Notebook

Introducing iGEM – starting to read about old projects.s
Dividing into groups and each group presented an old winning iGEM project.

Exploring the structure of iGEM competition.

Exploring the structure of iGEM competition.

Exam period – a break from iGEM 🙁

General

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

General

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

General

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

Brainstorming.

General

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

General

Second presenting of the 3 projects.

Deciding on an idea.

General

iGEM crash course on synthetic biology.

General

Final presentation and idea decision.

Planning future work.

Planning future work.

Planning future work.

Model

• We chose relevant equations for the kinetics model.

Model

• finish the basic kinetic model.

Human Practices

• 15.6 - hair donation event.

Model

• Meeting with dry lab team of Athens group.

Wet lab

Oracell

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

Exam period – a break from iGEM 🙁

Exam period – a break from iGEM 🙁

Exam period – a break from iGEM 🙁

Exam period – a break from iGEM 🙁

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.

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.

• Starters of CMV and K133 plasmids, and miniprep.

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.

• 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):

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):

Figure 3: Gel electrophoresis of reverse PCR to remove ximE from petduet 25.08.22

Desired length: 7990 bp.

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.

• Restriction of no_ximE_petduet with same restriction enzyme for self ligation.
• Transformation of self-ligated no_ximE_petduet.

Figure 4: Plates with petduet_no_ximE: regular + rest + control 26.08.22

• Colony PCR to ensure desired self-ligated product.

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.

• Digestion of K133, and run in low temp gel to separate desired backbone.
• Cut K133 from gel and purification using wizard kit.

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

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

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

Figure 6: Gel electrophoresis of CMV in low temp gel 28.08.22

• Purification using Wizard kit.

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

Figure 7: Gel electrophoresis of PcPT

Expected length: 1200 bp

• Restriction of all five prenyltransferase Gblocks.
• Restriction of PcPT amplified PCR product.

• Restriction of ximE gblock.

• 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.

• PCR for P2A oligos, with successful gel results.

Figure 8: Gel electrophoresis of P2A oligos annealing 31.08.22

Conc. 40.4 ng/μl

• Restriction of P2A

• PCR to amplify Blast from pTre.

First time we got unsuccessful results:

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:

Figure 10: Gel electrophoresis of Blast amplified from pTRE 1.9.22 - succeful

• Restriction of Blast amplified fragment with KpnI and HindIII.

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

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.

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

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

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

• 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:

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:

Figure 29: Gel electrophoresis of A133_P2A 1% gel 12.09.22

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

- digestion with NdeI
- purification of digested product

• Starters preparation:
- 3 starters of A133-P2A colony 5

• 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:

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:

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:

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:

Figure 34: Gel electrophoresis of PsPT cut with ageI 15.09.22

Digestion test to miniprep product of PsPT1 with AgeI.
Gel results:

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:

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:

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:

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

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:

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.

• Restriction of pETDuet_no_ximE_no_PcPT with SphI and NheI to prepare it for ligation with ximE g block.

• Minipreps of A133-P2A & pETDuet_no_ximE.

• We decided to try and cut A133_P2A 2 times (one enzyme after the other):
Restriction of A133_P2A NdeI.

• Restriction of A133_P2A (previously digested with NdeI) with NheI.

• 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:

Figure 40: Colony PCR for colonies suspected for no_ximE_no_PCPT, ximE and T7 2% gel 20.09.22

Figure 41: Colony PCR for colonies suspected for no_ximE_no_PCPT, ximE and T7 1% gel 20.09.22

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.

• 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.

• 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.

• 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:

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:

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:

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:

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)

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:

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

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.

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:

• 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.

• 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)

Figure 50: Low melting Gel electrophoresis of Pet9D 29.09.22

• Purification of restricted product with Geneaid.
Results:

• 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:

• 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:

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

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:

• Induced BL21 colony #2#5
• Restriction of PcPT Pcr with XbaI and NdeI
Cleaning with Geneaid.
Results:

• 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.

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.

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:

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:

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

Wet Lab

Cloning

• A133 P2A UDT Plates results:

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

Ran colony PCR, expected length 2,500 bp
Gel results:

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:

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.