel and purification using wizard kit.

Nanodrop results:

|  | Digested K133 |
| --- | --- |
| Conc. [ng/μl] | 22.9 |
| 260/280 | 1.94 |
| 260/230 | 1.48 |

• Restriction of Luciferase plasmid using KpnI, and cleaning using Wizard kit.

Nanodrop results:

|  | Digested Luciferase (KpnI) |
| --- | --- |
| Conc. [ng/μl] | 64.7 |
| 260/280 | 1.91 |
| 260/230 | 1.63 |

• Restriction of Luciferase plasmid using MluI, and cleaning using Wizard kit.

Nanodrop results:

|  | Digested Luciferase (MluI) |
| --- | --- |
| Conc. [ng/μl] | 34.5 |
| 260/280 | 1.93 |
| 260/230 | 1.30 |

• Restriction of CMV plasmid, and run in low temp gel:

![](https://static.igem.wiki/teams/4179/wiki/notebook/cmv-separation-from-backbone-low-temp.jpg)
Figure 6: Gel electrophoresis of CMV in low temp gel 28.08.22

• Purification using Wizard kit.

Nanodrop results:

|  | Digested Luciferase (KpnI) |
| --- | --- |
| Conc. [ng/μl] | 7.6 |
| 260/280 | 2.18 |
| 260/230 | 0.06 |

• PCR amplification of PcPT from petduet plasmid, successful gel run:

![](https://static.igem.wiki/teams/4179/wiki/notebook/amplified-pcpt-from-petduet-30-08-22.jpg)
Figure 7: Gel electrophoresis of PcPT

Expected length: 1200 bp

Nanodrop results:

|  | Amplified PcPT |
| --- | --- |
| Conc. [ng/μl] | 84 |
| 260/280 | 2.00 |
| 260/230 | 0.14 |

• Restriction of all five prenyltransferase Gblocks.

• Restriction of PcPT amplified PCR product.

Nanodrop results:

|  | PcPT | PsPT1 | FcPT | UDT | Chimera1 | Chimera2 |
| --- | --- | --- | --- | --- | --- | --- |
| Conc. [ng/μl] | 35.8 | 4.5 | 6.7 | 9.8 | 10.6 | 8.8 |
| 260/280 | - | 2.14 | - | - | 1.98 | 2.1 |
| 260/230 | - | 0.55 | - | - | 0.65 | 0.69 |

• Restriction of ximE gblock.

Nanodrop results:

|  | Digested ximE Gblock |
| --- | --- |
| Conc. [ng/μl] | 15.7 |
| 260/280 | 2.26 |
| 260/230 | 0.77 |

• T7 terminator annealing of two oligos.

Conc. 1828.1 ng/μl.

• Reverse PCR of no\_ximE\_petduet to remove PcPT. Repeated three times, with unsuccessful results.

• Preparation of backup starter of CMV plasmid, and miniprep.

Nanodrop results:

|  | CMV plasmid |
| --- | --- |
| Conc. [ng/μl] | 728 |
| 260/280 | 1.84 |
| 260/230 | 2.2 |

• PCR for P2A oligos, with successful gel results.

![](https://static.igem.wiki/teams/4179/wiki/notebook/p2a-annealing-pcr-31-08-22.jpg)
Figure 8: Gel electrophoresis of P2A oligos annealing 31.08.22

Conc. 40.4 ng/μl

• Restriction of P2A

Nanodrop results:

|  | Digested P2A |
| --- | --- |
| Conc. [ng/μl] | 20.9 |
| 260/280 | 2.02 |
| 260/230 | 1.14 |

• PCR to amplify Blast from pTre.

First time we got unsuccessful results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/amplification-of-blast-from-ptre-2-successful-1-9-22.jpg)
Figure 9: Gel electrophoresis of Blast amplified from pTRE 1.9.22 – unsucceful

Desired length: 736 bp

Repeated PCR to amplify Blast with one stage program, instead of two stage. Successful results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/amplification-of-blast-from-ptre-2-successful-1-9-22.jpg)
Figure 10: Gel electrophoresis of Blast amplified from pTRE 1.9.22 - succeful

• Restriction of Blast amplified fragment with KpnI and HindIII.

Nanodrop results:

|  | Digested Blast insert |
| --- | --- |
| Conc. [ng/μl] | 54.5 |
| 260/280 | 2.00 |
| 260/230 | 1.60 |

• Ligation of restricted P2A to both digested A133 and K133, and transformation.

Plate seedings:

![Digested A133 at Low temp.](https://static.igem.wiki/teams/4179/wiki/notebook/1-p2a-a133-p2a-k133-transformation-plates.jpg)

![Digested A133 at Low temp. after cut](https://static.igem.wiki/teams/4179/wiki/notebook/2-p2a-a133-p2a-k133-transformation-plates.jpg)

![Digested A133 at Low temp. after cut](https://static.igem.wiki/teams/4179/wiki/notebook/3-p2a-a133-p2a-k133-transformation-plates.jpg)

![Digested A133 at Low temp.](https://static.igem.wiki/teams/4179/wiki/notebook/4-p2a-a133-p2a-k133-transformation-plates.jpg)

![Digested A133 at Low temp. after cut](https://static.igem.wiki/teams/4179/wiki/notebook/5-p2a-a133-p2a-k133-transformation-plates.jpg)

![Digested A133 at Low temp. after cut](https://static.igem.wiki/teams/4179/wiki/notebook/6-p2a-a133-p2a-k133-transformation-plates.jpg)

Figure 11: Plates of A133-P2A and K133-P2A transformation

##### Oracell

• Splitting cells 1:10.

• Counting remaining cells. Results: 7.3\*10^6 cells/ml.

• Seeding calibration to know the concentration of cells we need to seed to get confluency in 6 walls plate.

## September

### [Week 36 - 4/9](#collapse9)

### Model

• Calculation of protein concentration in bacteria.

### Wet Lab

##### POC

• Running samples in HPLC for calibration curves.

![Digested A133 at Low temp.](https://static.igem.wiki/teams/4179/wiki/notebook/fig12-calib-curvve-for-dms.jpg)
Figure 12: Callibration curve for 7 -demethylsuberosin

![Digested A133 at Low temp. after cut](https://static.igem.wiki/teams/4179/wiki/notebook/fig13-calibration-curve-for-decursinol.jpg)
Figure 13: Calibration curve for decursinol

![Digested A133 at Low temp. after cut](https://static.igem.wiki/teams/4179/wiki/notebook/fig14-calibration-curve-for-umbelliferone.jpg)
Figure 14: Calibration curve for umbelliferone

##### Cloning

• Colony PCR of 20 colonies from each backbone, as well as controls:

![](https://static.igem.wiki/teams/4179/wiki/notebook/colony-p2a-k133-a133-1st-gel-04-09-2022.jpg)
Figure 15: Colony PCR for E. coli Top10 colonies suspected for K133-P2A and A133-P2A

![](https://static.igem.wiki/teams/4179/wiki/notebook/colony-p2a-k133-a133-2nd-gel-04-09-2022-3.jpg)
Figure 16: Colony PCR for E. coli Top10 colonies suspected for A133-P2A

Desired length:

P2A-A133: 1305 bp

P2A-K133: 1258 bp

• All K133 gel results were unsuccessful.
15 out of 20 colonies from A133 were successful.

• Prepared starters from colonies 5 and 16 of P2A-A133.

Nanodrop results:

|  | A133-P2A colony 5 | A133-P2A colony 16 |
| --- | --- | --- |
| Conc. [ng/μl] | 33.6 | 33 |
| 260/280 | 1.96 | 2.16 |
| 260/230 | 1.87 | 1.88 |

P2A – colonies 5 and 16.

• A133-P2A colonies 5 and 16 were send to sequencing (macrogen).

• 3 part ligation of Luciferase backbone, CMV and Blast.

• Transformation of Luciferase\_CMV\_Blast.

Transformation plates:

![](https://static.igem.wiki/teams/4179/wiki/notebook/1-luciferase-cmv0-blast-transformation-plates.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/2-luciferase-cmv0-blast-transformation-plates.jpg)

Figure 17: Plates for Luciferase+Blast+CMV regular and rest

• 1ST colony PCR to CMV+Blast transformation, 15 colonies were sampled.

Desired product: 1800 bp.

Undesired product: 450bp.

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/5-9-2022-gel-for-cmv-and-blast-bad-resolts.jpg)
Figure 18: Colony PCR for E. coli Top10 colonies suspected for CMV + Blast

Didn’t receive the desired product.

• Preparation of 2 starters – BFP plasmid, and miniprep.

Nanodrop results:

|  | BFP plasmid |
| --- | --- |
| Conc. [ng/μl] | 105 |
| 260/280 | 2.1 |
| 260/230 | 1.96 |

• 2nd repetition of 3 part ligation of Luciferase backbone, CMV and Blast.

• Transformation of Luciferase\_CMV\_Blast.

Transformation plates:

![](https://static.igem.wiki/teams/4179/wiki/notebook/1-liciferase-cmv-blast-transformation-attempt-2.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/2-liciferase-cmv-blast-transformation-attempt-2.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/3-liciferase-cmv-blast-transformation-attempt-2.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/4-liciferase-cmv-blast-transformation-attempt-2.jpg)

Figure 19: Plates for Luciferase+Blast+CMV regular and rest attempt #2 05.09.22

• 2nd colony PCR to CMV+Blast transformation, 16 colonies were sampled.

desired product: 1800 bp.

Undesired product: 450bp.

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/blast-cmv-ligation-6-9-22.jpg)
Figure 20: Colony PCR for E. coli Top10 colonies suspected for CMV + Blast #2 attempt 6.9.22

Received 5 colonies with the desired product.

• Prepared starters from colonies 7 and 20 of Luciferase-CMV-Blast.

Nanodrop results:

|  | Luciferase-CMV-Blast colony 7 | Luciferase-CMV-Blast colony 20 |
| --- | --- | --- |
| Conc. [ng/μl] | 686.1 | 935 |
| 260/280 | 1.8 | 1.87 |
| 260/230 | 2.13 | 2.22 |

• Preparation of 2 additional starters of both colonies(7 and 20 of Luciferase-CMV-Blast) for further preparation of glycerol stock.

• Glycerol stock preparation of colonies 7 and 20 of Luciferase-CMV-Blast.

• Both colonies (7 and 20 of Luciferase-CMV-Blast) were sent to sequencing with 2 primers for each.

Sequencing results:

Colony 7 – bad – doesn’t contain insert.

Colony 20 – good – will be further used for assay.

• Prepared starters from colonies 5 and 16 of P2A-A133 again.

Nanodrop results:

|  | A133-P2A colony 5 | A133-P2A colony 16 |
| --- | --- | --- |
| Conc. [ng/μl] | 36.5 | 51.6 |
| 260/280 | 1.92 | 1.9 |
| 260/230 | 1.82 | 2.03 |

• Digestion of A133-P2A – colonies 5 and 16 – with NdeI and NheI.

• Purification of digested A133-P2A colonies 5 and 16.

 \*while using the centrifuge the marker of each tube fell. From now the colonies are marked as colony 'A' and colony 'B'.

Nanodrop results:

|  | A133-P2A digested colony A | A133-P2A digested colony B |
| --- | --- | --- |
| Conc. [ng/μl] | 13.3 | 28.1 |
| 260/280 | 1.87 | 1.94 |
| 260/230 | 0.34 | 0.85 |

• Ligation of all 5 g-blocks and amplified PcPT from Pet-Duet plasmid with restricted A133-P2A,from both colonies A and B:

- UDT + restricted A133-P2A (X2 colonies)

- FcPT + restricted A133-P2A (X2 colonies)

- PsPT1 + restricted A133-P2A (X2 colonies)

- Chimera1 + restricted A133-P2A (X2 colonies)

- Chimera2 + restricted A133-P2A (X2 colonies)

- PcPT (from pETDuet) + restricted A133-P2A (X2 colonies)

• Transformation of all ligated products to top10 E.coli.

• Transformation plates:

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-ligation-of-gblocks-amplified-pcpt-1-min.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-ligation-of-gblocks-amplified-pcpt-2-min.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-ligation-of-gblocks-amplified-pcpt-3-min.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-ligation-of-gblocks-amplified-pcpt-4-min.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-ligation-of-gblocks-amplified-pcpt-5-min.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-ligation-of-gblocks-amplified-pcpt-6-min.jpg)

Figure 21: Plates for A133-P2A with Gblocks; UDT, FcPT, PsPT1, Chimera1, Chimera 2 and PcPT from pETDuet

• Colony PCR to all transformation plates (6 inserts X 2 colonies - A and B). Primers used: pSeq\_K133\_fwd and pSeq\_K133\_rev.

Desired product: 2510 bp.

Undesired product: 1305 bp.

Results:

-PcPT and PsPT1, colonies A and B:

![](https://static.igem.wiki/teams/4179/wiki/notebook/pcpt-pspt1-colonypcr-07-09-22.jpg)
Figure 22: Colony PCR for E. coli Top10 colonies suspected for PcPT and PsPT1 07.09.22

-FcPT and UDT, colonies A and B:

![](https://static.igem.wiki/teams/4179/wiki/notebook/fcpt-udt-colonypcr-07-09-22.jpg)
Figure 23: Colony PCR for E. coli Top10 colonies suspected for UDT and FcPT 07.09.22

-Chimera 1 and Chimera 2, colonies A and B:

![](https://static.igem.wiki/teams/4179/wiki/notebook/chimera1-chimera2-colonypcr-07-09-22.jpg)
Figure 24: Colony PCR for E. coli Top10 colonies suspected for Chimera1 and Chimera2 07.09.22

All colonies had major undesired band – 1300 bp and minor desired band – 2500bp.

• Starter preparation of Pet-Duet no ximE colony 10 (for sequencing) – with New\_ximE\_rev and New\_ximE\_fwd

Nanodrop results: 119.3 [ng/μl].

• Repetition of colony PCR:

Colonies from previous copy plate, chosen colonies:

PcPT – 4,5.

PsPT1 – 2,3.

FcPT – 6,7.

UDT – 1,4.

Chimera 1 – 4,5.

Chimera 2 – 1,8.

Additional colonies from original plates:

PcPT – 13,14.

PsPT1 – 15,16.

FcPT – 17,18.

UDT – 19,20.

Chimera 1 – 21,22.

Chimera 2 – 23,24.

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/colony-a133-p2a-pt-rerun-08-09-2022.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/colonypcr-for-a133-p2a-pt-8-9-22.jpg)

Figure 25: Colony PCR for E. coli Top10 colonies suspected for A133\_P2A\_PT 08.09.22

Most of the colonies had again major undesired band – 1300 bp and minor desired band – 2500bp.

• Starters preparation:

- One colony of each enzyme from plate 'B'

Nanodrop results:

|  | Conc. [ng/μl] |
| --- | --- |
| A133\_P2A Colony 5 | 7.8 |
| A133\_P2A Colony 16 | 7.7 |
| PsPT1 | 6 |
| PcPT | 3.8 |
| FcPT | 16.6 |
| UDT | 7.8 |
| Chimera1 | 3.5 |
| Chimera2 | 3.7 |

• Repeated digestion of A133-P2A with NdeI and NheI – both colonies (5 and 16).

Nanodrop results:

|  | A133-P2A digested colony 5 | A133-P2A digested colony 16 |
| --- | --- | --- |
| Conc. [ng/μl] | 2.7 | 3 |

• Repeated ligation of restricted A133-P2A with amplified PcPT from Pet-Duet, transformation (only positive rest plates).

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-pcpt-1.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-pcpt-2.jpg)

,Figure 26: Plates of A133\_P2A\_PcPT #5 & #16 09.09.2022

• Additional starters preparation and miniprep:

- Colony 5 and 16 of A133-P2A from glycerol stock

- PcPT – colony 2 from copy plate

- PsPT1 – colony 5 from copy plate

- FcPT – colony 3 from copy plate

- UDT – colony 8 from copy plate

- Chimera1 – colony 7 from copy plate

- Chimera2 – colony 1 from copy plate

Nanodrop results:

|  | A133-P2A colony 5 | A133-P2A colony 16 |
| --- | --- | --- |
| Conc. [ng/μl] | 1206 | 543 |
| 260/280 | 2.03 | 2.04 |
| 260/230 | 2.16 | 2.07 |

|  | UDT | FcPT | PsPT1 | PcPT | Chimera1 | Chimera2 |
| --- | --- | --- | --- | --- | --- | --- |
| Conc. [ng/μl] | 273 | 151.3 | 540.4 | 57.8 | 273.3 | 132.4 |
| 260/230 | 2.09 | 2.05 | 2.07 | 1.97 | 1.99 | 2.01 |
| 260/280 | 2.2 | 2.08 | 2.08 | 1.74 | 1.71 | 2 |

• Colony PCR to both transformation plates (colony 5 and 16 –ligated A133-P2A and PcPT).

Primers used: pSeq\_K133\_fwd and pSeq\_K133\_rev.

Desired product: 2510 bp.

Undesired product: 1305 bp.

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/colony-a133-10-09-2022.jpg)
Figure 27: Colony PCR for E. coli Top10 colonies suspected for A133\_P2A\_PcPT #5 and #16 10.09.22

All colonies had undesired product.

##### Oracell

• Splitting CHO cells 1:10 and 1:5.

• Counting cells. Results: 3.25\*10^6 cells/ml

• Pre-transfection for blue fluorescent protein (BFB) (protocol D).

• Flow cytometer.

• Prepare medium with BLAST for CHO cells selection

• CHO Transfection with our plasmid and PUC19 plasmid (without BLAST resistant) (protocol H).

• Replace the 8B f12 medium to 4B f12 and day after we replace 4B f12 medium to 8B f12 due to not enough cells dying.

##### Efficacy

• HaCaT cells defrost – split 1 million HaCaT cells into 3 plates (the cells didn’t grow).

### [Week 37 - 11/9](#collapse19)

##### General

Education: Teen’s workshop.

• Hosting Israel's mini jamboree with BGU and TAU iGEM Teams.

• Meeting with Prof.Yuval Shoham

##### Model

• Download and get to know the software for the mutation model.

• A preliminary model of ximE and DMS bond.

• MATLAB implementation of the fermentation model.

### Wet Lab

##### Cloning

• Digestion of A133-P2A in 2 steps, one for each enzyme as followed:

a. 1st Digestion with NdeI and purification – didn’t go well.

b. 2nd Digestion with NdeI and purification

Nanodrop results:

|  | A133-P2A colony 5 restricted with NdeI | A133-P2A colony 16 restricted with NdeI |
| --- | --- | --- |
| Conc. [ng/μl] | 5.5 | 12.4 |
| 260/280 | 17.1 | 1.69 |
| 260/230 | 0.04 | 0.16 |

c. 3rd Digestion with NdeI and purification (since nanodrop results were not high enough).

Nanodrop results:

|  | A133-P2A colony 5 restricted with NdeI | A133-P2A colony 16 restricted with NdeI |
| --- | --- | --- |
| Conc. [ng/μl] | 23.7 | 41.1 |
| 260/230 | 1.84 | 1.83 |
| 260/280 | 1.2 | 0.55 |
| 260/230 |  |  |

• Repeated nanodrop measure of A133-P2A, colonies 5 and 16 – got different results:

Nanodrop results:

|  | A133-P2A colony 5 | A133-P2A colony 16 |
| --- | --- | --- |
| Conc. [ng/μl] | 703.7 | 515 |
| 260/280 | 1.95 | 1.98 |
| 260/230 | 2.06 | 1.95 |

• Dilutions transformation - UDT:

- Dilutions of UDT miniprep as followed: 1st dilution of miniprep 1:50, then serial dilutions of 1:2 – 7 times.

- Transformation of each dilution and plating.

• Colony PCR of dilutions transformation:

Primers used: pSeq\_K133\_fwd and pSeq\_K133\_rev.

Desired product: 2510 bp.

Undesired product: 1305 bp.

Gel results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/colony-pcr-for-diluted-transformations-1-36-120922-tif.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/colony-pcr-for-diluted-transformations-37-41-120922-tif.jpg)

Figure 28: Colony PCR for E. coli Top10 colonies suspected for UDT – Dilution transformation 12.09.22

All colonies had undesired product.

• Preparation of LB agar mix for plates.

• Restriction test to A133-P2A plasmid:

- Digestion with NdeI – run in 1% agarose gel (550 ng of DNA per reaction)

- Digestion with NheI – run in 1% agarose gel (550 ng of DNA per reaction)

- Digestion with PstI – run in 1% agarose gel (550 ng of DNA per reaction)

- Uncut reaction – run in 1% agarose gel (550 ng of DNA per reaction)

- Digestion with NdeI+NheI – run in 2% agarose gel (500 ng of DNA per reaction)

- Digestion with NdeI+PstI – run in 2% agarose gel (500 ng of DNA per reaction)

- Digestion with NheI+PstI – run in 2% agarose gel (500 ng of DNA per reaction)

Gel results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-1-gel-restriction-12-9-22-3.jpg)
Figure 29: Gel electrophoresis of A133\_P2A 1% gel 12.09.22

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-2-gel-restriction-12-9-22-2-1.jpg)
Figure 30: Gel electrophoresis of A133\_P2A 2% gel 12.09.22

• Repeat A133-P2A digestion in 2 steps (from colony 5):

- digestion with NheI

- purification of digested product

Nanodrop results:

|  | A133-P2A colony 5 – digested with NheI |
| --- | --- |
| Conc. [ng/μl] | 52.6 |

- digestion with NdeI

- purification of digested product

Nanodrop results:

|  | A133-P2A colony 5 – digested with NheI+NdeI |
| --- | --- |
| Conc. [ng/μl] | 29.5 |

• Starters preparation:

- 3 starters of A133-P2A colony 5

Nanodrop results:

|  | A133-P2A colony 5 |
| --- | --- |
| Conc. [ng/μl] | 170 |
| 260/280 | 2.09 |
| 260/230 | 2.2 |

• Ligation of digested A133-P2A from (12.9) with digested PcPT from (30.8)

• Transformation of ligated product to top10 E.coli.

• Colony PCR to transformation plates.

Primers used: pSeq\_K133\_fwd and pSeq\_K133\_rev.

Desired product: 2510 bp.

Undesired product: 1305 bp.

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-pcpt-colony14-9-2.jpg)
Figure 31: Colony PCR for E. coli Top10 colonies suspected for A133\_P2A\_PcPT 14.09.22

• Reverse PCR of PcPT removal from Pet-Duet No XimE plasmid with new primers: new\_fwd\_no\_PcPT and new\_rev\_no\_PcPT.

Desired product 6500 bp.

Gel results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/amplifying-pcpt-out-petduet-no-xime-150922-2.jpg)
Figure 32: Gel electrophoresis of PcPT amplified out of pETDuet\_no\_XimE 15.09.22

• Repeat reverse PCR of PcPT removal from Pet-Duet No XimE plasmid and from original Pet-Duet, with new primers: new\_fwd\_no\_PcPT and new\_rev\_no\_PcPT.

Desired product 6500 bp.

Gel results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/33.jpg)
Figure 33: Colony PCR for E. coli Top10 colonies suspected for A133\_P2A\_PcPT #2 attempt 15.09.22

• PCR test for double bands colony of PsPT1: from miniprep product using Q5 and Taq polymerases. Primers used: pSeq\_K133\_fwd and pSeq\_K133\_rev.

Gel results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/34.jpg)
Figure 34: Gel electrophoresis of PsPT cut with ageI 15.09.22

Digestion test to miniprep product of PsPT1 with AgeI.

Gel results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/35.jpg)
Figure 35: Gel electrophoresis of PsPT cut with ageI 15.09.22

• 1st Repetition reverse PCR to remove PcPT from Pet-Duet No XimE – with 2 parts amplification.

Primers used for 1st fragment (NheI\_XimD\_AgeI): new\_fwd\_no\_PcPT and colony\_no\_ximE\_rev

Primers used for 2nd fragment (AgeI\_b.b\_Sph): new\_rev\_no\_PcPT and colony\_no\_ximE\_fwd

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/16-9-22-gel-of-pcr-for-no-pcpt-one-and-2stages.jpg)
Figure 36: Gel electrophoresis of pETDuet\_no\_XimE to remove PcPT 16.09.22

• 2nd repetition of reverse PCR to remove PcPT from pETDuet No XimE – with 2 parts amplification.

Primers used for 1st fragment (NheI\_XimD\_AgeI): new\_fwd\_no\_PcPT and colony\_no\_ximE\_rev

Primers used for 2nd fragment (AgeI\_b.b\_Sph): new\_rev\_no\_PcPT and colony\_no\_ximE\_fwd

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/gel-of-pcr-to-remove-pcpt-amplification-of-two-fragments-17-9-22.jpg)
Figure 37: Gel electrophoresis of pETDuet\_no\_XimE to remove PcPT 17.09.22

• Separating the amplification of fragments to 2 PCR machines (each with different parameters)

Primers used for 1st fragment (NheI\_XimD\_AgeI): new\_fwd\_no\_PcPT and colony\_no\_ximE\_rev

Primers used for 2nd fragment (AgeI\_b.b\_Sph): new\_rev\_no\_PcPT and colony\_no\_ximE\_fwd

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/38.jpg)
Figure 38: Gel electrophoresis of pETDuet\_no\_XimE to remove PcPT #2 attempt 17.09.22

• Sent A133+P2A+insert to sequencing (6 different inserts – 6 different PTs). We also sent pETDuet\_no\_ximE. Sequencing results for 6 different insert showed a lot of noise which is consistent with the fact that we saw multiple bands. PETDuet\_no\_ximE results were as expected.

##### Oracell

• Change medium to the transfected CHO cells

• Split untransfected CHO cells 1:10

• Change medium to transfected CHO cells

• Sorter for transfected CHO cells and freeze all remaining cells from the sorter.

##### Efficacy

• Defrosted HaCat without splitting (1 million cells to one 10 cm plate)

• Change medium to HaCat cells

### [Week 38 - 18/9](#collapse29)

### General

• Education: Children’s workshop for Ukrainian refugees.

### Wet Lab

##### Cloning

• Minipreps for pETDuet\_no\_ximE. We accidently used improper buffer so the results were compromised.

• We performed another attempt of reverese PCR to amplify PcPT out of pETDuet. We used a different enzyme - PrimeSTAR GXL. Same enzyme was used for a retry of the 2 fragments strategy.

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/39-new.jpg)
Figure 39: Gel electrophoresis of pETDuet\_no\_XimE to remove PcPT 18.09.22

• The gel showed a band in the expected length (~6000) so we moved on to clean it.

Nanodrop results:

|  | **PETDuet\_no\_ximE\_no\_PcPT** |
| --- | --- |
| Conc. [ng/μl] | 45.7 |
| 260/280 | 1.88 |
| 260/230 | 0.21 |

• Restriction of pETDuet\_no\_ximE\_no\_PcPT with SphI and NheI to prepare it for ligation with ximE g block.

Nanodrop results after clean:

|  | **PETDuet\_no\_ximE\_no\_PcPT cut** |
| --- | --- |
| Conc. [ng/μl] | 12.5 |
| 260/280 | 2.08 |
| 260/230 | 0.9 |

• Minipreps of A133-P2A & pETDuet\_no\_ximE.

Nanodrop results:

|  | **A133\_P2A #16** | **pETDuet\_no\_XimE #1** |
| --- | --- | --- |
| Conc. [ng/μl] | 218 | 296 |
| 260/280 | 1.94 | 1.92 |
| 260/230 | 1.83 | 1.88 |

• We decided to try and cut A133\_P2A 2 times (one enzyme after the other):

Restriction of A133\_P2A NdeI.

Nanodrop results:

|  | **A133\_P2A cut (NdeI)** |
| --- | --- |
| Conc. [ng/μl] | 59.2 |
| 260/280 | 1.97 |
| 260/230 | 2.01 |

• Restriction of A133\_P2A (previously digested with NdeI) with NheI.

Nanodrop results:

|  | **A133\_P2A cut (NdeI+NheI)** |
| --- | --- |
| Conc. [ng/μl] | 44.6 |
| 260/280 | 1.92 |
| 260/230 | 0.86 |

• Ligation of NdeI\_NheI\_A133\_P2A with PcPT\_cut.

• Transformation of A133\_P2A\_PcPT.

• Ligation of pETDuet\_no\_ximE\_no\_PcPT, T7 terminator and ximE g block.

• Transformation of pETDuet\_no\_PcPT\_ximE\_T7.

• Made starters from copy plates for FcPT & UDT.

• Colony PCR for both transformations (pETDuet\_no\_PcPT\_ximE\_T7 & A133\_P2A\_PcPT).

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/40.jpg)
Figure 40: Colony PCR for colonies suspected for no\_ximE\_no\_PCPT, ximE and T7 2% gel 20.09.22

![](https://static.igem.wiki/teams/4179/wiki/notebook/41-new.jpg)
Figure 41: Colony PCR for colonies suspected for no\_ximE\_no\_PCPT, ximE and T7 1% gel 20.09.22

![](https://static.igem.wiki/teams/4179/wiki/notebook/42.jpg)
Figure 42: Colony PCR for colonies suspected for A133\_P2A\_PcPT 20.09.22

most of the colonies of pETDuet\_no\_PcPT\_ximE\_T7 showed a positive band of ~650 bp.

The colony PCR of A133\_P2A\_PcPT showed only undesired band of ~1200 bp.

• Setting up starters from positive colonies of pETDuet\_no\_PcPT\_ximE\_T7.

• Restriction of TetR g block with SphI in preparation for ligation into the pETDuet\_no\_PcPT\_ximE\_T7 backbone.

Nanodrop results after clean:

|  | **TetR Gblock** |
| --- | --- |
| Conc. [ng/μl] | 6.5 |
| 260/280 | 2.17 |
| 260/230 | 0.6 |

• Mini preps of starters – 4 starters of each colony. 3 starters of each colony were loaded onto the same column while the 4th starter of each colony was loaded onto different column.

Nanodrop results:

|  | **Colony #2 (3 starters)** | **Colony #2 (1 starter)** | **Colony #4 (3 starters)** | **Colony #4 (1 starter)** |
| --- | --- | --- | --- | --- |
| Conc. [ng/μl] | 685.6 | 403.1 | 328 | 105.3 |

• Sending colonies #2 and #4 of pETDuet\_no\_PcPT\_ximE\_T7 to sequencing.

• Restriction of colonies #2 and #4 of pETDuet\_no\_PcPT\_ximE\_T7 with SphI in preparation for ligation with TetR g block.

• CIP reaction for digested backbones to prevent self ligation. Concentration was not measured between cleaning of restriction and CIP as it is not required according to CIP protocol.

Nanodrop results after clean of CIP reaction:

|  | **Colony #2** | **Colony #2** |
| --- | --- | --- |
| Conc. [ng/μl] | 11.3 | 36.9 |

• Ligation of pETDuet\_no\_PcPT\_ximE\_T7 colonies #2 and #4 and TetR g block.

• Transformation of ligation products.

• Setting up starters of A133\_rhlR.

• Colony PCR of pETDuet\_no\_PcPT\_ximE\_T7 colonies #2 and #4 and TetR g block transformations.

Primers used: Colony\_new\_XimE\_terminator\_fwd, Colony\_new\_XimE\_terminator\_rev.

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/petduet-tetr-gel1-22-9-1.jpg)
Figure 43: Colony PCR for colonies suspected for TetR insertion 22.09.22

desired product: 1482 bp.

Undesired product: 688 bp.

• Modified ligations:

- Repeated ligation of PcPT with A133-P2A – with high insert ratio (1:7 backbone:insert)

- 3 Part ligation of P2A, PcPT and A133 (cut with AgeI and NdeI)

• Transformation of both ligations to top10 E.coli.

Transformation plates:

![](https://static.igem.wiki/teams/4179/wiki/notebook/petduet-no-pcpt-xime-t7-tetr1.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/petduet-no-pcpt-xime-t7-tetr2.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/petduet-no-pcpt-xime-t7-tetr3.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/petduet-no-pcpt-xime-t7-tetr4.jpg)

Figure 44: Plates for pETDuet no PcPT ximE T7 TetR #2 & #4 rest + control

• Colony PCR of both transformations.

primers used: New\_fwd\_no\_PcPT, New\_rev\_no\_PcPT.

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/petduet-tetr-gel2-22-9-2-1.jpg)
Figure 45: Colony PCR for colonies suspected for TetR insertion 22.09.22

Desired product: 2500 bp.

Undesired product: 1300 bp.

• 3 part ligation gel results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/colony-a133-p2a-pcpt-brute-force-1-23-09-22.jpg)
Figure 46: Colony PCR for colonies suspected for A133\_P2A\_PcPT 1:7 ratio wrong primers 23.09.22

Unsuccessful because of wrong primers

• Reverse PCR of pETDuet\_no\_PcPT\_ximE\_T7

Used primers No\_T7\_ximD\_fwd\_mod\_hind & No\_T7\_ximD\_rev\_mod\_N with PrimeStar GLX Polymerase enzyme.

Results: 1 strong band of undesired product (1,600) and 1 weak band of unknown origin (~400)

![](https://static.igem.wiki/teams/4179/wiki/notebook/reverse-pcr-petduet-xime-ximd-no-t7-23092022.jpg)
Figure 47: Gel electrophoresis of pETDuet ximE ximD no T7 23.09.22

• Rerun Reverse PCR pETDuet\_no\_PcPT\_ximE\_T7, same mixes but 15 sec annealing at 68°C, 2 min elongation at 68°C, 30 cycles.

• Repeat colony PCR for A133-P2A-PcPT from copy plate, with different primers (first run was with incorrect primers)

Primers: pSeq\_K133\_fwd & pSeq\_K133\_rev

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/colony-a133-p2a-pcpt-brute-force-2-23-09-22.jpg)
Figure 48: Colony PCR for colonies suspected for A133\_P2A\_PcPT 1:7 ratio 23.09.22

• Transforming pETDuet\_no\_PcPT\_ximE\_T7 into BL21 from colony #2 and #4, seeded only rest, no control.

• Colony PCR to 2 transformation plates (pETDuet\_no\_ximE\_T7 to BL21) #2 and #4.

Results: got a band above 500

![](https://static.igem.wiki/teams/4179/wiki/notebook/transformatin-bl21-colonypcr-24-09-22.jpg)
Figure 49: Colony PCR for BL21 colonies suspected with pETDuet\_no\_ximE\_T7 24.09.22

• Making starters from BL21 – from plate #2 took colony #5, from plate #4 took colony #7.

##### Oracell

• Split untransfected CHO cells 1:10.

• Another transfection of CHO sells with our plasmid.

• Change medium to transfected CHO cells.

• Split transfected CHO cells.

##### Efficacy

• Split HaCat 1:10.

### [Week 39 - 25/9](#collapse43)

### General

• Zoom meeting with Shamna CEO.

• Zoom with fermentaion expert.

### • Workshop for Ukrainian children. • Meeting with Biology and biotechnology teachers to spread Escape Room.

### Wet Lab

• Making Glycerol stock from the starters Stored at -80°C

"BL21 – PETDuet no PcPT ximE T7 #2#5 amp 25/9"

"BL21 – PETDuet no PcPT ximE T7 #4#7 amp 25/9"

• Inducing starters with IPTG, placed 4 flasks in shaker.

• Added 1.5 of DMS to each of the 4 flasks, return to shaker.

• Restriction of A133 & Pet9D

• Run in low melt temp gel – Pet9D didn't run as expected

• Purifying A133 with Geneaid,

Results:

|  | **A133** |
| --- | --- |
| Conc. [ng/μl] | 39.4 |
| 260/280 | 1.92 |
| 260/230 | 0.8 |

• Isolation plating for future Real-Time PCR of: BL21 with no plasmid, BL21 #2#5 and BL21 #4#7 with plasmid (from glycerol stock)

• Made starters for A133 rhlR mCherry(AMP), and Pet9D (KAN)

Mini prerp of A133 and Pet9D.

Nanodrop results:

|  | **A133** | **Pet9D** |
| --- | --- | --- |
| Conc. [ng/μl] | 264.2 | 319.2 |
| 260/280 | 2.14 | 2.21 |
| 260/230 | 2.29 | 2.37 |

• Prepared LB Agar for plates

• Restriction Pet9D with EcoRI and SphI

• Low melting agar separation of backbone (in 1.5% gel) was successful
(3504 bp)

![](https://static.igem.wiki/teams/4179/wiki/notebook/50.jpg)
Figure 50: Low melting Gel electrophoresis of Pet9D 29.09.22

• Purification of restricted product with Geneaid.

Results:

|  | **Pet9D** |
| --- | --- |
| Conc. [ng/μl] | 4.9 |
| 260/280 | 2.63 |
| 260/230 | 0.63 |

• Preparation of new xim design G-Block (2 from twist and 1 from IDT)

Resuspention of LacI(1308bp), XimD(1628bp) and TetR\_ximE(1460bp) – final concentration 20 ng/μl

• Restriction of G-Blocks; TetR\_ximE with bglII+SphI, LacI with NdeI+BglII, ximD with NdeI+EcorI.

• Cleaning digestion products with Geneaid

Results:

|  | **XimD** | **LacI** | **TetR\_XimE** |
| --- | --- | --- | --- |
| Conc. [ng/μl] | 26.3 | 23.6 | 19.6 |
| 260/280 | 1.9 | 1.9 | 1.92 |
| 260/230 | 1.15 | 1.22 | 1.3 |

• 4-Part Ligation of Pet9D(50 ng), TetR\_ximE(62.5ng), LacI(56ng), ximD(69.7ng)

• Starters preparation of #2#5 BL21 and #4#& BL21

• PCR for PcPT amplification from PETDuet(7 ng/μl) with new primers: PcPT\_XbaI\_rev, PcPT\_fwd

• Mini prep for pet9D.

Results:

|  | **Pet9D** |
| --- | --- |
| Conc. [ng/μl] | 290.3 |
| 260/280 | 2.22 |
| 260/230 | 0.40 |

• Gel 1% run of PcPT PCR – length ~1,400bp

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/pcpt-pcr-30-9-22.jpg)
Figure 51: Gel electrophoresis of PcPT 30.09.22

• Made glycerol stock to plack-V called mb27 Can b

• Restriction of Pet9D with EcoRI and SphI.

• After cleaning nanodrop results:

|  | **Pet9D** |
| --- | --- |
| Conc. [ng/μl] | 60.9 |
| 260/280 | 1.79 |
| 260/230 | 0.88 |

• Induced BL21 colony #2#5

• Restriction of PcPT Pcr with XbaI and NdeI

Cleaning with Geneaid.

Results:

|  | **PcPT** |
| --- | --- |
| Conc. [ng/μl] | 37.3 |
| 260/280 | 2.02 |
| 260/230 | 0.31 |

• Ligation of A133[NdeI, XbaI](50ng) with PcPT (55.6ng).

• Making stater for Pet9D from glycerol stock.

• Transformation for PETDuet into BL21 (amp).

Result: growth in negative control and rest plates.

![](https://static.igem.wiki/teams/4179/wiki/notebook/pet9d-into-bl21-1.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/pet9d-into-bl21-2.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/pet9d-into-bl21-3.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/pet9d-into-bl21-4.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/pet9d-into-bl21-5.jpg)

Figure 52: Plates of Pet9D in BL21

• Transformation for TetR\_ximE\_LacI and ximD into Top10 (kan).

Results: growth in regular and rest.

• Transformation for A133\_PcPT into Top10 (amp).

Results: didn’t grow at all.

![](https://static.igem.wiki/teams/4179/wiki/notebook/tetr-xime-laci-ximd-into-top10-1.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/tetr-xime-laci-ximd-into-top10-2.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/tetr-xime-laci-ximd-into-top10-3.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/tetr-xime-laci-ximd-into-top10-4.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/tetr-xime-laci-ximd-into-top10-5.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/tetr-xime-laci-ximd-into-top10-6.jpg)

Figure 53: plates of TetR\_ximE LacI ximD into Top10

• Colony PCR for Pet9D ximD

Primers: fwd\_9d\_ximD, rev\_9d\_ximD

Expected length 1199 bp, Tm 58.8°C, elongation time: 2min, 15 sec

• Colony for Pet9D LacI

Primers: fwd\_9d\_TetR, rev\_9d\_LacI

Expected length 2010 bp, Tm 58.8°C, elongation time: 2min, 15 sec

• Colony PCR for PETDuet in BL21

Expected length 1600 bp, Tm 56.3°C, elongation time: 1min, 45 sec

• Gel run of Pet9D 4 part ligation – all colonies had the right band using fwd/rev\_9d\_ximD. Colony #8 had a weak band from LacI, TetR primers. -> repeat colony only for #8 and run over night

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/1-10-petduet-in-bl21.jpg)
Figure 54: Colony PCR for BL21 colonies suspected for pETDuet 01.10.22

• Prepared Starter from colony #8

• PETDuet BL21 gel run – got expected length in 1,600 bp.

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/1-10-pet9d-terr-laci-ximd-xime.jpg)
Figure 55: Colony PCR for Top10 colonies suspected for pet9D TerR lacI XimD XimE 01.10.22

Prepared starter from colony #7, glycerol #2#5 amp, Pet9D #8 kan, 18 starters from #2#5 & #4#7, Luciferase starter, col #8 Pet9D 4 part ligation.

• Ran our cultures in HPLC to try and identify production of decursinol. The method used on 29/09 did not show decursinol or DMS peaks – probably should try other extraction method.

##### Oracell

• Seeding cells into 96 well plate named: Copy plate1 22.09.2022.

• Dividing Trypsin stock to aliquots.

• Copy plate from copy plate1.

• Preparation for sorter to sort cells according to their purity to achieve sorting for one cell per well .

• Freezing cells after counting.

• Seed 16 wells with untransfected cells in the test plate.

##### Efficacy

• Testing the decursin effect on HaCaT cells after treating cells with TNF-Alpha that induces apoptosis to cells. We seeded cells in 2 plates with 24 wells and incubated with 3 different concentrations of decursin () and 100 ng/ml TNF-alpha.

• Run samples in FACS, we got strange results, the precentage of apoptotic cells was higher in the untreated samples comparing it to the treated samples with TNF-Alpha.

• Change medium for HaCaT cells

• Split HaCaT 1:2

## October

### [Week 40 - 2/10](#collapse10)

### Wet Lab

• Ran gel 1% for ximD and LacI
Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/ximd-laci-2-10-1.jpg)
Figure 56: Gel electrophoresis of XimD LacI 02.10.22

• BL21 (after induction) glycerol stock plate (real-time PCR Backup) placed in -80°C freezer

• RNA extraction to BL21 #2#5, #4#7, and BL21 without plasmid.

• RNA 1% gel run in 70v for 40 minutes showed nothing (bad results)

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/igem-2nd-rna-pcr.jpg)
Figure 57: Gel electrophoresis of pETDuet RNA 02.10.22

• Continue to CDNA reaction, stored at -20°C

• Prepared 18 new starters from glycerol stock for new RNA extraction

• Induction of PETDuet\_PcPT\_ximE\_ximD with IPTG with colony #7

• RNA extraction

• Reverse Transcriptase reaction to make cDNA

• Preparation of qPCR mix for BL21 no plasmid's cDNA

• Restriction of A133 with NdeI and XbaI (Plasmid from 29/9, conc. 264 ng/μl)

• Inactication of NdeI and XbaI in 65°C for 20 minutes.

• CIP reaction for restricted A133

• Inactication of CIP enzyme in 80°C for 2 minutes.

• Ligation of A133 and PcPT (conc. 37 ng/μl)

• Transformation to Bl21 and Top 10

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-pcpt-bl21-neg.jpeg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-pcpt-bl21-rest.jpeg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-pcpt-bl21-self-ligation.jpeg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-pcpt-bl21-uncut.jpeg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-pcpt-top10-neg.jpeg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-pcpt-top10-rest.jpeg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-pcpt-top10-uncut.jpeg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-pcpt-top10.jpeg)

Figure 58: Plates of A133\_PcPT BL21 & Top10 transformation 06.10.22

Got bad results/ no growth

• Prepared 2% gel and ran A133 cip to test restriction

Results: got the wanted length, 2 bands.

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-cip-1-test-7-10.jpg)
Figure 59: A133 cip restriction test 1% gel 07.10.22

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-cip-2-test-7-10.jpg)
Figure 60: A133 cip restriction test 2% gel 07.10.22

• Prepared 2 starters for original PETDuet in BL21 and BL21 non-transformed

• Allocated starters to Erlenmeyer flasks of 50ml lb and induced with iptg

• Transformed A133 P2A PcPT overnight ligation

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/20221008-164542.jpg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/20221008-164600.jpg)

Figure 61: Plates for A133 P2A PcPT ligation 07.10.22

Ligation of UDT cut with nheI,ndeI (from 30.8) and A133-P2A cut with ndeI, nheI (19.9) ran ligation overnight

• Transformed ligation product of A133\_P2A\_UDT into Top10

• Ran colony PCR to A133\_PcPT

![](https://static.igem.wiki/teams/4179/wiki/notebook/igem-a133-p2a-overnight-ligation-lower-expo-8-10-22.jpg)
Figure 62: Gel electrophoresis of A133 P2A overnight ligation 08.10.22

Bad results

• Ran our cultures in HPLC to try and identify production of decursinol. On 02/10 we tried another extraction method that did not show decursinol but did show DMS peak – we deduce that the method is adequate.

• Ran the culture of PETDuet\_PcPT\_ximE\_ximD in HPLC – no decursinol/umbelliferone/DMS peaks.

##### Oracell

• Preparation for Luciferase decursin assay- It is a pre-experiment in order to make sure that the Luciferase reporter gene emits luminescence.

Table 1: Luminescene results

![](https://static.igem.wiki/teams/4179/wiki/notebook/63.jpg)

We can see from the results that there is a luminescence emmited from the cells after adding the luciferene in different concentration to each well (cell seeded in (A1-A4,B1-B4))

• 1:10 split(plate nemed:CHO P.42 F12 Untrans 3.10.2022 iGEM) and seed two rows(A1-12,B1-12) a in 96 well plate-These are the control wells for the assay test

• Transfer transfected cells from 96 cell plate to 10 cm plate(the 10 cm plate named best luci 2.10.2022 iGEM F12 B4)

• 1:10 split for transfected cells(plate name: best luci 2.10.2022 iGEM) , count and seed the third and the fourth rows (C1-12,D1-12)in same 96 wells plate (the same plate with two rows untransfected cells)

• The first assay test: goal: test the optimal incubation time for transfected cells with decursin-we incubated the cell with decurisn for 1,7.5,12,24 hours and tested the luminescence.

![](https://static.igem.wiki/teams/4179/wiki/notebook/fig-63.jpg)
Figure 63: Luminescence of transfected CHO cells.

• 1:5split cell-untransfected cells to achieve faster confluence.

Efficacy:

• Change medium for HaCaT cells

### [Week 41 - 9/10](#collapse20)

### Wet Lab

##### Cloning

• A133 P2A UDT Plates results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-udt-rest.jpeg)

![](https://static.igem.wiki/teams/4179/wiki/notebook/a133-p2a-udt-control.jpeg)

Figure 64: Plates of A133 P2A UDT control + rest 08.10.22

Ran colony PCR, expected length 2,500 bp

Gel results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/igem-a133-p2a-udt-9-10.jpg)
Figure 65: colony PCR for A133\_P2A-UDT 09.10.22

• Put up starts for pETDuet\_no\_PcPT\_XimD\_XimE\_T7 in BL21, and A133\_rhlR\_mCherry in Top10 from glycerol stock.

• Ran an experiment for the production of decursinol with petduet\_PcPT\_XimD\_XimE.
Bacteria were incubated with UMB and the medium was extracted at 1,8,24 hours. Later on the extracts were analyzed by HPLC.

##### Oracell

• Seeding transfected and untransfected (control) cells in 96 well plate.+Add decursin in two specific times (1hr and 24 hr).

• Test the effect of decursin on the luminescence of the Luciferase reporter gene.

Results:

![](https://static.igem.wiki/teams/4179/wiki/notebook/fig-66.png)
Figure 66: Decursin incubation time calibration.

##### Efficacy

• Seed HaCaT cells in 24 well plate with TNF-Alpha and decursin

• Test the effect of decursin on HaCaT cells using Annexin dye and run in FACS.

### [Week 42 - 16/10](#collapse30)

### [Week 43 - 23/10](#collapse44)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}The Design-Build-Test cycle is an engineering approach the iGEM strongly promotes and encourages teams
to use as a template throughout the advancement of their project, and this is what we did. This page presents the
iterations that our team has gone through using this approach. {% endblock %}
{% block page\_content %}

### Intro

Every **engineering** process consists of multiple milestones that together build the final system. Engineering a
biological system is not straightforward and often requires many customizations and fine tunings to achieve optimization.
In Fig.1 you can see the **engineering cycle** that describes the main stages in every **engineering process**. In our
case, our end system is an *E. coli* BL21 strain which has two plasmids that express three enzymes: Umbelliferone-6-prenyl-transferase, XimD, and XimE.
Read more in our  [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page.

![cycle](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic1.png)

**Figure 1** : Engineering success cycle

### U6PT plasmid

---

In order to build our entire biological system, we worked on both plasmids in parallel. **Design, build, test, learn and
re-design**. This was our strategy to tackle all the wet lab obstacles. Starting with the challenging cloning of the first
enzyme Umbelliferone-6-prenyl-transferase (U6PT):

![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic9-desighn.png) | 1st Design |

U6PT genes are plant sourced. Thus, in order to enlarge our chances of success, six different U6PT genes were used; four of which originate from different plant organisms
and two were constructed by us using BLAST and docking computational tool, for more details,
read in more in our  [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page.

All were designed to be
cloned into a plasmid that has mCherry reporter gene downstream to the P2A sequence (fig.2-fig7).

![Pcpt](https://static.igem.wiki/teams/4179/wiki/engineering-succes/3-untitled-slide.png)

**Figure 2** :A133-P2A-PcPT

![Pspt1](https://static.igem.wiki/teams/4179/wiki/engineering-succes/4-untitled-slide.png)

**Figure 3** :A133-P2A-PsPT1

![Fcpt1](https://static.igem.wiki/teams/4179/wiki/engineering-succes/5-untitled-slide.png)

**Figure 4** :A133-P2A-FcPT1

![Udt](https://static.igem.wiki/teams/4179/wiki/engineering-succes/6-untitled-slide.png)

**Figure 5** :A133-P2A-UDT

![ChimeraI](https://static.igem.wiki/teams/4179/wiki/engineering-succes/7-untitled-slide.png)

**Figure 6** :A133-P2A-ChimeraI

![ChimeraII](https://static.igem.wiki/teams/4179/wiki/engineering-succes/8-untitled-slide.png)

**Figure 7** :A133-P2A-ChimeraII

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic8-build.png) | 1st Build |
| --- | --- |

Step1: cloning the P2A sequence upstream to the mCherry sequence into A133 plasmid.
Step2: cloning All U6PTs genes upstream to the P2A sequence.

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic10-test.png) | 1stTest |
| --- | --- |

Each cloning was **verified using colony PCR**. The results have shown **positive insertion for the P2A sequence** (fig.8-Colony PCR gel for the verification of P2A cloning) but **negative results for all the U6PTs genes** (fig.9-Colony PCR gel for the verification of All U6PT cloning).

![cycle](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pcr8.png)

**Figure 8** :-Colony PCR gel for the verification of P2A cloning (desired product-1300bp)

![cycle](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pcr9.png)

**Figure 8** : Colony PCR gel for the verification of All U6PT cloning (desired product-2500bp, undesired product-1300bp)

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic11-learn.png) | 1st Learn |
| --- | --- |

The ligation was not successful.

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic9-desighn.png) | 2nd Re-design |
| --- | --- |

We decided to tweak the ligation protocol, using different ligation **ratios** between the insert and the backbone (1:7 rather than 1:3).

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic10-test.png) | 2nd Re-Test |
| --- | --- |

Another negative Colony PCR results- Self ligation products

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic11-learn.png) | 2nd Re-Learn |
| --- | --- |

The P2A sequence might hinder the ligation of the U6PT genes.

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic9-desighn.png) | 3rd Rre-design |
| --- | --- |

Ligation of three fragments: The digested backbone (A133), one of U6PT genes and the P2A sequence. Thus, the two inserts will be cloned simultaneously.

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic10-test.png) | 3rd Re-Test |
| --- | --- |

Colony PCR results have shown self-ligation products only. A digestion test also was conducted to make sure
that all the required restriction sites are found in the backbone. Indeed, all of them were found but they are very close (402bp).

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic11-learn.png) | 3rd Re-Learn |
| --- | --- |

We have learned that in order to get the desired restricted product. A two satge of digestion should be done in order to avoid a steric hindrance. After sending the colony PCR products for sequencing, it was shown that the cloning of U6PT genes
interrupts the sequencing process. Meaning, Sanger sequencing failed after less than 400 bases (fig.10-sequencing results).

![sequencing_results](https://static.igem.wiki/teams/4179/wiki/engineering-succes/fig-10-sequencing-results.png)

**Figure 10** :sequencing results

To further investigate this situation, we set aside five of the six U6PTs, and continued with PcPT, for the purpose of pinpointing the problem more efficiently.

PcPT was the only U6PT out of the six that was not ordered as a DNA fragment, rather it was amplified from a plasmid using PCR.

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic9-desighn.png) | 4th Re-design |
| --- | --- |

We have re-designed the new genetic U6PT circuit in a way that will allow us to clone the PcPT gene into the same target backbone but after digestion of the mCherry sequence out of the plasmid(fig.11-PcPT genetic circuit)

![sequencing_results](https://static.igem.wiki/teams/4179/wiki/engineering-succes/9-untitled-slide.png)

**Figure 11** :A133-PcPT

Step1: Amplifying the PcPT gene with new restriction sites.
Step2: A133 backbone digestion with the new restriction enzymes.
Step3: Ligation of two fragments.
Step4: Transformation

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic10-test.png) | 4th Re-Test |
| --- | --- |

As mentioned previously ,every cloning of a specific fragment was verified using Colony-PCR. However,we have gotten undesierd prodcuts.

Unfurtionatly,Many steps were not accomplished, and many others were skipped. But we are aware of the fact that the process of engineering a biomanufacturing machine like ours is full of obstacles that we did not have enough time to tackle. However, we've created additional designs and future plans to continue with. To read more about our future plans [click here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D#BLIP)

.

---

### ximD\_ximE plasmid:

As mentioned in the [Design page,](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) the second reaction in our synthetic pathway is mediated by two enzymes, ximD and ximE. Both enzymes work together to convert 7-demethylsuberosin (DMS) into decursinol.

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic9-desighn.png) | 1st Design |
| --- | --- |

The two genes of interest: ximD and ximE were designed to be cloned into the same plasmid under the regulations of Lac and Tet respectively as shown in fig.12.

![cycle](https://static.igem.wiki/teams/4179/wiki/engineering-succes/new/fig-12-initial-ximd-xime.png)

**Figure 12** :initial map of ximD & ximE plasmid

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic8-build.png) | 1st Build |
| --- | --- |

We started with pET\_Duet\_PcPT\_ximD\_ximE plasmid. The building of this circuit was done as shown below:
Step1: Reverse PCR to amplify the ximE gene out from the plasmid. This step was done in order to replace the gene of ximE with the same gene but under inducible regulation.
Step2: Reverse PCR with primeSTAR GXL polymerase to amplify the PcPT out from the plasmid. This step was done multiple times using Q5 polymerase, but no successful amplifications were achieved.
To solve this problem, we looked for a polymerase with the ability to amplify long amplicons. Eventually, primeSTAR GXL polymerase was the solution. It is the most high-fidelity polymerase that
is used for challenging targets like: high GC content, long amplicons and excess template[[1](#ref1)].
Step3: Ligation of 3 fragments - pET\_Duet backbone, ximE, T7 Terminator.

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic10-test.png) | 1st Test |
| --- | --- |

Tests were ran using ODE and statistical models based on Michales Menten equations as described on the  [Modeling page.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)

Modeling results have shown positive correlation between the concentration of decursinol (the desired product) and the concentration of XimE enzyme (fig.13).
![Model](https://static.igem.wiki/teams/4179/wiki/engineering-succes/fig-12-more-dec.svg)

**Figure 13** : Modeling results([ximD]<[ximE])

![Model](https://static.igem.wiki/teams/4179/wiki/engineering-succes/11-untitled-slide.png)

**Figure 14** : DEC:MAR Vs. XimE Molar Fraction

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic11-learn.png) | 1st Learn |
| --- | --- |

XimE should be expressed constitutively and not under any inducible regulation.

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic9-desighn.png) | 2nd Re-design |
| --- | --- |

To fulfill a constitutive expression for XimE enzyme, TetR repressor was not cloned into ximD\_ximE plasmid as shown in fig.13.

**To sum up**, according to the final design for the second plasmid, ximD is expressed inducibiliy under the Lac operator, however ximE is expressed constitutively to achieve maximal biomanufacturing yield of decursinol. This in accordance to our model that predicts higher decursin to marmesin ratio when the molar fraction of XimE increases.

![cycle](https://static.igem.wiki/teams/4179/wiki/engineering-succes/11-untitled-slide.png)

**Figure 15** : final ximD & ximE circuit

| ![](https://static.igem.wiki/teams/4179/wiki/engineering-succes/pic10-test.png) | 2nd Re-Test |
| --- | --- |

Real time qPCR assessment was run, following a transformation step into *E. coli* BL21 DE3. The purpose of this test is to determine the mRNA levels of each enzyme. XimE enzyme is constitutively expressed However, ximD expression is controled under the Lac regulation. So we added an IPTG molecule to induce the enzyme's expression. The qPCR results are presented in fig.16.

![](https://static.igem.wiki/teams/4179/wiki/assets/qpcr-bar-plot.png)

**Figure 16:** qPCR results

**In the scope of iGEM**, it was clear to us that achieving all of our milestones is a challenge due to the lack of time. Functionality tests and other modelling results were planed to be implemented in the designs of our genetic circuit. In our [future plants](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27future%27%29%20%7D%7D#BLOP) page, all of our next steps and future proposed improvmments are presented.

{% endblock %}
{% block references %}

1. Jia, H., Guo, Y., Zhao, W., & Wang, K. (2014). Long-range PCR in next-generation sequencing: Comparison of six enzymes and evaluation on the MiSeq sequencer. Scientific Reports, 4. <https://doi.org/10.1038/srep05737>

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}In this page we accumulated the results we got from all parts of the project, including cloning, transformation, transfection, and all various assay methods used along the way. {% endblock %}
{% block page\_content %}

## Introduction

---

In our project we had three different fronts of experimental efforts done simultaneously: biomanufacturing, measurement and efficacy tests.

On this page, we present the results achieved thus far in each one of them.

[[![](https://static.igem.wiki/teams/4179/wiki/results/biomanufacturing.svg)

##### Biomanufacturing](#bio)](#join)

[[![](https://static.igem.wiki/teams/4179/wiki/results/measurement.svg)

##### Measurement](#mes)](#join)

[[![](https://static.igem.wiki/teams/4179/wiki/results/efficacy.svg)

##### Efficacy test](#ef)](#join)

### Biomanufacturing

---

Our goal is to produce decursinol from umbelliferone using bacteria, which subsequently become decursin via a chemical
esterification.
As detailed in the [design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page , we planned to clone three enzymes
into two separated circuits. We chose to work with *E. coli* BL21 DE3 strain as the bacterial cell factory that
will implement these plasmids, as it expresses the T7 polymerase and therefore employs the T7 promoter for transcription.

#### Umbelliferone-6 Prenyltransferase

As mentioned in the [design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page, to convert umbelliferone to DMS we
need the action of the mediating enzyme Umbelliferone-6 prenyltransferase (U6PT). We planned to clone six different
variants of U6PT enzymes to A133\_rhlR\_tdPP7\_mCherry plasmid upstream to an mCherry gene with mediating P2A self-cleaving
peptide sequence. The cloning process contains two stages: a) cloning of the P2A sequence and,

b) cloning of each one of the designed U6PT enzymes.

##### Cloning

P2A was cloned between AgeI and NdeI restriction sites. The ligated plasmid was transformed into *E. coli* TOP10.

![FirstPic](https://static.igem.wiki/teams/4179/wiki/results/results-picture-3-new.png)

**Figure 1: Colony PCR of first stage A133-P2A plasmid transformation.** Eight colonies from the transformed plate and
a single colony from no-insert control plate were picked and underwent colony PCR. A no colony reaction was measured as well (negative). Expected length
for positive clones and no-insert control were 1305 and 1941 bp, respectively. Results were made with a 1% agarose gel and DNA stained with ethidium bromide (EtBr).

Apart from two sampled colonies, all colonies had the desired fragment length of 1305bp, indication for successful cloning.

The subsequent cloning of U6PT enzymes was unsuccessful as of the time of the wiki-freeze deadline, despite many strategies that we have attempted. The enzyme was to be cloned between NdeI and NheI restriction sites, which sequencing results confirmed are present. To read more about our different strategies as part of the Design-Build-Test cycle refer to our [Engineering Success](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) page.

#### XimD & XimE

##### Cloning

The two enzymes are required to turn 7-dimethyl suberosin (DMS) into decursinol, as detailed in the [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page. After amplifying out XimE and PcPT from PetDuet plasmid in two stages, we have successfully cloned into the plasmid a new XimE sequence, which contains the desired regulation.

![SecondPic](https://static.igem.wiki/teams/4179/wiki/results/results-picture-1.png)

**Figure 2: Colony PCR final XimD and XimE plasmid transformation.** 16 colonies from the transformed plate and a single colony from no-insert control plate were picked and underwent colony PCR. A no colony reaction was measured as well (neg. control). Expected length for positive clones and no-insert control were 677 and 150 bp, respectively. Results were made with a 2% agarose gel and DNA stained with ethidium bromide (EtBr).

Apart from one sampled colony, all colonies had the desired fragment length of 677bp, indication for successful cloning.

##### Quantitative PCR

Following the successful XimD+XimE design cloning process, the final plasmid was transformed into *E. coli* BL21 DE3 strain. The next step was conducting a qPCR assay for two main purposes: 1) evaluation of XimD and XimE transcription. 2) assessment of our LacI-based regulatory system effect.

![SecondPic](https://static.igem.wiki/teams/4179/wiki/results/qpcr-results.png)

**Figure 3: (A-C) Amplification curves of XimD, XimE and housekeeping gene transcripts.** 5ml *of E. coli* BL21 cells were allowed to grow over night in the presence (induced) or absence (non-induced) of 1mM IPTG, before RNA extraction. RNA was then subjected to 1h of DNase and converted to cDNA. Finally, samples underwent quantitative PCR with gene-specific primers. IPTG presence resulted in the induction of XimD (A) by 14-fold (D), while XimE transcription remains relatively unchanged (B, D). rssA was used as a housekeeping gene. The threshold for analysis was set at 0.3. Horizontal red line: threshold. Dashed lines: Ct value for each sample. NTC: non-template control. The XimD-XimE plasmid was used as a positive control. **D. Fold-change of XimD and XimE transcription.** The Ct values acquired correspond to the initial total RNA value levels. Hence, the values were normalized to the Ct values of endogenous housekeeping gene, rssA, a known housekeeping gene [[1](#ref1)]. This figure was created with the assistance of user-created functions [[2](#ref2)][[3](#ref3)].

Referring to each one of our set purposes:

1) Our assay confirmed both genes were transcribed.

2) As described on our [design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page, the XimD gene was cloned under LacI regulation. Thus, when introducing IPTG to the system we expected transcription levels to rise. Induction level was revealed to be 14-fold after six h of incubation with a one mM of IPTG (figure 3D).
Interestingly, XimE seemed to have also been affected in the IPTG-induced culture, marking a ~3-fold increase in transcription (figure 3D). A possible explanation is the proximity of the XimD and XimE genes in our construct. When we introduce IPTG to the system, the Lac repressor detach from the Lac operator regulating the XimD gene[[4](#ref4)]. This in turn, permits the recruitment of T7 RNA polymerase to the XimD T7 promoter. Since the XimE promoter is compatible with T7 RNA polymerase as well, the IPTG induction might have allowed the T7 polymerase to bind to the relatively proximal XimE promoter, increasing its transcription levels.

### Measurement

---

#### OraCell

##### Cloning

To construct our new measurement tool, we planned to generate a stable clonal cell line, which harbors in its genome a commercially available system with a Luciferase reporter. The system is susceptible to perturbations in the Hippo pathway, a cascade which decursin was found to interact with [[4](#ref4)][[5](#ref5)]. To integrate the plasmid, we had to insert a selection marker gene that could pressure cells to randomly integrate the system into their genome. We chose the blasticidin resistance gene (BlastR), which confers resistance to a toxin for mammalian cells [[5](#ref5)][[6](#ref6)].

BlastR was cloned under a constitutive CMV promoter. We successfully assembled the Luc\_blast\_oracell plasmid from a backbone and 2 inserts simultaneously (promoter and BlastR).

![SecondPic](https://static.igem.wiki/teams/4179/wiki/results/results-picture-2.png)

**Figure 4:** Colony PCR of Luc\_blast\_oracell transformation. 25 colonies from the transformed plate were picked and underwent colony PCR using a ready-to-use mix (Hylabs, #EZ3006/7/636). A no colony reaction was measured as well. Expected length for positive clones and no-insert control were 1781 and 450 bp, respectively. Results were made with a 1% agarose gel and DNA stained with ethidium bromide (EtBr). A commercial ladder (L) was used (NEB, #3232).

Colonies 6,7,9,11,20,21 and 24 had the desired fragment length of 1781bp, indication for successful cloning. Colonies 5,8,10,12,13,18,19,22,23 and 25 had expected self-ligation fragment length of 450bp. Rest of the colonies had no amplification.

##### Linear Polyethylenimine (PEI) Calibration

Before transfecting the cells, we calibrated the PEI transfection reagent, to find the best concentration that will show low cellular death and high transfection efficiency.

![](https://static.igem.wiki/teams/4179/wiki/results/picture2-iser.png)

**Figure 5: PEI calibration.**CHO cells were transfected with a BFP-containing plasmid using increasing amounts of PEI. X h later cells were taken to a flow cytometer BD™ LSR II flow cytometer and gated for FSCxSSC (viability gate), out of which gated for SSC\_AxSSC\_H (single events gate, ~100% of viable events), and out of which gated for BFP (fluorescence gate). 7.2μg of PEI was chosen as the optimal condition for transfection (dotted line), yielding high viability and strong transfection efficiency. Viability percentages are of the total number of events, fluorescence percentages are of the total number of single events.

Cell viability dropped gradually until 7.5μg PEI that was tested. We chose 7.2μg as the working PEI amount based on optimal fluorescence signal with minimal drop in viability (Figure 5).

##### Luminescence of Transfected Cells

![](https://static.igem.wiki/teams/4179/wiki/results/picture1-iser.png)

**Figure 6: Luminescence of transfected CHO cells**. CHO cells were transfected with plasmid containing Luciferase enzyme. Following selection and proliferation, transfected cells were lysed and following the addition of luciferin, the cells were analyzed for bioluminescence in Synergy H1 Hybrid Multi-Mode Microplate Reader. Untransfected CHO cells and Buffer underwent the same process and were analyzed for bioluminescence, serving as negative controls. In addition, bacterial cells expressing Luciferase and producing luciferin were diluted 1:10 and analyzed for bioluminescence serving as a positive control. Transfected CHO cells show a clear bioluminescent signal, similar to the positive control, Luciferase expressing *E. coli* 10-beta, and clearly higher than the two negative controls, buffer with luciferin and untransfected CHO cells with luciferin. From these results, we were able to conclude that the CHO cells retained the entire construct, both the blasticidine resistance gene and the Luciferase enzyme and successfully expressed both.

![](https://static.igem.wiki/teams/4179/wiki/results/decursin-time-calibration.png)

**Figure 7: A preliminary decursin incubation time calibration assay**. Cells were incubated with decursin with various time points ranging from 1-22 hours before being lysed. Then, luciferin was added and bioluminescence was measured. In addition, there were two types of negative control used, OraCell cells that were incubated without luciferin and CHO cells that do not express Luciferase. A control OraCell cells were incubated without decursin for 22h. Number of replicates = 1.

OraCell cells showed a periodic behavior of decursin’s effect on Luciferase expression via the Hippo pathway over time. Additionally, following a 22-hour incubation time with decursin, the luminescence was markedly decreased compared to the same incubation time without decursin. This result also showed that the OraCell cells showed a clear bioluminescent signal compared to the negative controls, the same cells without the addition of luciferin and CHO cells that do not express Luciferase.

![](https://static.igem.wiki/teams/4179/wiki/results/decursin-16-hour-time-experiment.png)

**Figure 8: Decursin incubation time assay**. OraCell cells were incubated with and without decursin at various time points ranging from 1-16 hours, lysed, luciferin was added, and bioluminescence was measured. The cells without decursin served as the positive control, to see how much decursin impacted the cells’ bioluminescence.

This result reinforced the trend shown previously (Figure 7), that there’s a periodic oscillation of decursin’s impact on the Hippo pathway over time. Interestingly, cells without decursin presented a decrease in luminescence between 10 and 16 hours of incubation. Number of replicates = 3.

![](https://static.igem.wiki/teams/4179/wiki/results/calibration-curve-decursin-3-and-16-hour-experiment.png)

**Figure 9: Decursin dose-response assay at 3 and 16 hours**. OraCell cells were incubated with increasing concentrations of decursin ranging from 0-240 µM for either 3 or 16 hours. The cells were then lysed, luciferin was added, and bioluminescence was measured. The luminescence was normalized to the no-decursin control for each time point. Number of replicates = 3.

Following a 3-hour incubation with decursin, there was no clear trend to be discerned from the calibration curve. However, at the 16h assay, increasing the decursin concentration resulted in increase in luminescence. This result along with others (Figures 7 and 8), might suggest how decursin impact the Hippo pathway within the OraCell system, and by extension the Hippo pathway as a whole. Our working theory is that decursin is capable of increasing or decreasing Luciferase expression as explained on our [Measurement](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D) page.

#### HPLC

As mentioned on our [measurement page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D), high-performance liquid chromatography (HPLC) is a sensitive method for the detection and quantification of chemical compounds. We plan to use HPLC to validate enzymes' functionality through detection of the different metabolites from the [decursin pathway](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D#opening).

![](https://static.igem.wiki/teams/4179/wiki/results/calibrationn.png)

**Figure 10: (A-C) Calibration Curves of Umbelliferone, 7-Demethylsuberosin and Decursinol.** Samples of umbelliferone (A) at 0.25, 0.5, 0.75 and 1 mM, 7-demethylsuberosin (B) at 0.75, 1, 1.25 and 1.5 and decursinol (C) at 0.1, 0.2, 0.3, 0.4 and 0.5 mM were dissolved in 20% acetonitrile (ACN) and measured. Note that the value for 1.25 mM 7-demethylsuberosin was excluded. H2O (a) and ACN (b) were used as mobile phase with a gradient: zero min 20% b, five min 20% b, 30 min 80% b and 40 min 20% b. A linear regression was fitted to the values. Umbelliferone, 7-demethylsuberosin and decursinol samples were measured at wavelengths of 194, 332 and 205 nm, and showed an average retention time of 10.069, 23.86 and 15.475 min, respectively.

As a part of our efforts to get large-scale production of decursin, we plan to detect and quantify decursinol production using the pETDuet-PcPT-XimD-XimE plasmid, mentioned on our [design page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D). This will act as a positive control and as a baseline which we aim to improve.

### Efficacy Test

---

##### PAMPA

The permeability of the decursin molecule was measured to assess the safety of our planned shampoo formulation. For this purpose, we used the Parallel Artificial Membrane Permeability Assay, a cell-free, safe, inexpensive, and high throughput method that mimics passive diffusion through the main barrier to permeation through the scalp, the stratum corneum (SC).

![](https://static.igem.wiki/teams/4179/wiki/poc/123.png)

**Figure 11: Decursin’s low permeability.** Decursin's permeability was tested using a PAMPA kit, by introducing the metabolite to a system comprised of Skin Mimic solution and two plates with a membrane between them. Decursin was added to the donor well and absorbance was measured in the accpetor well following 18 h incubation at a 330 nm wavelength. The absorbance was converted to permeability by using a known equation provided by the kit. The controls used are the standards that are supplied with the PAMPA kit. Methyl Paraben’s, Propyl Paraben’s and Theophylline’s absorbance was measured following 18 h incubation at 260, 260 and 270 nm wavelength respectively[[10](#ref10)].

This result showed that decursin has low permeability similar to theophylline. Following this result, we learned that the decursin has low permeability, meaning in our final formulation we could more easily optimize and adjust the formulation. In addition, when comparing to the permeability of theophylline, which is delivered topically, we can see that decursin has the ability to penetrate to the necessary depth.

As a part of the PAMPA assay, a calibration curve of decursin was also performed. The concentration of increasing amounts of decursin is plotted against absorbance, proving that the decursin that succeeded in permeating the membrane of the PAMPA was able to be quantified using absorbance measurements (Figure 12).

![](https://static.igem.wiki/teams/4179/wiki/results/calibration-curve.svg)

**Figure 12: Calibration curve of decursin**. Increasing concentrations of decursin were added to the donor plate and measured after 18 h in the acceptor plate. Absorbance was measured at 330 nm.

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{% block references %}

1. Peng, S., Stephan, R., Hummerjohann, J., & Tasara, T. (2014). Evaluation of three reference genes of Escherichia coli for mRNA expression level normalization in view of salt and organic acid stress exposure in food. FEMS microbiology letters, 355(1), 78-82.
2. Víctor Martínez-Cagigal (2022). Shaded area error bar plot (https://www.mathworks.com/matlabcentral/fileexchange/58262-shaded-area-error-bar-plot), MATLAB Central File Exchange. Retrieved October 8, 2022.
3. Douglas Schwarz (2022). Fast and Robust Curve Intersections (https://www.mathworks.com/matlabcentral/fileexchange/11837-fast-and-robust-curve-intersections), MATLAB Central File Exchange. Retrieved October 8, 2022.
4. Marbach, A., & Bettenbrock, K. (2012). lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA. Journal of biotechnology, 157(1), 82-88.
5. Han, Y. (2019). Analysis of the role of the Hippo pathway in cancer. Journal of translational medicine, 17(1), 1-17.
6. Kimura, M., Takatsuki, A., & Yamaguchi, I. (1994). Blasticidin S deaminase gene from Aspergillus terreus (BSD): a new drug resistance gene for transfection of mammalian cells. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 1219(3), 653-659

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