Team:Technion-Israel/Notebook

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Notebook

This timeline includes experiments and results.
Click on the date to see the notes!

01 June
02 June
03 June
04 June
07 June
08 June
09 June
10 June
11 June
15 June
16 June
17 June
21 June
22 June
23 June
25 June
30 June
01 July
03 Aug
04 Aug
05 Aug
06 Aug
09 Aug
10 Aug
11 Aug
12 Aug
13 Aug
17 Aug
18 Aug
19 Aug
23 Aug
24 Aug
25 Aug
26 Aug
27 Aug
30 Aug
31 Aug
01 Sep
02 Sep
03 Sep
06 Sep
07 Sep
08 Sep
09 Sep
10 Sep
12 Sep
13 Sep
14 Sep
15 Sep
16 Sep
17 Sep
21 Sep
22 Sep
23 Sep
24 Sep
25 Sep
04 Oct
05 Oct
06 Oct
07 Oct
08 Oct
11 Oct
12 Oct
13 Oct
14 Oct
15 Oct
18 Oct
19 Oct
22 Oct

June 1st

Bacillus group -Preliminary sporulation experience (Shany & Dor)

Protocol - LB agar

•LB preparation and autoclave + LB plates (with and without antibiotic AMP).
•Taking the Bacillus subtilis 168 from Yuval Shoham's lab and plate them on LB agar.
  •An 500 ml Erlenmeyer with 100% Glycerol was sent to outoclave.
June 2nd

Bacillus group - Preliminary sporulation experience (Shay)

•Plating Bacillus subtilis 168 on to two agar plates with and without antibiotics (Fig.1.)

Fig.1. B. subtillistilis 168 LB plate without antibiotics
June 3rd

Bacillus group -Preliminary sporulation experience (Shay)

Protocol - Starter

• Peaking colonies from agar plates and making 2 starters with 5 ml each.
• Incubate overnight (O/N) on 37°C and shaking at 200 rpm.

Gel group (Yara, Zixuan, Noa)

Protocol - Hydrogel Preparation

• Dissolve Pluronic F-127 with DDW.
• Leave in the shaker at 4℃ overnight.
June 4th

Bacillus group -Preliminary sporulation experience (Amir & Shay)

Protocol - Glycerol stock, Spore purification (DSM preparation)

• Preparing 22 tubes with 200 μL (100 Glycerol 100 Bacillus subtilis 168 starter) for stocking in -80°C.
• Preparation of sporulation medium + autoclave.
• Plates with the Bacillus subtilis 168  stored at 4°C.

Gel group (Yara, Zixuan, Noa)

Protocol - Examination of Gelation Time

• Determine the gelation time of created hydrogel at two temperatures (Figure 1).
• Test and image the reversible transformation between liquid and gel (Figure 2, Figure 3).

Fig.1. Position of Plate Heater for Gelation Time Determination

Fig.2,3. Hydrogel in Gel Form (left) and Hydrogel in Liquid Form (right)
June 7th

Bacillus group -Preliminary sporulation experience (Tomer)

Protocol - Starter

• Prepare 2 starters with LB (no antibiotics) for B. subtillis 168 colonies that were grown in an LB plate (large colonies), grow O/N. 
• Near fire, 5 ml of LB were added to two 50 ml Falcons, colony was added and copied before. left O/N in shaker: 37°C, 250 rpm.

LB plate with B. subtillis 168 strain
June 8th

Bacillus group -Preliminary sporulation experience (Dor)

Protocol - sporulation, Spore purification (DSM preparation), LB agar, Starter

• DSM preparations.
• sporulation procedure.
• LB agar + Chloramphenicol. (CM) 
• Weizmann institute update: Arrival of B. subtillis PY79 & pDG364 in E.Coli . 
1. Two colonies were taken from the pDG364 plate and inoculated into 5 ml LB with either CM or AMP antibiotic (5 µl). Kept at 4°C. 
2. Starter creation - One colony from the PY79 strain was taken and added to LB.

Fig.1. PY79 plate from Weizmann
June 9th

Bacillus group -Preliminary sporulation experience (Amir, Shany & Shay)

Protocol - Glycerol stock, Miniprep 

• Preparations of 8 Glycerol stocks of B. subtillis PY79 and one Glycerol stock of E.coli with pDG364. Stored at -80°C.
• Preparation of plates LB with CM, stored at 4°C.
• Extraction of pDG364 plasmid from E.coli via "Pureyield TM" miniprep system kit.
• NanoDrop Results: 

Concentration

451.1 ng/μl 

280/260

1.89

280/230

1.1
June 10th

Bacillus group -Preliminary sporulation experience (Dor)

Protocol - Glycerol stock

• Preparation of 2 Glycerol stocks for the B. subtilis 168 and B. Subtilis PY79. 
June 11th

Bacillus group -Preliminary sporulation experience (Amir, Shany & Shay)

Protocol - sporulation

• Beginning off sporulation protocol for PY79.
• Spores of B. subtillis 168 checked in light microscope {*40} and compared to 168 starter from 10/06/20. 

 Fig.1. Spores of B. subtillis 168
June 15th

Bacillus group -Preliminary sporulation experience (Shany & Dor)

Protocol - sporulation

• sporulation of B. subtillis PY79 checked on light microscope{*40}.
• Continue sporulation protocol for pY79: steps 3-5 with a change of using 10 ml DDW (instead of 200 ml).

Fig.1. B.subtillis PY79 spores.
June 16th

Bacillus group -Preliminary sporulation experience (Dor, Shany & Amir)

Protocol - sporulation

• Sporulation - Step 6 (10 ml DW instead of 200 ml).
• Examined spores under microscope.
• The results were compared with purified spore's, Conclusion: more washing steps are nedded. 
• ~65% spores/overall. 

Fig.1. B.subtillis PY79 spores.
June 17th

Bacillus group -Preliminary sporulation experience (Shany & Shay)

Protocol - sporulation

• sporulation - Step 6.
• Examined spores under microscope.

Fig.1. B. subtillis PY79 spores
June 21st

Bacillus group -Preliminary sporulation experience (Zixuan)

• Two LB plates were plated with B. subtillis PY79 from Glycerol stock, Incubated at 37°C.
June 22nd

Bacillus group -Preliminary sporulation experience (Yara & Saar)

Protocol - sporulation, starter

• Restart sporulation of 168 protocol, due to DSM contamination.  
• Started sporulation of PY79.
June 23rd

Bacillus group -Preliminary sporulation experience (Zixuan)

Protocol - Starter

• Starter - 5 ml LB and a PY79 colony. 
June 25th

Bacillus group -Preliminary sporulation experience (Amir, Dor)

Protocol - sporulation

• Sporulation: Steps 3-5 - 3 washes + centrifugation for 10 min at 10000 G, at the 4th wash an example was taken for examination with electron microscope, as show in the pictures.   

Fig.1. Electron microscope image of B. subtillis PY79, spores after 4th wash.
June 30th

ACE2 group -Vector creation (Inbal)

• Starter for yeast with p416-FEC-mCore plasmid - 5 ml LB + 5 μl AMP + Pinch of yeast.
• Incubate O/N, 30°C
July 1st

ACE2 group -Vector creation (Ilana)

Protocol - Miniprep (NucleoSpin kit ,page 13)

• Miniprep (NucleoSpin kit): Extraction of p416-FEC-mCore plasmid from yeast.
• Plasmid concentration via NanoDrop: 560 ng/μl.
• PCR - Linearization and amplification of the plasmid with specially design primers that add His tag.
• Verifying the PCR reaction in 1% Agarose gel.
• Conclusion & Result: The reaction failed
Aug 3rd

Gel group (Niv, Noa)

Protocol - Microgel Preparation (Step 1)

• Nitrilotriacetic acid (NTA) monomers preparetion - Dripping the Acryloyl chloride and toluene mixtureto the L-lysine NaOH solution, while stirring using separatory funnel and ice bath (Figure 1).
• Turn on the lyophilization device before leaving.

fig.1.: Dripping System used in Monomer Preparation
Aug 4th

Bacillus group - Cloning (Amir, Shay)

Protocol - LB agar, Chemical transformation, Glycerol stock

• LB+AMP plates creation.
• Chemical transformation of pBS1C into E.coli Top10cc, plate on AMP+LB agar plates.
• Bacillus Glycerol stock making.

Gel group (Niv, Noa)

Protocol - Microgel Preparation (Step 2: 10-18).

• The remaining solution in the separatory funnel was directly poured into the beaker since the funnel got stuck during the night and didn’t finish the dripping process.
• Toluene in the solution was evaporated using a rotary evaporator, DDW was added afterward to re-dissolve the solids.
• The solution was filtrated by a filter column with Dowex paste (Figure 1).
• The product was packed in falcons and sent for lyophilization (Figure 2).

Fig.1.: Filtration of Solution via Column with Dowex.

Fig.2.: Solutions Packed in Falcons and Placed in Lyophilization Device.
Aug 5th

Bacillus group - Cloning (Amir, Shay)

Protocol - Starter

• Starter E.coli Top10  with PBS1C_LacZ.
Aug 6th

Bacillus group - Cloning (Shay, Dor, Shany, Hadas)

Protocol - Glycerol stock, miniprep ,PCR

• Glycerol stock of PBS1C_LacZ in E.coli Top10.
• DNA extraction (miniprep) using NucleoSpin Plasmid EasyPure kit.
• Nanodrop Results:

 

pBS1C_LacZ_mRFP

B1-2 (stored in -20°C)

Concentration (ng/μl )

181.8

174.3

280/260

1.91

1.91

280/230

1.81

2.11

• PCR on the miniprep samples.
• PCR verification in 1% agarose gel.
• No expected PCR product were shown in the gel for all 5 samples.

ACE2 group -Vector creation (Ilana & Hadas)

Protocol - PCR (new reagent) , Restriction

• Linearization and amplification of the plasmid with specially design primers that add His tag.
• Restriction map was made to verify the vector.
• 1% agarose gel - Verifying both PCR reaction and restriction map.

• Conclusion & Result:
1. The p406-FAV-mCore1 plasmid we extracted at 01/06 was cut as expected, therefore the plasmid is OK.
2. The PCR reaction (with new reagents) failed.
Aug 9th

Bacillus group - Cloning (Amir, Shay)  

Protocol - Gel electrophoresis

• Run agarose gel with PCR product from R.PCR pBS1C_LacZ
• One band (3000 bp) was detected

ACE2 group -Vector creation (Ilana & Hadas)  

• 1st PCR (R test) - A known primer for our plasmid (Alper 14) was used to determinate whether R primer is OK.
• 2nd PCR (F test) - A known primer for our plasmid (Alper 9) was used to determinate whether F primer is OK.
• 1st Gel (R test) - Verifying PCR reaction. Conclusion & Result: R primer – according to expectations
• 2nd Gel (F test) - Verifying PCR reaction. Conclusion & Result: Failed.
Aug 10th

Bacillus group - Cloning (Amir, Shay, Tomer, Dor)

Protocol - PCR , Gel Electrophoresis, Purification, Gibson assembly , Chemical transformation

• PCR for pBS1C plasmid:
1. Noa: R.PCR with primeSTAR GXL DNA polymerase.
2. Amir and Shay: R.PCR with Q5 polymerase
• Noa – bands have been seen on the expected size ~7.8Kb
• Noa - NanoDrop Result.

pBS1C

Concentration (ng/μl)

66.9

280/260

1.86

280/230

1.29

• Amir and Shay – band has been seen on the expected size ~7.8Kb
• Amir and Shay – Nanodrop Results:

pBS1C

Concentration (ng/μl)

28.2-30

280/260

1.97-1.92

280/230

1.3-1.34

• Gibson Assembly for pBS1C+ACE2/SYB15/SYB68/control
• Chemical transformation of the Gibson products to E.coli, plate and left for O/N at 37°C.

ACE2 group -Vector creation (Shanny & Ilana)

• PCR (F test) - A known primer for our plasmid (Alper 9) was used to determinate whether F primer is OK.
• Gel (F test) - Verifying PCR reaction.
• Conclusion & Result: Failed.

Gel group (Niv, Noa)

Protocol -Microgel Preparation (Step 2: 19-34)

• The created emulsion was sonicated (Figure 2).
• The air inside the round bottom flask was pumped out (Figure 3).
• Nickel solution was prepared (Figure 4).

Fig.2. Sonication of Created Emulsion.

Fig.3. Pumping Out Air from Round Bottom Flask.

Fig.4. Nickel Solution Preparation.
Aug 11th

Bacillus group - Cloning (Shay, Dor, Amir)

Protocol - Colony PCR, Gel electrophoresis

• Colony PCR for E.coli Top10cc with pBS1C_ACE2/Syb15/Syb68.
• Gel electrophoresis Results:
1. Syb15+syb68: noticed bands as expected (~600 bp).
2. ACE2: no visible bands.
• Starters: 5 ml LB + 5 μl AMP. Two colonies from syb15 and two from syb68.

ACE2 group -Vector creation (Ilana& Hadas)

• PCR (R test & AGAP preparation) - Amplification of 1st needed fragment for the AGAP (R->16 cut).
• Gel (R test & AGAP preparation) - Verifying PCR reaction (R-16 creation).
• PCR Cleaning (R test) - Cleaning the amplified 1st fragment (R-16).
by a commercial protocol (page 6).
• NanoDrop: 160.7 ng/ml , 260/280: 1.78, 260/230: 1.89 .

Gel group (Niv, Noa, Zixuan)

Protocol - Microgel Preparation (Step 2: 19-34)

• Repeat the steps that have been done yesterday.
• Ammonium persulfate (APS) solution was prepared fresh this time.
• Duration of polymerization reaction was extended to 1.5 hour and the solution was suspended in methanol for 1 hour.

Aug 12th

Bacillus group - Cloning (Amir, Shay)

Protocol - Glycerol stock, miniprep, colony PCR

• Glycerol stock: PBS1C_syb15 + Top10, PBS1C_syb68 + Top10
• DNA extraction - Miniprep - PBS1C_SYB15 + SYB68.
• NanoDrop - Elution volume:

	colony	Concentration (ng/μl)	280/260	280/230
Syb68-50μl	3'.5	160.8	1.83	1.58
Syb68-50μl	3.2	123	1.71	0.86
Syb15-30μl	2'.3	204.4	1.84	1.54
Syb15-30μl	2.8	215.7	1.79	1.21

• Colony PCR for PBS1C_ACE2
  Index for plates & colonies:
  1--> PBS1C_ACE2 rest
  1'--> PBS1C_ACE2 100 μl
  4--> Copy plate2 (from today)
  4'--> Copyplate1 (from yesterday)
  N.C--> Negative Control
• Gel Results: suspicious colonies: 1.3, 1.7, 1'.8, 1'.9, 1.16, 1.18

Aug 13th

Bacillus group - Cloning (Amir, Shay)

Protocol - miniprep, Glycerol stock

• DNA extraction - Miniprep pBS1C_ACE2.
• NanoDrop concentration-

colony	Concentration (ng/μl)	280/260	280/230
1'.8	163	1.92	1.77
1.16	163.5	1.89	1.52
1'.9	162.6	1.9	1.96
1.3	211.7	1.87	1.85
1.7	236.3	1.81	1.49
1.18	205.3	1.81	1.69

• Glycerol stock for colonies: 1.3, 1.7, 1'.8, 1'.9, 1.16, 1.18.

Aug 17th

Bacillus group - Cloning (Amir, Shay, Shany)

Protocol - Restriction, Sanger sequencing, gel electrophoresis

• Restriction reaction top10 colonies with pBS1C_ACE2 was done to verify the existence of the insert.
• Dilution (for Sanger sequencing).
• Gel electrophoresis Results:

Sybodies group - Amplification of the plasmid (Shanny & Ella)

Protocol - Chemical transformation to E.coli Top10CC

Transformation of pET-9d plasmid into TOP10 E.coli.
Aug 18th

Bacillus group - Cloning (Amir, Shay, Shany)

Protocol - Gibson assembly, Chemical transformation

• Gibson assembly to ACE2 (insert) and pBS1C (vector).
• Sanger sequencing for: SYB15_pBS1C - colony 2.8, SYB15_pBS1C colony 2.'3, SYB68_pBS1C colony 3.2, SYB68_pBS1C colony 3.'5.
• Gibson products transformed to TOP10.

Sybodies group - Amplification of the plasmid (Shanny & Ella)

• Nanodrop for the original plasmid tube - Conc. 0.5 ng/μl.
• Preparing plates with LB + Kan.
• Starter of TOP 10 with pET-9D.
Aug 19th

Bacillus group - Cloning (Amir, Shay, Shany)

Protocol - colony PCR, Gel electrophoresis

• 35 colonies (pBS1C _ACE) were taken colony PCR.
• Gel electrophoresis was preformed to PCR products.

Sybodies group - Amplification of the plasmid (Shanny & Ella)

Protocol - DNA extraction (Miniprep), Glycerol stock

• Miniprep for the plasmid.
• Nanodrop for the plasmid - Conc.128.7 ng/μl.
• Glycerol stock for the TOP10 cells with the plasmid inside.
Aug 23rd

Bacillus group - Cloning (Ella, Shanny A)

Protocol - Starter

• Starter for top10 colonies with pBS1C _ACE2.

Sybodies group - Ligation of the insert and the plasmid (Shanny & Ella)

Protocol - PCR, DNA Cleaning, Agarose Gel Electrophoresis

• PCR of the Syb sequences.
• Gel Electrophoresis as varification.

Aug 24th

Bacillus group - Cloning (Shay, Shany)

Protocol - Glycerol stock, miniprep

• Glycerol stock.
• DNA extraction – Miniprep.
• Nanodrop concentration:

colony	Concentration (ng/μl)	260/280	260/230
1.3	134.9	1.94	2.05
1.4	85.9	2.00	2.05
1.5	127.6	1.95	2.00
1.9	149.9	1.92	1.96
1.10	200.8	1.84	1.36
1.11	183.5	1.92	2.03
1.17	198.4	1.88	1.88
1.18	158.3	1.94	1.76

• Samples were taken to sequencing with R_colonyPCR primer.
• Sanger sequencing for: pBS1C_ACE2 colonies 1.18, 1.17, 1.11, 1.10, 1.9, 1.5, 1.4, 1.3.

Sybodies group - Ligation of the insert and the plasmid (Shanny & Ella)

Protocols- Restriction, ligation, Chemical transformation of E.Coli Top10CC

• Restriction:   1. Syb15/Syb68 and Nco1-HF + BamH1-HF.
  2. pET-9d plasmid and Nco1-HF + BamH1-HF.
  3. control - Nco1-HF + BamH1-H without templet
• Purification
• Nanodrop concentrations: Syb15: 9.2 ng/μl.
   Syb68: 3 ng/μl.
   PET 9D: 3.8 ng/μl.
• Nanodrop: Syb68: 32.7 ng/μl.
• Ligation with Syb15/Syb68 and pET-9d plasmid.
• Transformation of the ligation product to E.coli TOP10.

Aug 25th

Sybodies group - Ligation of the insert and the plasmid (Shanny & Ella)

Protocol - Restriction, ligation, Chemical transformation of E.Coli Top10CC

• We got growth in the control plate, so we did another Restriction with Nco1 from Fishman's lab, with ~1 ug DNA
  1. Syb15/Syb68 and Nco1-HF + BamH1-HF.
  2. pET-9d plasmid and Nco1-HF + BamH1-HF.
  3. control - Nco1-HF + BamH1-H without template
• Nanodrop: Syb15: 23.5 ng/μl
  Syb68: 26.0 ng/μl
  PET 9D: 8 ng/μl
• Cleaning the DNAs
• Ligation with Syb15/Syb68 and pET-9d plasmid.
  We put 50ug pET 9D + 21.63ug syb15 ; 50ug pET 9D + 23.26ug syb68
• Transformation of the ligation product to E.coli TOP10.
• Transformation of pET-9d plasmid to TOP10 for another amplification.
Aug 26th

ACE2 group -Vector creation (Ilana& Hadas)

Protocol - PCR (R->F), PCR (R->9)

• Linearization and amplification of the plasmid with specially design primers that add His tag, This time with GXL polymerase.
• Amplification of the 2nd fragment needed for AGAP (F-9) while adding His tag to the sequence.

Sybodies group - Ligation of the insert and the plasmid (Shanny & Ella)

Protocol - Chemical transformation of E.Coli Top10CC

• Mini-prep for 3 starters.
• Nanodrop:
  Starter 1: conc.- 58.2 ng/μl
    260/280- 1.87
    260/230- 1.28
  Starter 1: conc.- 92.2 ng/μl
    260/280- 1.94
    260/230- 0.71
  Starter 3: conc.- 92.2 ng/μl
    260/280- 1.90
    260/230- 1.68
• We got growth in the control plates.
Aug 27th

ACE2 group -Vector creation (Ilana& Hadas)

Protocol - Agarose Gel Electrophoresis, PCR cleaning

• Gel Electrophoresis to Verifying both PCR reactions (R-9 creation and R-F creation).
• Conclusion & Result: All Results are as axpected: F->9 ~4300bp, 9->1 ~3300bp, R->F ~6000bp
• Cleaning of the amplified PCR products: 2nd (F-9) and R-F fragments.
• Amount of PCR product - R->F: 46.1 μl, F->9: 44 μl
• NanoDrop concentration: R->F: 60.5 (ng/μl), 260:280 1.7, 260:230 1.4
F->9: 199.0(ng/μl), 260:280 1.81, 260:230 2.19
Aug 30th

Sybodies group - Restriction map (Shanny & Ella)

Protocol - Restriction

• In order to confirm the plasmid content, we did restriction map.

Gel group (Niv, Noa, Yara, Zixuan)

Protocol - Microgel Preparation (Step 1), Thaw and Establish 293-F Cells

• The dripping process wasn’t continuous because of the several meeting we had in this day.

Aug 31st

Bacillus group - Cloning (Amir, Shay, Dor)

Protocol - PCR, gel electrophoresis, B. subtillis competent cells stock, transformation

• PCR for ACE2 g_block
• Gel electrophoresis for PCR was made
• Gel Results: No bands in the control, 2 bands of 500 bp instead of 2500 bp
• For B. subtillis Competent cells protocol: Stocks for the sp + spitzen salts + autoclave

Sybodies group - Ligation of the insert and the plasmid- again (Shanny & Ella)

Protocol - Chemical transformation of E.coli Top10CC

• Miniprep for the starters:

	concentration	260/280	260/230
1	199ng/μl	1.90	2.09
2	118.1ng/μl	2.06	1.83
3	161.4ng/μl	1.94	1.92
4	126.9ng/μl	2.02	1.82
5	103.9ng/μl	1.99	1.72

Gel group (Niv, Yara, Zixuan)

Protocols- Microgel Preparation (Step 2: 10-18)

• The washing of Dowex before using was not so successfμl since the pH was still acidic even after many times of washing. Therefore, we decided to use the pH of DDW as a standard and wash the Dowex till its pH got close to the standard.
• For lyophilization, the solution was separated to 11 falcons with ~20ml each. These falcons were covered with a piece of wipe and rubber band and were quickly frozen using liquid nitrogen. After they were solidified, we used chew sticks to make small holes on the wipe. Then the falcons were sent for lyophilization.

Sep 1st

Bacillus group - Cloning (Amir, Shay)

Protocols- PCR, Gel Eletrophoresis

• PCR for ACE2 G-block with GXL polymerase instead of Q5

ACE2 group -YFP production (Ilana& Hadas)

Protocols- PNK (R->F), ligation (R->F PNK), heat shock transformation

• PNK- Phosphate addition to linearized fragment (PCR product), in order to close it to circμlar plasmid later on.
• Ligation in order to Close the plasmid to circμlar form, to enable transformation.
• Transformation of the circμlar plasmid to top10 E.coli as shuttle vector.
Sep 2nd

Bacillus group - Cloning (Amir, Shay, Shany)

Protocols- B. subtillis competent cell (sp1 preparation), PCR, Gel electrophoresis

• SP1 preparation (300ml)
• PCR for ACE2 g block with GXL polymerase instead of Q5
• preparation for competent PY79
• Gel electrophoresis for PCR was made
• Gel Results: ~500bp product was seen clearly and some very weak bends of ~2500bp were also detected.

ACE2 group -YFP production (Ilana& Hadas)

Protocols- Plate counting, F2 primer dilution, Colony PCR, Gel electrophoresis

• Plats counting: Lig1 – 100μl 4 colonies, rest 42 Colonies. Lig 2 – 100μl 8 colonies, rest 52 Colonies
• Dilution for F2 primer was done.
• Colony PCR was done to Verify if the transformation of the plasmid was done properly
• Gel electrophoresis was done to verifying the colony-PCR.
• Conclusion & Result: Colonies #1 & #2 are with 700bp band (unexplainable), colonies #3, #5 & #10 are with smear (unexplainable).
• Colony PCR with GXL was done to Verify if the transformation of the plasmid after the failure of the colony PCR with PCR mix.

Sybodies group - Ligation of the insert and the plasmid- again (Shanny & Ella)

Protocols- Restriction, Ligation

• Restriction: 1. Syb15/Syb68 and Nco1-HF + BamH1-HF – 1ug from each sybody and 2ug from the plasmid
2. pET-9d plasmid and Nco1-HF + BamH1-HF- 2ug
3. control - Nco1-HF + BamH1-H without template
• Cleaning the DNAs.
• Nanodrop:

concentration

260/280

260/230

PET 9D

20.9ng/μl

1.97

1.2

Syb15

25.4ng/μl

1.97

1.25

Syb68

24.1ng/μl

2.07

1.17

• Ligation O/N in 16C with Syb15/Syb68 and pET-9d plasmid.
Sep 3rd

Bacillus group - Cloning (Amir, Shay, Shany, Dor)

Protocols- Soft agar, PCR

• Soft agar for the PCR products from 02/09/2020: No Results were detected
• Purified the rest of the cut (c) sample (20 μl).
• Starter of the Bacillus from 02/09/20 – O.D was 0.6- too low for the protocol. It was decided to throw the starter and start with plating before setting a starter.

ACE2 group -YFP production (Ilana& Hadas)

Protocols-gel electrophoresis

• Gel running for verifying the colony-PCR.

• Colony picked: 1,2,5 from copy plate ,15-20 from lig1 and lig2 plate 1% agarose.

• Conclusion & Result: Colonies #1, #2 and #5 – same as previous Result. Colonie #19 is with 700bp band (unexplainable), colonies #20 & #21 are with smear (unexplainable).

Gel group (Niv, Yara, Zixuan)

Protocols- Cell Count, Cell Cμlture Maintenance, Microgel Preparation (Step 2: 19-34)

• The number of cells was determined via both cell counting and fluorescence-activated cell sorting (FACS).
• Monomer products were collected from the falcons after lyophilization, and the weight was about 1.41gr.
Sep 6th

Bacillus group - Cloning (Dor, Shany)

• Plate prepration
• Plate B. subtillis PY79 on two LB plates 37°C O/N for competent protocol.

ACE2 group -YFP production (Hadas)

Protocols- Starters

• Starter for one of the unexplainable colonies was made in order to extract the plasmid and sequence it.

Sybodies group - Ligation of the insert and the plasmid- again (Shanny & Ella)

Protocols-Chemical transformation of E.Coli Top10CC

• Transformation of the ligation product into E.coli TOP10.

Gel group (Niv, Yara, Zixuan)

Protocols-Microgel Preparation (Step 2: 19-34)

• The APS concentration was changed to 0.15gr/ml this time.
• Stirring after starting to add the monomer solution lasts for almost 2hr.
Sep 7th

Bacillus group - Cloning (Shay, Shany)

Protocol - PCR, Gel, transformation of cots to top10

• amplify the ACE2 segments that were cut/ stabbed from gel and purified on 3.9 by PCR
• Run in 1% gel. Expected 2500 band was not detected
• Transformation of pUC57_cotC/G to Top10 :
• Sp1 solution was infected.
• Prepared sp1 for B. subtillis competent protocol was done
• Prepared 5 ml starter for B. subtillis PY79 for competent protocol in sp1.

ACE2 group -YFP production (Hadas)

Protocol - Miniprep (NucleoSpin kit), sequencing, PNK (R->F), ligation (R->F PNK)

• Miniprep for extraction of the plasmid from the unexplainable colony to sequence it.
• Plasmid concentration via NanoDrop: #1 – 827.2 (ng/μl), 1.87 (260,280), 2.21 (260/230). #2 – 55.8 (ng/μl), 2.02 (260,280), 0.65 (260/230).
• Sending the plasmid from the unexplainable colony to sequencing.
• Sequencing Results: His tag was added as expected but 5 bases were eliminated up-stream to the His-tag.
• YFP production – Second attempt of cloning: Transformation of p4160-FEC-mCore1 with His tag to E.coli, in order to produce YFP.
• PNK - Phosphate addition to linearized fragment (PCR product), in order to close it to circμlar plasmid later on.
• Ligation in order to Close the plasmid to circμlar form, to enable transformation.

Sybodies group - Verification of the insertion (Shanny & Ella)

Protocol -Colony PCR

• Colony PCR to make sure the ligated plasmid enter the E.coli TOP10.
  1. Syb15 + pET 9D- wanted length 555bp
  2. Syb68 + pET 9D- wanted length 773bp
• Primer dilution:
  F - pET colony PCR- 29.2 nmol + 293μl UPW= 0.1 nmol/μl
  R - syb68 colony PCR- 31.8 nmol + 318μl UPW= 0.1 nmol/μl
  R - syb15 colony PCR- 29.8 nmol + 298μl UPW= 0.1 nmol/μl

• We took colonies 3,17,18.19 for mini prep.

• We took colonies 9,10,17.18 for mini prep.

Gel group (Niv, Yara)

Protocol -Cell Count, Cryopreservation, Microgel Preparation (Step 2, 35)

• The cμltured cells were counted again and were frozen before transfection.
• Centrifuged the falcon of yesterday for 3 min, 4000 rpm. Supernatant was discarded without touching the precipitation. Add DDW until volume of 40ml.
• Repeat the above-mentioned steps times.
• We didn't discard all of the solution because the precipitation wasn't stable. After that, we placed the falcon with a holed cap in the hood for a day. And day after, we placed it in the lyophilization device.

Sep 8th

Bacillus group - Cloning (Shay, Shany)

Protocols- Bacillus competent cells, Chemical transformation.

• Starter for B. subtillis PY79 for competent protocol was done:
one colony of PY79 from palate in sp1 , one colony of PY79 from palate in LB
• B. subtillis from –80°C Glycerol stock was transformed to LB and sp1 Continued transformation of pUC57_cotC/G • Smears were detected in all the plates.

ACE2 group -YFP production (Hadas)

Protocols- Heat- Shock transformation

• Transformation of the circμlar plasmid to Top10 E.coli as shuttle vector.

Sybodies group - Verification of the insertion (Shanny & Ella)

Protocols- Mini prep

• Amplification of the ligated plasmid from the selected colonies
• Miniprep for the starters:

	concentration	260/280	260/230	Comments	Answer from sequencing
Syb15- 3	106.1ng/μl	1.83	1.38	Sent to sequencing- 1F1CKAA024 (first sequencing)	The His tag is damaged- only 4 his instead of 6
Syb15- 17	76.7ng/μl	1.75	0.95
Syb15- 18	91.4ng/μl	1.83	1.38	Sent to sequencing- 1F1CKAA025 (first sequencing)	Mixed template
Syb15- 19	88.3ng/μl	1.88	1.58	Sent to sequencing- 1F1CKAA011 (second sequencing)	Not completed sequence
Syb68- 9	120.8ng/μl	1.83	1.27	Maybe infected
Syb68- 10	108.2ng/μl	1.87	1.63	Sent to sequencing- 1F1CKAA026 (first sequencing)	Mixed template
Syb68- 17	94.9ng/μl	1.85	1.54	Sent to sequencing- 1F1CKAA027 (first sequencing)	Mixed template
Syb68- 18	90.8ng/μl	1.79	1.09	Sent to sequencing- 1F1CKAA015 (second sequencing)	Not completed sequence

Gel group (Niv, Yara)

Protocols- Transfection

• The cμltured cells were transfected with designed plasmids.

Sep 9th

Bacillus group - Cloning (Shay, Shany)

Protocols- Competent B. subtillis

• Groth (measured as O.D 600 nm) Competent B. subtillis (internet protocol)

Time (min)	Dilute (PY79)	O.D (PY79)	Dilute (WT)	O.D (WT)
0	1	0.006	1	0.021
60	1	0.0492	1	0.0504
90	1	0.0557	1	0.0559
120	1	0.0705	1	0.0858
150	1	0.1421	1	0.1075
180	1	0.1926	1	0.2751
210	1	0.2570	1	0.3901
240	1	0.3491	1	0.4629
270	1	0.4567	1	0.4393

ACE2 group -YFP production (Hadas)

Protocols- plate counting, colony PCR, gel electrophoresis

• Plats counting: Lig3 – 100μl 0 colonies, rest 1 Colonies. Lig 4 – 100μl 1 colonies, rest 2 Colonies. Control – 0 colonies.

Fig.1. LB agar + ampicillin plate circμlar plasmid to top10 E.coli

• Colony PCR for Verifying the appropriate transformation of the plasmid.
• Gel running for Verifying the colony-PCR.
• Conclusion & Result: All reaction failed.

Gel group (Niv, Zixuan)

• As verification methods of the transfected cells, Plate Reader and FACS were used to determine both the total cell counts and the number of cells with fluorescence.
• According to the FACS Results, one day after transfection, 62.2% of the cells in tested sample were alive, and 79.9% among these cells were single cells while 20.4% of these single cells had fluorescence.
• However, no red color can be observed visually in the medium.

Sep 10th

Bacillus group - Cloning (Amir, Shay, Shany)

Protocol - chemical transformation, transformation to bacillus

• Transformation of pUC57_cotC/G to Top10 was done according to the protocol and was plate on LB+AMP plates. 2 ug of plasmids were used.
• Transformation of pBS1C_syb15\68 to B. subtillis py79 was done according to protocol. Was plated on LB+CM plates for 37°C O/N incubation.

Gel group (Niv, Zixuan)

Protocol - Cell Cμlture Maintenance

• The transfected cells were again taken for Plate Reader and FACS analysis.
• According to FACS Results, two days after transfection, 70.5% of the cells in tested sample were alive, and 89.2% among these cells were single cells while 44.9% of these single cells had fluorescence.
• Again, no red color can be observed visually in the medium.
• The Microgel product of 2nd attempt was recovered after lyophilization (Figure 13).
Sep 12th

ACE2 group -YFP production (Hadas)

Protocol - starters (for sequencing)

• Starter for one of the unexplainable colonies was made in order to extract the plasmid and sequence it.
Sep 13th

Bacillus group - Cloning (Amir, Shay, Shany)

Protocol - Colony PCR

• Colony PCR was done to 60 colonies For PY79_PBS1C_SYB15/68
• Positive colonies were detected for syb68 colonies: 3.5.13, 3.5.1,3.5.2, 3.5.15,3.2.7: 
• No reasμlts for syb15 colonies
• For TOP10_Puc57_cotC/G:  Some colonies were detected in rest plates.
• No colonies were detected in control of Top10 

ACE2 group -YFP production (Hadas)

Protocols- Miniprep (NucleoSpin kit), Sequencing, PCR (R-16 creation before cutting)

• Extraction of the plasmid from the unexplainable colony to sequence it.
• Concentrations (NanoDrop):
   #1 – 92.4(ng/μl), 1.98 (260,280), 1.45 (260/230).
   #2 – 92.8 (ng/μl), 1.84 (260,280), 1.73 (260/230).
   #3 – 55.1 (ng/μl), 1.98 (260,280), 1.03 (260/230).
   #4 – 125.2 (ng/μl), 1.85 (260,280), 1.93 (260/230).
   #5 – 272.6 (ng/μl), 1.93 (260,280), 1.64 (260/230).
• Sending the plasmid from the unexplainable colony to sequencing.
• Sequencing Results: Unconclusive sequence, probably more than 1 colony was picked.
• ACE production – fragments preparation:
Transformation of p4160-FEC-mCore1 with His tag and ACE2 instead of YFP to W303 yeasts strain, in order to produce ACE2 protein. The method used is Any-gene-any-plasmid (AGAP),Ttree fragments (with 20-40 bases overlapping) are needed in 1ug amount: R-16 cut (without the YFP), F-9 and ACE2 variant (from G-block). • 1st PCR Amplification of 1st needed fragment for the AGAP (R->16 cut).
• Expected Results - Revers test: 1600bp, -Control:0bp, +Control:1100bp.
• Gel running for Verifying PCR reaction.

• Conclusion & Result: Reaction failed

Gel group (Niv, Zixuan)

Protocol - Gravity System for His-tagged Protein Purification, Magnetic System for His-tagged Protein Purification

• The transfected cells were again taken for Plate Reader and FACS analysis.
• According to FACS Results, five days after transfection, 42.7% of the cells in tested sample were alive, and 96.1% among these cells were single cells while 48% of these single cells had fluorescence.
• Although still no color change can be visually detected, according to the Results from Plate Reader and FACS, it’s reasonable to conclude that the cells have started to secrete fluorescent proteins into the medium.
• The samples were split into two, one of them was purified via gravity system and the other was purified with magnetic beads.

Protein Purification with Gravity System

Protein Purification via MagneHis™ Protein Purification System

Sep 14th

Bacillus group - Cloning (Amir, Shany)

Protocols- Glycerol stock, miniprep, plate reader experiment, Restriction assay

• Glycerol stock and miniprep were prepared from TOP10_Puc57_cotC/G starters. 
• Nanodrop Concentrations of DNA extraction - miniprep: 

	[c] ng/μl	260/280	260/230
PUC57_cotC_1	108.8	1.88	2.03
PUC57_cotC_2	46.3	1.91	1.69
PUC57_cotG_1	69	1.96	2.00
PUC57_cotG_2	70.7	1.70	0.81

• Restriction of plasmid pBS1C_SYB15/68 and CotC/G inserts: 
• 20 μl of restriction Results were loaded on gel and expected lengths were cut from gel 
• CotC1/2 were not detected 
• Nanodrop Concentrations of DNA extraction - Miniprep for gel Results was done: 

	[c] ng/μl	260/280	260/230
PBS1C_syb15_2.3	21.4	2.14	0.71
PBS1C_syb15_2.8	14.4	2.50	0.41
PBS1C_syb68_3.2	16.2	2.23	0.69
PBS1C_syb68_3.5	17.5	2.12	0.85
cotG1	9.1	2.34	0.54
cotG2	8.1	3.14	0.44

ACE2 group -ACE production – fragments preparation (Hadas,Shay)

Protocol - primer dilution, PCR (R->16 & G- block), gel electrophoresis, PCR cleaning

• Primers dilution was done to Alper 16, Alper 10, Gb_F, and Gb_R.
• 2nd PCR Amplification of 1st needed fragment for the AGAP (R->16 cut).
• Expected Results- R->16: 1600bp, -Control: 0bp, +Control – 1100bp
• PCR Amplification and size verification of three G-blocks.
• Gel running for Verifying both PCR reactions.

• Conclusion & Result: All R->16 reactions are as expected (1600bp), +Control for R-16 is as expected as well (1100bp), G-blocks are all according to expectations (2600bp) and have few by-product (to be expected from that polymerase).
• R-16 (PCR product) cleaning for restriction.

Amount of PCR product:

PCR tube	R-16	R-16	R-16	X1	V1	Vneg
Volume [μl]	38.4	39.5	30	34	38	36

• NanoDrop Concentration:

	R-16	R-16	R-16	X1	V1	Vneg
ng/μl	75.1	49.9	46.9	129.6	109.4	93.3
260/280	1.8	1.78	1.74	1.88	1.89	1.9
260/230	1.53	1.2	1.3	2.02	1.87	1.65

Mix of all three R-16 reactions :59.4 ng/μl, 260/280 2.84, 260/230 1.24
• 3rd PCR Amplification of 1st needed fragment for the AGAP (R->16 cut). In order to create 1ug of cut product, we set up few PCR reactions followed by few restriction reactions.
• R-16 (PCR product) cleaning for restriction later on.
• NanoDrop Concentrations:

	1	2	3	4	5	6
ng/μl	38.1	63.8	51.6	55.6	57.8	55.7
260/280	1.67	1.74	1.72	1.73	1.71	1.76
260/230	1.34	1.71	1.62	1.72	1.77	1.62

Mix of all:54.8ng/μl, 260/280 1.76, 260/230 1.61

Sybodies group - Verification of the insertion (Shanny & Ella)

Protocols- Colony PCR

• Amplification of the ligated plasmid from the selected colonies
• Miniprep for the starters:

	concentration	260/280	260/230	Comments	Sequencing
Syb15- 14	81.8ng/μl	1.97	1.75	Sent to sequencing- 1F1CKAA008	Not completed sequence
Syb15- 15	80.0ng/μl	1.88	1.45	Sent to sequencing	Perfect!
Syb15- 1	89.7ng/μl	1.95	1.53	Sent to sequencing	Perfect!
Syb15- 16	79.7ng/μl	1.91	1.59
Syb15- 2	48.4ng/μl	1.91	1.42
Syb15- 6	63.5ng/μl	1.86	1.31
Syb15- 10	65.2ng/μl	1.90	1.31
Syb68- 14	80.1ng/μl	1.96	1.55	Sent to sequencing	Perfect!
Syb68- 15	85.2ng/μl	1.93	1.49	Sent to sequencing	Not completed sequence
Syb68- 16	63.0ng/μl	1.97	1.55	Sent to sequencing	Not completed sequence

• Colony PCR- another 19 colonies for each sybody

Gel group (Niv, Zixuan)

• After purification, the samples were loaded into SDS-page gel. PM2600 ExcelBand (3 colors, high range) was used as protein ladder and two concentrations of bovine serum albumin (BSA) were used as protein standards.

Sep 15th

Bacillus group - Cloning (Amir, Shany)

Protocol - Ligation, plate reader, Miniprep.

• Ligation of syb15/68 to pBS1C vector.
• transformation to E. coli Top10. 
• Plate reader experiment: According to protocol for E. coli. 
• No florescence was detected. 
• New stocks of syb68/15 were made
• NanoDrop Results

	Concentration (ng/μl)	260/280	260/230
PBS1C_syb15-1	166.5	1.74	1.11
PBS1C_syb15-2	100.7	1.79	1.32
PBS1C_syb68-1	188.3	1.79	1.32
PBS1C_syb68-2	187.7	1.70	0.95

ACE2 group - ACE production – fragments preparation (Hadas)

Protocol - Restriction (R->16 for AGAP), gel electrophoresis, Cleaning from gel

• 1st Restriction was done (R-16, AGAP).
• Gel electrophoresis.
• NanoDrop Concentration:
8.5 ng/μl, 1.82 260/280, 0.51 260/230
9.7 ng/μl, 2.14 260/280, 0.56 260/230

Gel group (Niv, Zixuan)

• Results of SDS-page were obtained.

Fig.1. SDS-Page Results

Sep 16th

Bacillus group - Cloning (Amir, Shany, Shay)

Protocol - Transformation to B. subtillis, colony PCR, Restriction, Miniprep

• Transformation of syb_15/68 to PY79  
• Colony PCR for pBS1C+CotG+SYB15/68
• Restriction of Cots and pBS1C_syb15
• Gel electrophoresis. 
• DNA extraction. (The miniprep for the restricted products was thrown to bin by accident.) 

ACE2 group - ACE production – fragments preparation (Hadas)

Protocol - Restriction (R->16 for AGAP), gel electrophoresis, Cleaning from gel

• 2nd Restriction was done (R-16, AGAP).
• Gel electrophoresis.
• NanoDrop Concentration: 11ng/μl, 2.33 260/280, 0.62 260/230

Gel group (Niv, Zixuan)

Protocol - Dialysis

• Dialysis of purified proteins.

Fig.1. Dialysis System

Sep 17th

Bacillus group - Cloning (Amir, Shany, Shay)

Protocol - - Transformation to B. subtillis, miniprep

• Transformation results of PY79_pBS1C_syb15/68: again, colonies were detected in negative control of PY79 without plasmid.
• NanoDrop concentration- Miniprep stocks were prepared from 16/09/2020 starters: 

	[c] ng/μl	260/280	260/230
9-pBS1C_syb68_3.2_cotG	754.1	1.88	2.32
PBS1C_syb68_1	251	1.63	0.87
Puc57_cotc_1	156.6	1.88	1.83
Puc57_cotg_1	165.7	1.48	0.57
PBS1C_syb15_1	116.7	1.78	1.19
4-pBS1C_cotg_syb15	36.1	1.64	0.65
PBS1C_syb68_2	107.2	1.85	1.54
Puc57_cotg_2	10.4	1.63	0.52
2-pBS1C_cotc_syb15	764.8	1.88	2.31
PBS1C_syb15_2	298.1	1.65	0.96
PBS1C_syb68_3	160.2	1.85	1.47
Puc57_cotc_2	202.5	1.39	1.17
Puc57_cotg_3	57.5	1.52	0.60
PBS1C_syb68_4	135.8	1.82	1.31
1-pBS1C_cotg_syb15	669	1.9	2.31
Puc57_cotg_4	44.2	1.7	0.9
Puc57_cotc_3	188.2	1.85	1.57
Puc57_cotc_4	220.5	1.78	1.21

ACE2 group - ACE production – fragments preparation (Hadas)

Protocols- PCR (F->9 creation for AGAP & R->16), gel electrophoresis, PCR cloning

• 1st PCR reaction to create the 2nd
• 4th PCR for fragment amplification (R->16 cut).
• Gel electrophoresis.

• Conclusion & Result: All F->9 reactions are as expected (4300bp), reactions #2, #3, #4, #6, #7, #8 & #9 in R-16 are as expected (1600pb), reactions #1 & #5 in R-16 failed.
• NanoDrop Concentration:

	F9 1	F9 2	F9 3	R16 2	R16 3	R16 4	R16 6	R16 7	R16 8	R16 9	R16 10
ng/μl	189.6	30.1	163.2	147.5	26.4	21.9	21.9	31.5	31.9	21.1	24.9
260/280	1.87	1.51	1.88	1.87	1.65	1.54	1.54	1.54	1.62	1.48	1.54
260/230	2.05	1.28	2.03	2.03	0.85	0.76	0.76	1.24	0.61	1.04	1.14

Gel group (Zixuan)

Protocols- Protein Concentration

• Concentration determination via Nanodrop post-dialysis, Blank: Dialysis buffer (Table 1).
• Concentration with Amicon, and concentration determination via Nanodrop post-concentration.
• Final concentrations were presented as well (Table 2). Blank: Dialysis buffer.

Table 1: Protein Concentration after Dialysis, Measured by Nanodrop

Name	Concentration (mg/ml)
BSA 1:100	0.005
hACE2-tdPP7-His	0.246
hACE2-mCherry-tdPP7-His	0.179
hACE2-mCherry-tdPP7-His’	0.015

(There were two values of the hACE2-mCherry-tdPP7-His sample since the volume was separated into two Eppendorf.)

Table 2: Protein Concentration after Concentrating with Amicon, Measured by Nanodrop

Name	Concentration (mg/ml)
BSA 1:100	0.013
hACE2-tdPP7-His	1.117
hACE2-mCherry-tdPP7-His	0.108

Sep 21st

Bacillus group - Cloning (Dor)

Protocol - Chemical transformation

• Top10 grew in CM -> Contaminated CM stock -> New CM stock was prepared.
   • Plates of LB + CM were prepared.  
• Colony isolation and plating from –80°C freezer of B. subtillis PY79 (Weizmann institute).  
• Chemical transformation of the competent B. subtillis PY79 from the –80°C freezer (09/09/2020).  
• The plasmid used is from Sari the pBS1c_lacZ.  

ACE2 group - ACE production – fragments preparation (Hadas)

Protocol - PCR (R->16), gel electrophoresis, PCR cleaning

• 5th PCR for fragment amplification. (R->16 cut).
• Gel electrophoresis.
• Conclusion & Result: All R-16 are as expected (1600pb) with by-products.
• NanoDrop Concentration:

PCR tube	1	2	3	4	5	6	7	8	9	10
ng/μl	76.8	57.9	84.2	73.6	60.6	67.3	92.2	66.9	81.8	71.8
260/280	1.70	1.69	1.72	1.71	1.71	1.71	1.74	1.74	1.76	1.79
260/230	1.69	1.46	0.65	1.75	1.34	1.34	1.92	1.89	1.89	1.77
Volume	21.5	22	20.5	22	26	26	25	23.5	23.5	22
ng	1651	1273	1726	1619	1575	1749	2305	1572	1922	1579

Sep 22nd

Bacillus group - Cloning (Amir, Shany)

Protocol - Restriction, Soft gel, Miniprep, Ligation

• Restriction: Puc75_cotC, PUC57_cotG_1, PBS1C_68_1, PBS1C_15_1.
•  NanoDrop concentration: DNA extraction – Miniprep:

	[c] ng/μl	260/280	260/280
CotC	1.8	1.86	0.2
PBS1C_syb15	30.7	1.92	0.45
PBS1C_syb68	7.6	1.98	0.71

• Ligation was done to the PCR product and CotC insert
•  Second restriction for the verification of pBS1C_cotG_syb15/68 followed by gel electrophoresis.
• Transformation results from yesterday were obtained.  

ACE2 group - ACE production – fragments preparation (Amir)

Protocol - Restriction, Gel electrophoresis

• Gel electrophoresis.
• Conclusion & Result: The 6000 is probably the full plasmid since it got cut to 4000bp and 2000bp as it should. It is possible to clean the 900 bend.

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol - Miniprep

• 2 starters for 1 successful sequenced colony of each Sybody.

Sep 23rd

Bacillus group - Cloning (Amir, Shany)

Protocol - ligation, chemical transformation, starter

• Transformation of ligation product
• Starters were made:  Top10_pUC57_cotG_1, Top10_pUC57_cotG_2, Top10_pUC57_cotG_3, Top10_pUC57_cotG_4, Top10_pUC57_cotC_1, Top10_pUC57_cotC_2, Top10_pUC57_cotC_3, Top10_pUC57_cotC_4, Top10_pBS1C_LacZ_1,  Top10_pBS1C_LacZ_2. 

ACE2 group - ACE production – fragments preparation (Hadas)

Protocol - Restriction (R->16 for AGAP), gel electrophoresis, Cleaning from gel

• 3rd Restriction was done to R-16 for AGAP
• Gel electrophoresis.
• Conclusion & Result: Electrodes were connected in the wrong way, all DNA left the gel

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol - Miniprep

• Nanodrop:

	concentration	260/280	260/230
pET9D+Syb15 1	96.5ng/μl	1.87	1.60
pET9D+Syb15 2	97.1ng/μl	1.91	1.61
pET9D+Syb68 1	77.5ng/μl	1.91	1.44
pET9D+Syb68 2	114.3ng/μl	1.91	1.66

Sep 24th

Bacillus group - Cloning (Amir, Dor)

Protocol - Miniprep, Restriction, Gel electrophoresis, Ligation

• NanoDrop concentration: Miniprep stocks were prepared from yesterday's starters: 

	[c] ng/μl	260/280	260/230
Puc57_cotC_1*	146.6	1.7	1.12
Puc57_cotC_2*	138	1.82	1.58
Puc57_cotC_3*	85.1	1.73	1.18
Puc57_cotC_4*	196.1	1.8	1.46
Puc57_cotG_1*	34.4	1.79	1.25
Puc57_cotG_2*	87.1	1.68	0.94
Puc57_cotG_3*	163.2	1.74	1.18
Puc57_cotG_4*	104.4	1.76	1.39
Pbs1c_lacZ_1	106.2	1.75	1.23
Pbs1c_lacZ_2	119.3	1.75	1.25

• Restriction for PBS1C_syb15/68+puc57_cotC/G 
•  20 or 40 μl of restriction results were loaded on gel and expected lengths were cut from gel.
•  Results- CotC barely visible lane 2.   
• NanoDrop concentration - PCR cleaning:

	Puc57_cotC_3	Pbs1C_syb68_3	Puc57_cotG_3*	PBS1C_syb15_2
Weight after gel cut (mg)	184.6	101.6	153.5	166.5
Conc. (ng/μl)	4.1	55.1	6.6	45.9
280/260	1.54	1.85	2.47	1.9
280/230	0.1	1.59	0.41	0.19

• Ligation was done with: PBS1C_syb15_cotC_3, PBS1C_syb15_cotg_3*, PBS1C_syb68_ cotc_3. And: N.C- PBS1C_syb15, N.C- PBS1C_syb68, PBS1C_syb68_ cotg_3* 
• Transformation of ligation product to E. coli DH5a competent bacteria. (from Noa) 
• Transformation products were plated to amp plates and incubated at 37°C o/n. 

ACE2 group - ACE production – fragments preparation (Ilana, Yara & Hadas)

Protocol - PCR (R->16), gel electrophoresis, PCR cleaning, Restriction (R->16 for AGAP), gel electrophoresis, Cleaning from gel

• 6th PCR to amplify the 1st needed fragment for the AGAP (R->16 cut).
• Gel electrophoresis.

• Conclusion & Result: All R-16 are as expected (1600pb) with by-products.
• R-16 (PCR product) cleaning for restriction.
• NanoDrop Concentration:

	ng/μl	260/280	260/230	μl	ng	Number of restriction reaction
1	79.1	1.79	1.76	18.5	1463	1
2	68.7	1.70	1.71	24.6	1690	1
3	72.2	1.70	1.74	25.1	1812	1
4	63.9	1.63	1.76	22.5	1437	2
5	63.5	1.27	1.77	24.8	1571	2
6	65.6	1.70	1.77	26.7	1751	3.4 from 2 23 from 3
7	70.4	7.63	1.74	26.4	1858	3
8	66.3	1.15	1.79	26.2	1737	3
9	657.6	1.71	1.77	25.9	1750	3
10	63.0	1.75	1.77	25.3	1594	4
11	66.3	1.71	1.75	26.1	1730	4
12	65.9	1.48	1.78	25.8	1687	4
13	65.2	1.74	1.75	26	1695	5
14	61.7	1.60	1.79	25.6	1579	5
15	67.4	1.68	1.76	25.1	1691	5
16	66.3	1.41	1.78	25.7	1704	6
17	59.6	0.99	1.78	24.7	1472	6
18	65	0.92	1.75	24.7	144	6
19	63.8	1.63	1.7	21.6	1429	6.6 from 6 15.8 from 7
20	64.3	1.70	1.81	22.4	1601	7
21	57.4	1.1	1.76	249	1469	7
22	62.6	1.69	1.74	25.6	1571
23	42.5	1.48	1.74	25.3	1075	7

• 4rd Restriction was done to R-16 for AGAP
• Gel electrophoresis.
• Conclusion & Result: 900 bands are cuttable.
• Cleaning 900bp band from gel.
• Gel amount :

	1	2	3	4
Gel amount [gr]	0.5	0.59	0.6	0.51
Membrane binding solution [μl]	500	590	600	510

Sep 25th

Bacillus group - Cloning (Zixuan)

• Plate results from 24/09/2020

Fig.1. DH5a_pBS1C_SYB15_CotG_3*, 24/09/2020, AMP

Fig.2. DH5a_pBS1C_SYB68_CotG_3*, 24/09/2020, AMP

Fig.3. DH5a_pBS1C_SYB68_CotC_3, 24/09/2020, AMP

Fig.4. DH5a_pBS1C_SYB15, Negative control, 24/09/2020, AMP

Fig.5. DH5a_pBS1C_SYB15_CotC_3, 24/09/2020, AMP

Fig.6. DH5a_pBS1C_SYB68, Negative control, 24/09/2020, AMP
Oct 4th

ACE2 group - ACE production – fragments preparation (Noa & Shanny)

Protocol - - Plating

• Plating W303 yeasts (Leo2, Trp1, Ura3, Ade1, His3) on YPD.
• Two strains (YA619 and YA204) where plated in the same plate.

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol - - Transformation into KRX

• Dilution:
pET9D+Syb15 2 : original concentration- 97.1ng/μl
  1:10-> 9.71ng/μl
  1:100-> 0.97ng/μl

pET9D+Syb68 2: original concentration- 114.3ng/μl
  1:10-> 11.43ng/μl
  1:100-> 1.14ng/μl

• Transformation was done with 1, 10, 50 ng ligated plasmid from each Sybody.
Oct 5th

Bacillus group - Cloning (Amir)

Protocol - colony PCR,gel

• Colony PCR for ligated colonies- suspected for the final plasmid-cotg/c +Sb# 15/68
• Index: for colonies numbers
1-20: DH5a_PBS1C_cotc_syb15
21-40: DH5a_PBS1C_cotc_syb68
41: DH5a_PBS1C_cotg_syb15
42-60: DH5a_PBS1C_cotg_syb68
61: N.C DH5a_PBS1C_syb15
62: N.C DH5a_PBS1C_syb68
63: N.C-No colony
• Results: no bands have been observed at the expected length
• Starters were made from colonies numbers: 3,4,6,11,16,19,21,32,33,43,45,47,50,51 ,58,59-suspected colonies. For a restriction verification.

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol - Guidelines for Protein Expression in KRX Strains

• Only in 1ng ligated plasmid transformation the colonies were isolated.
• 4 starters for each ligated Sybody in LB+Kan.
• Prepared 1L TB for the over-expression of itself.

Fig.3. LB agar+KAN pET9D_syb15

Gel group (Yara, Zixuan)

Protocol - Microgel Preparation (Step 2, 19-34)

• Aggressive stirring was used instead of sonication.

Oct 6th

Bacillus group - Cloning (Amir)

Protocol -Miniprep, Restriction

• Miniprep was made for 16 suspected colonies as preparation for the restriction verification
• NanoDrop concentration:

Type	Colony number	[c] ng/μl	260/280	260/230	DNA volume for restriction(1500 ng) (μl)
Cotc_syb15	3	126.8	1.83	1.62	11.8
Cotc_syb15	4	79.3	1.8	1.57	18.9
Cotc_syb15	6	116	1.74	1.12	12.9
Cotc_syb15	11	156.4	1.69	1.02	9.6
Cotc_syb15	16	146.2	1.73	1.19	10.2
Cotc_syb15	19	95.5	1.85	1.71	15.7
Cotc_syb68	21	81.8	1.83	1.68	18.3
Cotc_syb68	32	112.2	1.85	1.97	13.4
Cotc_syb68	33	145.7	1.65	0.81	10.3
Cotg_syb68	43	115.6	1.77	1.22	13
Cotg_syb68	45	172.7	1.61	0.79	8.7
Cotg_syb68	47	102.2	1.81	1.47	14.7
Cotg_syb68	50	118.4	1.72	1.24	12.7
Cotg_syb68	51	144.3	1.75	1.14	10.4
Cotg_syb68	58	126.3	1.8	1.42	11.9
Cotg_syb68	59	93.3	1.81	1.71	16

• Restriction was made with ECORI enzyme for all the plasmids mentioned above + 2 plasmids of: Pbs1c_syb15 and Pbs1c_syb68 (without insert)
• +control plasmids: UNCUT = UN_Pbs1c_syb15 and UN_Pbs1c_syb68 Results: no band was found at the expected length. But there are bands that might be correct for the PBS1C_cotc_syb15 or PBS1C_cotc_syb68.

ACE2 group - ACE production – fragments preparation (Hadas & Inbal)

Protocol - Concentration, starter, OD measurement, plate preparation, transformation

• Concentrating a mix of all R-26 cut to reduce the volume. • Mixing the tubes:

1

2

Mixing tubes

R-16 cut I 15.9 23.5 μl

R-16 cut II 15.9 24μl

R-16 cut II 24.9 22.9μl

R-16 cut 16.9 8.3

R-16 cut I 24.9 21.3μl

R-16 cut III 24.9 21.2μl

R-16 cut IV 24.9 22.6μl

R-16 cut 16.9 13.6

Total volume

78.7μl

• Concentrations:

	1	2
ng/μl	72.9	96
260/280	2.1	1.93
260/230	0.41	0.65
volume	7.3	11.2
ng	532.17	1075.2

• Creating starters of the yeast for transformation
• Measured OD:

	13:10	14:10	15:10	16:15	17:35	19:10
YA619	Start	0.029	0.008	0.026	0.056	0.053
YA204	Start	0.027	0.016	0.022	0.034	0.012

• Starters OD measurement -Concentration check before dilution.

	09:10
YA619	0.674
YA204	0.097

• Starter dilution- Getting to logarithmic OD (0.4-0.8)
• Measured OD:

10:00

11:00

11:58

13:00

14:00

15:00

16:00

18:20

YA619

0.098

0.096

0.102

0.165

0.212

0.482

(Diluted)

0.672

(Diluted)

YA204

0.041

0.026

0.032

0.136

0.159

0.356

0.963
(Diluted)

• Selecting the 619 for transformation, for generation time calculation we chose only the last two measurements that are certainly in the log phase:

• Generation time is: 1/0.3542=2.8 (1/hour)
• Plate preparation by adding AA to the YNB plates.
• Transformation- AGAP with the V1.
• Plating 50μl from each.

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol - Guidelines for Protein Expression in KRX Strains

• Growth of KRX with ligated plasmid in 0.5L of TB, induced to express the pET9D plasmid by Rhamnose.

Sybody	time	OD	Temp
68	14:30	0.043	37
68	15:50	0.448	37
68	16:25	0.802	37
15	16:25	0.712	37
68	17:10	1.143	25
15	17:10	1.113	25

• The rhamnose was add, to induce creation of Sybodies.

Gel group (Yara, Zixuan)

Protocol - Microgel Preparation (Step 2, 35)

• Centrifuged and discarded the supernatant. Add DDW until volume of 45ml.
• Repeat the washing step for six times.
• Not all of the solution was discarded because the precipitation wasn't stable. After that product was lyophilized.

Oct 7th

Bacillus group - Cloning (Amir)

Protocol - sporulation, Starter

• Sanger sequencing of suspected colony with pBS1C_cotC_SYB15 and 2 colonies of pBS1C_cotC_SYB68 • Preparation of new stock solutions for sporulation protocol • Plate of PY79 was incubated with a colony from –80℃ glycerol stock for further starter.

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol - Protein extraction

• OD of the solution wasn't checked at the beginning so 20 OD was used for the calculation.
• Concentration, before column-
Sb# 15- 82.9mg/ml
Sb# 68- 84.9mg/ml

Fig.1. homogenizer

Fig.2. The cell extract after homogenization

Oct 8th

Bacillus group – Proof of concept – spores in hydrogel(Amir)

Protocol - sporulation

• First step of sporulation protocol

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol -Purifying Polyhistidine proteins (His-tag)

• Two resins were compared by testing 15ml extract proteins from each Sybody with each.
• The flowthrough for each samples were collected (four- 2 for each Sybodies in each of two resin brands)- F (flow), W (wash), E1-4 (Elution).

• Nanodrop concentrations:
  BSA 1:100- 0.081mg/ml
  Elution buffer Cube- 0.676mg/ml
  Elution buffer Promega- 0.740mg/ml

Sample	Syb68 Promega	Syb68 Cube	Syb15 Promega	Syb15 Cube
F	79.018mg/ml	78.896mg/ml	76.756mg/ml	78.556mg/ml
W	8.818mg/ml	27.126mg/ml	2.171mg/ml	4.468mg/ml
E1	11.943mg/ml	3.990mg/ml	2.674mg/ml	1.823mg/ml
E2	1.370mg/ml	2.320mg/ml	1.943mg/ml	1.762mg/ml
E3	1.319mg/ml	1.365mg/ml	1.500mg/ml	1.107mg/ml
E4	1.164mg/ml	0.918mg/ml	1.431mg/ml	0.914mg/ml

• The bolded ones were run in the gel

Fig.1. A disposable column containing the Nickel bound beads

Gel group (Yara)

Protocol - Microgel imaging via microscope

• The sample that was prepared on 06.10.20 was taken from the lyophilizer and was transmitted to a clean Eppendorf.
• A 10 µL of DDW was added to the remaining product on the walls of the falcon of the samples that were prepared in 07.09.2020 and 06.10.2020 and a 2 µL of that was sent to microscope imaging

Fig.2. Microgel image of the 2nd product

Oct 11th

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol -SDS PAGE

• The expected size of the Sybodies are-
Sb# 15-13.12kilodalton
Sb# 68-14.26kilodalton
• The heaviest product among all the samples is a 98,885Da molecular weight band containing the T7 RNA polymerase.

Fig.3. SDS page
Oct 12th

Gel group (Yara, Zixuan)

Protocol - Binding Evaluation of Microgel Beads and microscopy

• Only the 2nd Microgel product was used.
• Only one concentration of GFP (20μg/600μl) was used.

Fig.1. Microscopic image of microgel-GFP complex.
Oct 13th

Sybodies group - Another Over-expression of the Sybodies in KRX cells (Yara)

Protocol - Guidelines for Protein Expression in KRX Strains

• 2 starters- control and Sb# b15.
Oct 14th

Bacillus group - Proof of concept – spores in hydrogel (Amir)

Protocol - DPA, Germination

• MOP buffer preparation for DPA protocol
• Started germination protocols
• A total of 8 Cuvettes for each temperature (4°C or 37°C) were prepared- 3 triplicates with hydrogel and 3 triplicates with DDW.
• All falcons were measured O.D(600) (time 0 min)
• 8 Cuvettes were taken to the fridge (4°C)
• 8 to the incubator in the lab (37°C)
• O.D(600 nm) was measured in intervals of 15 min and 60 min

Sybodies group - Another Over-expression of the Sybodies in KRX cells (Shanny)

Protocol - Guidelines for Protein Expression in KRX Strains

• Growth of KRX with Sb# 15 ligated plasmid and control without plasmid in 0.5L of TB, induced to express the pET9D plasmid by Rhamnose.

Sybody

time

OD

Temp

control

14:30

0.997

37

control

14:50

1.076

25

15

14:50

1.024

25

• Rhamnose was add, to induce in sybodies' creation.

Oct 15th

Bacillus group - Proof of concept – spores in hydrogel (Shany)

• O.D(600 nm) was measured for yesterday's experiment at 13:55 (24 hr)
• Started sporulation protocol: 1 colony from 7.10 PY79 was incubate in 5 ml DSM. After 3.5 hr, the O.D(600nm) reached 0.156 so starter was diluted to 3 Erlenmeyers ; 1:100 – 500 μl from starter was transformed to 50 ml DSM Erlenmeyer over the weekend

Temp	#	O.D for 600nm
Time (min)		0	15	30	45	60	1440
4°C Gel	1	0.553	0.670	0.533	0.684	0.400	0.480
	2	0.562	0.680	0.56	0. 560	0.508	0.460
	3	0.597	0.750	0.58	0.630	0.62	0.486
37°C Gel	4	0.393	0.519	0.520	0.520	0.513	0.791
	5	0.395	0.521	0.524	0.525	0.670	0.698
	6	0.430	0.536	0.534	0.531	0.590	0.744
4°C DW	1*	0.669	0.678	0.678	0.676	0.673	0.720
	2*	0.673	0.684	0.684	0.687	0.678	0.632
	3*	0.544	0.546	0.545	0.545	0.542	0.566
37°C DW	4*	0.685	0.684	0.686	0.690	0.695	0.781
	5*	0.685	0.700	0.698	0.703	0.710	0.799
	6*	0.516	0.514	0.514	0.516	0.519	0.476

Sybodies group - Another Over-expression of the Sybodies in KRX cells (Shanny)

Protocol - Protein extraction

• OD measurement for calcμlating the amount of cells:

Tube	OD
Syb15 1:1	2.519
Control 1:1	2.577
Syb15 1:10	1.054
Control 1:10	1.114

• I made HEPES 1M, pH 7.5 for the Promega beads.

Oct 18th

Bacillus group - Proof of concept – spores in hydrogel (Shany, Dor)

Protocol - sporulation, DPA

• sporulation , steps 3-5
• DPA staining

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol - - Purifying Polyhistidine proteins (His-tag)

• Again, I compered two brands of resin, one from Promega and one from Cube biotech. I tested 10ml extract proteins from each sample with each method. • I collected samples of the flowthrough for each sample (four samples- 2 for each sample in each of two resin brands)- F (flow), W (wash), E1-4 (Elution). • Nanodrop concentrations: Elution buffer Cube- 0.676mg/ml Elution buffer Promega- 0.674mg/ml

Sample	Control Promega	Control Cube	Syb15 Promega	Syb15 Cube
F	55.842mg/ml	54.492mg/ml	121.002mg/ml	120.488mg/ml
W	1.878mg/ml	6.383mg/ml	6.077mg/ml	6.533mg/ml
E1	14.369mg/ml	0.916mg/ml	5.272mg/ml	2.261mg/ml
E2	5.067mg/ml	2.392mg/ml	3.734mg/ml	7.856mg/ml
E3	4.570mg/ml	1.146mg/ml	3.156mg/ml	1.659mg/ml
E4	5.585mg/ml	0.840mg/ml	3.256mg/ml	0.972mg/ml

• The bolded ones were run in the gel

Oct 19th

Bacillus group - Proof of concept – spores in hydrogel (Shany, Dor, Amir)

• sporulation, starter of 5 ml DSM and 1 colony of py79 was made. When O.D(600 ml)=618, 250 ml of starter were transformed to 50 ml dsm in 3 erlenmeyers.
• Starter of 5 ml DSM and 1 colony of py79 was made.
• DPA dying

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol -SDS PAGE

• The expected size of the Sybodies are-
Syb15-13.12kilodalton
Syb68-14.26kilodalton
• The highest band in all the samples wells is a 98,885Da molecμlar weight band containing the T7 RNA polymerase.
Oct 22nd

Sybodies group - Over-expression of the Sybodies in KRX cells (Shanny)

Protocol - Western Blot