iGEM 2020 — Team Technion-Israel

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# Team:Technion-Israel/Notebook

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Notebook

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

1. [01 June](#0)
2. [02 June](#0)
3. [03 June](#0)
4. [04 June](#0)
5. [07 June](#0)
6. [08 June](#0)
7. [09 June](#0)
8. [10 June](#0)
9. [11 June](#0)
10. [15 June](#0)
11. [16 June](#0)
12. [17 June](#0)
13. [21 June](#0)
14. [22 June](#0)
15. [23 June](#0)
16. [25 June](#0)
17. [30 June](#0)
18. [01 July](#0)
19. [03 Aug](#0)
20. [04 Aug](#0)
21. [05 Aug](#0)
22. [06 Aug](#0)
23. [09 Aug](#0)
24. [10 Aug](#0)
25. [11 Aug](#0)
26. [12 Aug](#0)
27. [13 Aug](#0)
28. [17 Aug](#0)
29. [18 Aug](#0)
30. [19 Aug](#0)
31. [23 Aug](#0)
32. [24 Aug](#0)
33. [25 Aug](#0)
34. [26 Aug](#0)
35. [27 Aug](#0)
36. [30 Aug](#0)
37. [31 Aug](#0)
38. [01 Sep](#0)
39. [02 Sep](#0)
40. [03 Sep](#0)
41. [06 Sep](#0)
42. [07 Sep](#0)
43. [08 Sep](#0)
44. [09 Sep](#0)
45. [10 Sep](#0)
46. [12 Sep](#0)
47. [13 Sep](#0)
48. [14 Sep](#0)
49. [15 Sep](#0)
50. [16 Sep](#0)
51. [17 Sep](#0)
52. [21 Sep](#0)
53. [22 Sep](#0)
54. [23 Sep](#0)
55. [24 Sep](#0)
56. [25 Sep](#0)
57. [04 Oct](#0)
58. [05 Oct](#0)
59. [06 Oct](#0)
60. [07 Oct](#0)
61. [08 Oct](#0)
62. [11 Oct](#0)
63. [12 Oct](#0)
64. [13 Oct](#0)
65. [14 Oct](#0)
66. [15 Oct](#0)
67. [18 Oct](#0)
68. [19 Oct](#0)
69. [22 Oct](#0)

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1. ## 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.
2. ## June 2nd

   ### Bacillus group - Preliminary sporulation experience (Shay)

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

   ![sometext](https://static.igem.org/mediawiki/2020/b/bb/T--Technion-Israel--notebook1.png)
3. ## 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.
4. ## 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).

   ![sometext](https://static.igem.org/mediawiki/2020/6/66/T--Technion-Israel--notebookfig1.jpg)

   ![sometext](https://static.igem.org/mediawiki/2020/f/fc/T--Technion-Israel--notebookfig2%2C3.png)
5. ## 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.

   ![sometext](https://static.igem.org/mediawiki/2020/4/43/T--Technion-Israel--notebook2.png)
6. ## 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.

   ![sometext](https://static.igem.org/mediawiki/2020/9/97/T--Technion-Israel--june8fig1.png)

   Fig.1. PY79 plate from Weizmann
7. ## 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 |
8. ## 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.
9. ## 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.

   ![sometext](https://static.igem.org/mediawiki/2020/2/2e/T--Technion-Israel--june11fig1.png)

    Fig.1. Spores of *B. subtillis* 168
10. ## 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).

    ![sometext](https://static.igem.org/mediawiki/2020/9/9b/T--Technion-Israel--june15fig1.png)

    Fig.1. *B.subtillis* PY79 spores.
11. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/d/d2/T--Technion-Israel--june16fig1.png)

    Fig.1. *B.subtillis* PY79 spores.
12. ## June 17th

    ### Bacillus group -Preliminary sporulation experience (Shany & Shay)

    **Protocol -** sporulation

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

    ![sometext](https://static.igem.org/mediawiki/2020/a/a9/T--Technion-Israel--june17fig1.png)

    Fig.1. *B. subtillis* PY79 spores
13. ## June 21st

    ### Bacillus group -Preliminary sporulation experience (Zixuan)

    • Two LB plates were plated with **B. subtillis** PY79 from Glycerol stock, Incubated at 37°C.
14. ## 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.
15. ## June 23rd

    ### Bacillus group -Preliminary sporulation experience (Zixuan)

    **Protocol -** Starter

    • Starter - 5 ml LB and a PY79 colony.
16. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/0/00/T--Technion-Israel--june25fig1.png)

    Fig.1. Electron microscope image of **B. subtillis** PY79, spores after 4th wash.
17. ## 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
18. ## July 1st

    ### ACE2 group -Vector creation (Ilana)

    **Protocol -** Miniprep ([NucleoSpin kit](https://www.takarabio.com/assets/documents/User%20Manual/NucleoSpin%20Plasmid%20EasyPure%20DNA%20Purification%20User%20Manual_Rev_02.pdf) ,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
19. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/e/e8/T--Technion-Israel--aug3fig1.png)

    fig.1.: Dripping System used in Monomer Preparation
20. ## 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).

    ![sometext](https://static.igem.org/mediawiki/2020/5/5d/T--Technion-Israel--aug4fig1.png)

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

    ![sometext](https://static.igem.org/mediawiki/2020/7/79/T--Technion-Israel--aug4fig2.png)

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

    ### *Bacillus group* - Cloning (Amir, Shay)

    **Protocol -** Starter

    • Starter *E.coli* Top10  with PBS1C\_LacZ.
22. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/d/df/T--Technion-Israel--NEB_Ladder.png)

    ### 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.

    ![sometext](https://static.igem.org/mediawiki/2020/9/96/T--Technion-Israel--Ladder.Black.png)
23. ## 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

    ![sometext](https://static.igem.org/mediawiki/2020/1/1b/T--Technion-Israel--Ladder.red.png)

    ### 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.

    ![sometext](https://static.igem.org/mediawiki/2020/6/65/T--Technion-Israel--Ladder_Reverse_Negative.png)

    ![sometext](https://static.igem.org/mediawiki/2020/5/5c/T--Technion-Israel--Ladder_Forward_positive_nedative_ladder.png)
24. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/1/18/T--Technion-Israel--Negative_sample2_sample2_sample1_Ladder.png)

    ### 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).

    ![sometext](https://static.igem.org/mediawiki/2020/0/0b/T--Technion-Israel--Sonication_of_emulsion.png)

    **Fig.2.** Sonication of Created Emulsion.

    ![sometext](https://static.igem.org/mediawiki/2020/c/c1/T--Technion-Israel--Pumping_air.png)

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

    ![sometext](https://static.igem.org/mediawiki/2020/1/15/T--Technion-Israel--Nickel_solution.jpeg)

    **Fig.4.** Nickel Solution Preparation.
25. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/4/48/T--Technion-Israel--gel_2800bp.png)

    ![sometext](https://static.igem.org/mediawiki/2020/4/49/T--Technion-Israel--gel_570bp.png)

    ![sometext](https://static.igem.org/mediawiki/2020/f/f0/T--Technion-Israel--gel_600bp.png)

    ### 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 .

    ![sometext](https://static.igem.org/mediawiki/2020/7/74/T--Technion-Israel--Ladder_Rrest_Positive_Negative.png)

    ### 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.
26. ## 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

    ![sometext](https://static.igem.org/mediawiki/2020/e/ef/T--Technion-Israel--2-Log_Ladder_2800bp.png)
27. ## 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.
28. ## 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:

    ![sometext](https://static.igem.org/mediawiki/2020/5/51/T--Technion-Israel--2-Log_Ladder_460bp_3066bp.png)

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

    **Protocol -**  Chemical transformation to *E.coli Top10CC*

    Transformation of pET-9d plasmid into TOP10 E.coli.
29. ## 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.
30. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/4/4b/T--Technion-Israel--2-Log_Ladder_2800bp_C1-C4.png)

    ![sometext](https://static.igem.org/mediawiki/2020/2/22/T--Technion-Israel--2-Log_5.1-5%27.14.png)

    ### 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.
31. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/7/73/T--Technion-Israel--2-Log_Ladder_372bp_400bp.png)
32. ## 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*.
33. ## 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.
34. ## 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.
35. ## 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

    ![sometext](https://static.igem.org/mediawiki/2020/2/24/T--Technion-Israel--2-Log_Ladder_4300bp_6000bp.png)
36. ## Aug 30th

    ### Sybodies group - Restriction map (Shanny & Ella)

    **Protocol -** Restriction

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

    ![sometext](https://static.igem.org/mediawiki/2020/1/16/T--Technion-Israel--2-Log_Ladder_4338bp_4302bp.png)

    ### 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.
37. ## 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

    ![sometext](https://static.igem.org/mediawiki/2020/5/58/T--Technion-Israel--2-Log_Ladder_2524bp.png)

    ### 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.
38. ## Sep 1st

    ### Bacillus group - Cloning (Amir, Shay)

    **Protocols-** PCR, Gel Eletrophoresis

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

    ![sometext](https://static.igem.org/mediawiki/2020/9/9c/T--Technion-Israel--2-Log_Ladder_2524bp_negative.png)

    ### 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.
39. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/0/0c/T--Technion-Israel--2-Log_Ladder_Red_2524bp.png)

    ### 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.

    ![sometext](https://static.igem.org/mediawiki/2020/8/83/T--Technion-Israel--2-Log_4300bp.png)

    ### 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.
40. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/e/ed/T--Technion-Israel--Ladder_ACE2_cut_ACE2_STAB.png)

    ### ACE2 group -YFP production (Ilana& Hadas)

    **Protocols-**gel electrophoresis

    • Gel running for verifying the colony-PCR.

    ![sometext](https://static.igem.org/mediawiki/2020/4/41/T--Technion-Israel--2-Log_Ladder_4300bp.png)

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

    ![sometext](https://static.igem.org/mediawiki/2020/8/89/T--Technion-Israel--2-Log_Ladder_4300bp_negative.png)

    • 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.
41. ## 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.
42. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/f/f9/T--Technion-Israel--Ladder_ACE2-CUT_ACE2-STAB_neg_ladder.png)

    ### 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.

    ![sometext](https://static.igem.org/mediawiki/2020/1/11/T--Technion-Israel--2-Log_Ladder_555bp.png)

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

    ![sometext](https://static.igem.org/mediawiki/2020/7/73/T--Technion-Israel--2-Log_Ladder_773bp.png)

    ### 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.
43. ## 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.
44. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/a/a0/T--Technion-Israel--LB_Amp_top10.png)

    **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.

    ![sometext](https://static.igem.org/mediawiki/2020/3/33/T--Technion-Israel--2-Log_Ladder_4300bp_no-bands.png)

    ### 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.
45. ## 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).
46. ## 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.
47. ## 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

    ![sometext](https://static.igem.org/mediawiki/2020/d/d3/T--Technion-Israel--Fig1_Fig2_600bp.png)

    ![sometext](https://static.igem.org/mediawiki/2020/a/a6/T--Technion-Israel--Fig3_Fig4_600bp.png)

    ### 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.

    ![sometext](https://static.igem.org/mediawiki/2020/a/a6/T--Technion-Israel--2-Log_Ladder_1600bp_1100bp.png)

    • 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.

    ![sometext](https://static.igem.org/mediawiki/2020/5/5a/T--Technion-Israel--Gravity_system.png)

    Protein Purification with Gravity System

    ![sometext](https://static.igem.org/mediawiki/2020/d/d9/T--Technion-Israel--MagneHis.png)

    Protein Purification via MagneHis™ Protein Purification System
48. ## 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 |

    ![sometext](https://static.igem.org/mediawiki/2020/3/3d/T--Technion-Israel--2-Log_Ladder_233bp_7164bp_663bp_7134bp.png)

    ### 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.

    ![sometext](2-Log_Ladder_1600bp_1100bp_2600bp)

    • 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

    ![sometext](https://static.igem.org/mediawiki/2020/f/fa/T--Technion-Israel--First_gel_555bp_773bp.png)

    ![sometext](https://static.igem.org/mediawiki/2020/0/00/T--Technion-Israel--Second_gel_773bp.png)

    ### 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.
49. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/a/ae/T--Technion-Israel--SDS-Page_results.png)

    **Fig.1.** SDS-Page Results
50. ## 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.)

    ![sometext](https://static.igem.org/mediawiki/2020/e/ef/T--Technion-Israel--2-Log_Ladder_1339bp_1369bp.png)

    ![sometext](https://static.igem.org/mediawiki/2020/e/e1/T--Technion-Israel--2-Log_green-rectangle.png)

    ### 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.

    ![sometext](https://static.igem.org/mediawiki/2020/1/13/T--Technion-Israel--Dialysis_system.png)

    **Fig.1.** Dialysis System
51. ## 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 |

    ![sometext](https://static.igem.org/mediawiki/2020/c/c2/T--Technion-Israel--2-Log_Ladder_9176bp_1125bp_7905bp_3066bp.png)

    ### 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.

    ![sometext](https://static.igem.org/mediawiki/2020/b/bf/T--Technion-Israel--2-Log_Ladder_F9_R16_negative.png)

    • 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 |
52. ## 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 |

    ![sometext](https://static.igem.org/mediawiki/2020/f/fe/T--Technion-Israel--2-Log_Ladder_R16.png)
53. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/3/3f/T--Technion-Israel--2-Log_Ladder_green_rectangles.png)

    ### 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.
54. ## 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 |
55. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/5/5e/T--Technion-Israel--2-Log_Ladder_4green_rectangles.png)

    ### 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.

    ![sometext](https://static.igem.org/mediawiki/2020/c/c7/T--Technion-Israel--2-Log_Ladder_R16-1600bp.png)

    • 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 |
56. ## Sep 25th

    ### Bacillus group - Cloning (Zixuan)

    • Plate results from 24/09/2020

    ![sometext](https://static.igem.org/mediawiki/2020/1/1a/T--Technion-Israel--Fig1_DH5a.png)

    **Fig.1.** DH5a\_pBS1C\_SYB15\_CotG\_3\*, 24/09/2020, AMP

    ![sometext](https://static.igem.org/mediawiki/2020/0/00/T--Technion-Israel--Fig2_DH5a.png)

    **Fig.2.** DH5a\_pBS1C\_SYB68\_CotG\_3\*, 24/09/2020, AMP

    ![sometext](https://static.igem.org/mediawiki/2020/8/83/T--Technion-Israel--Fig3_DH5a.png)

    **Fig.3.** DH5a\_pBS1C\_SYB68\_CotC\_3, 24/09/2020, AMP

    ![sometext](https://static.igem.org/mediawiki/2020/a/a8/T--Technion-Israel--Fig4_DH5a.png)

    **Fig.4.** DH5a\_pBS1C\_SYB15, Negative control, 24/09/2020, AMP

    ![sometext](https://static.igem.org/mediawiki/2020/9/98/T--Technion-Israel--Fig5_DH5a.png)

    **Fig.5.** DH5a\_pBS1C\_SYB15\_CotC\_3, 24/09/2020, AMP

    ![sometext](https://static.igem.org/mediawiki/2020/f/fd/T--Technion-Israel--Fig6_DH5a.png)

    **Fig.6.** DH5a\_pBS1C\_SYB68, Negative control, 24/09/2020, AMP
57. ## 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.
58. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/e/ee/T--Technion-Israel--2-Log_Ladder_1-35_36.png)

    ![sometext](https://static.igem.org/mediawiki/2020/e/e6/T--Technion-Israel--2-Log_Ladder_1-35_6_8-26.png)

    ### 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.

    ![sometext](https://static.igem.org/mediawiki/2020/c/cd/T--Technion-Israel--LB_KAN.png)

    **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.
59. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/0/02/T--Technion-Israel--2-Log_Ladder_1-19.png)

    ### 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 | 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 | 0.159 | 0.356 | 0.963 |

    ![sometext](https://static.igem.org/mediawiki/2020/5/5d/T--Technion-Israel--Graph_starter_dilution_1-19.png)

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

    ![sometext](https://static.igem.org/mediawiki/2020/3/34/T--Technion-Israel--Graph_linear_curve_1-19.png)

    • 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.
60. ## 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

    ![sometext](https://static.igem.org/mediawiki/2020/7/77/T--Technion-Israel--Homogenizer.png)

    **Fig.1.** homogenizer

    ![sometext](https://static.igem.org/mediawiki/2020/0/04/T--Technion-Israel--cell_extract.png)

    **Fig.2.** The cell extract after homogenization
61. ## 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

    ![sometext](https://static.igem.org/mediawiki/2020/2/28/T--Technion-Israel--Nickel_bound_beads_column.png)

    **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

    ![sometext](https://static.igem.org/mediawiki/2020/3/39/T--Technion-Israel--Microgel_imaging_2nd.png)

    **Fig.2.** Microgel image of the 2nd product
62. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/8/81/T--Technion-Israel--Firs_gel_%28BZ_side%29.png)

    ![sometext](https://static.igem.org/mediawiki/2020/e/e8/T--Technion-Israel--Second_gel_%28other_side%29.png)

    ![sometext](https://static.igem.org/mediawiki/2020/2/28/T--Technion-Israel--SDS_page.png)

    **Fig.3.** SDS page
63. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/9/9e/T--Technion-Israel--microgel-GFP_complex.png)

    **Fig.1.** Microscopic image of microgel-GFP complex.
64. ## 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.
65. ## 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.
66. ## 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.
67. ## 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
68. ## 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.

    ![sometext](https://static.igem.org/mediawiki/2020/3/32/T--Technion-Israel--Excel_band_PM2600.png)

    ![sometext](https://static.igem.org/mediawiki/2020/1/14/T--Technion-Israel--Excel_band_PM2600_sybody15.png)
69. ## Oct 22nd

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

    **Protocol -** Western Blot

    ![sometext](https://static.igem.org/mediawiki/2020/9/9f/T--Technion-Israel--PageRμler_Marker.png)

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Department of Biotechnology & Food Engineering

Technion – Israel Institute of Technology

Haifa 32000, Israel

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# Team:Technion-Israel/Engineering

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     Implementation](https://2020.igem.org/Team%3ATechnion-Israel/Implementation)
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    Success](https://2020.igem.org/Team%3ATechnion-Israel/Engineering)
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* [*B. subtilis* Spore Surface Display](#pi)
* [Engineering the vector](#eng)
* [Decoy proteins](#dp)
* [Insertion into **B. subtilis** genome](#ins)
* [Anchor protein cloning](#anc)
* [Sporulation](#sp)
* [Sybodies – planning, cloning, and purifying](#syb)

**Research begins with an idea; the design cycle then begins.**

![](https://static.igem.org/mediawiki/2020/c/cf/T--Technion-Israel--ENG_SUCC_diag1.png)

**Fig.1.** The engineering success cycle

In the diagram, the “learning” step and the “improvement” step are emphasized since we believe they are what sets apart a **good** engineering process from a **great** engineering process. In research as in life, we fall so we can learn to pick ourselves up. With each failure, we learned from our mistakes and with each success we thought about ways to improve our results for the next time.

# *B. subtilis* Spore Surface Display

### Research

We first conducted a literature review searching for suitable "decoy proteins" against SARS-CoV-2. This review led us to the cell entry mechanism of SARS-CoV-2 (the hACE2 receptor), and to information about the Sybodies (Sb#15 and Sb#68) [[1]](#r1) [[2]](#r2) [[3]](#r3).
For surface display, **B. subtilis** spore display (BSSD) was found to be the most suitable technique for displaying functional heterologous proteins anchored to bacteria [4][5] .

### Design

6 different plasmids were designed to be integrated to *B. subtilis* (**B. subtilis**) PY79. Featuring CotC or CotG as anchor proteins, linker and one of the decoy proteins: ACE2, Sb#15 and Sb#68 [[2]](#r2) [[4]](#r4) [[5]](#r5).

![](https://static.igem.org/mediawiki/2020/5/5f/T--Technion-Israel--bacillus_plasmids_final.png)

**Fig.2.** The different plasmids

# Engineering the vector

The cloning was planned as follows:

1. The “decoy proteins” will be cloned to a linearized pBS1C\_lacZ vector using Gibson assembly and transformed to **E. coli** TOP10 cc.
2. Sequences which encode the Anchor (Cot) proteins will be inserted to the vector using restriction enzymes.
3. The final vector will be inserted to *B. subtilis* PY79’s genome

All steps are verified by colony PCR and sequencing with optimized conditions for each plasmid

# Decoy proteins

### Test

The cloning of Sb#15/68 and the pBS1C\_LacZ vector was verified using colony PCR. However, colony PCR did not yield positive results for the ACE2 insertion.

### Re-test

We planned another verification test using restriction enzymes in order to examine the cloning by detecting the unique restriction pattern of the plasmid (restriction map). However, the pattern was not detected.

### Re-plan

Due to the limitation of parameter-changing in the Gibson assembly method, the Colony PCR’s conditions were the ones to be adapted: temperatures, duration time and number of cycles were changed several times. Yet, the cloning of ACE2 and pBS1C\_LacZ was not verified.

### Re-test

Transformation of pBS1C\_syb15/68 and pBS1C\_ACE2 was checked by Sanger sequencing. The results confirmed that the vector was transformed into the bacteria but without the ACE2 fragment. However, positive cloning of the Sybodies and the vector was verified.

### Research

Another cloning attempt using restriction enzymes was planned.

### Re-Plan

Since the combination of the Sybodies has showed substantial inhibition of SARS-CoV-2’s interaction with ACE2 [[1]](#r1), we decided to continue with pBS1C\_syb15 and pBS1C\_syb68 to the second cloning step - adding the anchor proteins to the final plasmid.

# Insertion into **B. subtilis** genome

### Plan

Genomic insertion of pBS1C\_syb15 and pBS1C\_syb68 into **B. subtiis** using corresponding insertion protocols and colony PCR.

### Test

After insertion, colony PCR was conducted to verify the insertion of pBS1C\_syb68 to *B. subtilis’*s genome. The insertion of pBS1C\_syb15 was not verified.

### Research

Other verification methods, such as amylase test and colony PCR are being investigated at the moment for future research and development.

# Anchor protein cloning

### Plan

Insertion of the anchor proteins, CotC and CotG into the pBS1C\_ACE2/syb15/syb68 vector using restriction enzymes.
The anchor proteins and vectors will be digested, separated using electrophoresis, and ligated.

### Test

No anchor proteins were detected in electrophoresis gel.

### Re-Plan

A larger amount of anchor proteins was digested.

### Re-Test

Anchor proteins were detected in electrophoresis gel.

### Verification

Colony PCR results did not confirm the ligation.

### Re-Plan

Ligation temperature and duration were adjusted. Since the Cot protein sequences are short (~250-700bp), larger amounts of DNA fragments were ligated.

### Verification

Verification attempt using restriction enzymes was done. Results of restriction map were inconclusive.

### Re-Test

Suspected colonies from restriction-map attempt were sent for Sanger sequencing. But the sequencing results implied that the anchor proteins and vector were not cloned.

### Re-Plan

Different approaches, such as using Gibson assembly, have been investigated.

# Sporulation

### Research

Sporulation was conducted for *B. subtilis* PY79’s bacteria, With the goal of testing the germination ratio of B. subtilis PY79 in the chosen hydrogel of the final product.

### Plan

Three germination tests were performed:

1. Analyzing germination protocol
2. Assessment of Dipicolinic acid (DPA)
3. Spore Staining

### Test

Germination and DPA tests verified the stability of spores in the hydrogel compared to water. However, staining test indicated that there was a large amount of living bacteria presented in the gel at time zero.

### Re-Plan

Sporulation protocol requires modification in order to achieve a product consisting of at least 90% spores. In the future, we hope to use the non-germinating *B. subtilis* mutant strain that was synthesized by the 2012 Munich iGEM group [[6]](#r6) to avoid possible germinations.

# Sybodies – planning, cloning, and purifying

As mentioned in the [Sybodies Result page](https://2020.igem.org/Team%3ATechnion-Israel/Results#syb) and the [Design page](https://2020.igem.org/Team%3ATechnion-Israel/Design), Sybodies were chosen as decoy proteins against SARS-CoV-2 due to their advantages as binders. As far as we know, we are the first and only iGEM team to use these synthetic nanobodies in the iGEM competition.
We came across the Sybodies while searching for a way to destroy the virus once it attaches to the gel in our product. To learn more about the ACE2-Spike protein binding mechanism, we consulted with Assistant Prof. Yotam Bar-On from the Technion and Dr. Ron Diskin from the Weizmann institute. Both experts suggested us to look for novel antibodies and Dr. Diskin advised us to expand our research to the “bioRxiv” preprints.
We were able to find two possible proteins for our project: Sybodies [[3]](#r3) and Human mAb 47D11 [[7]](#r7). Both options are antibodies while the first is a nanobody (more information in the [Sybodies Result page](https://2020.igem.org/Team%3ATechnion-Israel/Results#syb)) and the latter is a human antibody. The advantage of the Human mAb 47D11 antibody is that it would not compete with the binding of the spike protein to the ACE2 receptor expressed at the human cell surface [[7]](#r7), so it can be used together with our ACE2 baits. The disadvantage is the lack of information about it. Nanobodies, on the other hand, are highly researched and are reported to be functionally expressed in bacteria [[8]](#r8) [[9]](#r9) [[10]](#r10). Because of this, we chose to continue with the Sybodies.

The most relevant work we encountered is the preprint prepared by Walter et al [[3]](#r3). After careful consideration and consultation with the writers, we chose two Sybody sequences. Detailed information can be found in the [Sybodies Result page](https://2020.igem.org/Team%3ATechnion-Israel/Results#syb). At this point, we started to design our genetic constructs and entered the lab with a well written plan and complete protocols. The general plan is described in Fig.1.

![](https://static.igem.org/mediawiki/2020/e/e2/T--Technion-Israel--ENG_SUCC_diag.png )

**Fig.3.** Scheme illustrating steps of the general production process of the Sybodies.

After performing restriction and ligation cloning on the Sybody sequences and the PET-9d backbone, bacterial growth was observed in the control plates. The immediate culprits that came to mind were the restriction enzymes. We repeated the cloning process with enzymes from another laboratory and different suppliers, but it did not solve the problem. The next possible issue that came to mind was a possible issue with the plasmid. To check it, we ran a restriction verification test of the plasmid and saw that it exhibited the expected patterns on the gel. We decided to continue with our colonies to colony PCR, to see whether the Sybody sequences have been ligated into the plasmid backbone successfully despite the growth in the control plates. As shown in the [Sybodies Result page](https://2020.igem.org/Team%3ATechnion-Israel/Results#syb), the ligation of the sequences was indeed successful. Our conclusion was that the backbone had a high tendency to perform self-ligation even though different restriction enzymes were used. In addition to colony PCR, the ligated plasmid sequences were also confirmed by Sanger sequencing.
The next step in the Sybody creation was transformation into KRX *E. coli*, a strain of **E. coli** suitable for over-expression of proteins [[11]](#r11). After protein extraction and purification, the Sybodies’ presence and size were examined by running an SDS-PAGE and a Western-Blot, both tests resulted in positive verifications. The complete results can be found in the [Sybodies Result page](https://2020.igem.org/Team%3ATechnion-Israel/Results#syb)  and both of [Sybody 15](http://parts.igem.org/Part%3ABBa_K3653002)  and [Sybody 68](http://parts.igem.org/Part%3ABBa_K3653003) were registered as new Parts in the iGEM part registry.

**References**

1. Brielle ES, Schneidman-Duhovny D, Linial M. The SARS-CoV-2 Exerts a Distinctive Strategy for Interacting with the ACE2 Human Receptor. Viruses. 2020;12(5):497. doi:10.3390/v12050497
2. Walter JD, Hutter CAJ, Zimmermann I, et al. Sybodies targeting the SARS-CoV-2 receptor-binding domain. bioRxiv. 2020. doi:10.1101/2020.04.16.045419
3. Zimmermann I, Egloff P, Hutter CAJ, et al. Generation of synthetic nanobodies against delicate proteins. Nat Protoc. 2020;15(5):1707-1741. doi:10.1038/s41596-020-0304-x
4. Lin P, Yuan H, Du J, Liu K, Liu H, Wang T. Progress in research and application development of surface display technology using Bacillus subtilis spores. Appl Microbiol Biotechnol. 2020;104(6):2319-2331. doi:10.1007/s00253-020-10348-x
5. Guoyan Z, Yingfeng A, Zabed HM, et al. Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications. J Microbiol Biotechnol. 2019;29(2):179-190. doi:10.4014/jmb.1807.06066
6. iGEM team Munich 2012, How do sporulation & Germination Work, 2012
7. Wang C, Li W, Drabek D, et al. A human monoclonal antibody blocking SARS-CoV-2 infection. Nat Commun. 2020;11(1):2251. doi:10.1038/s41467-020-16256-y
8. De Genst E, Silence K, Decanniere K, et al. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci. 2006;103(12):4586-4591. doi:10.1073/pnas.0505379103
9. Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol. 2005;23(9):1126-1136. doi:10.1038/nbt1142
10. Muyldermans S. Nanobodies: Natural Single-Domain Antibodies. Annu Rev Biochem. 2013;82(1):775-797. doi:10.1146/annurev-biochem-063011-092449
11. Promega Corporation: Hartnett J, Gracyalny J, Slater MR. The Single Step (KRX) Competent Cells: Efficient Cloning and High Protein Yields. Promega Notes. 2006;94(August):27-30

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Department of Biotechnology & Food Engineering

Technion – Israel Institute of Technology

Haifa 32000, Israel

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# Team:Technion-Israel/Experiments

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Here are the protocols we used in our experiments.
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Gel

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# Team:Technion-Israel/Results

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* [General Overview](#pi)
* [Gels](#gel)
* [*Bacillus subtilis* Spore](#bac)
* [Mutated ACE2](#ace)
* [Sybodies](#syb)

# General Overview

In this page, detailed information will be provided to guide you through our adventure in the lab. Until this very moment, we have managed to purify and verify the Sybodies as well as created both the Microgel and the hydrogel. Since a relatively large number of experiments have been conducted, it’s reasonable to conclude that our designed “decoy proteins” can certainly be linked with other components as planned and eventually assemble ACT.

# Gels

[Overview](#nav-geloverview)
[Key Achievements](#nav-gelkey)
[Introduction](#nav-gelintro)
[Aims](#nav-gelaims)
[Results](#nav-gelresults)

Various natural and synthetic polymers, such as xyloglucan and dextran, have thermo-responsive properties [[1]](#r1). Hydrogels made of these polymers have been applied in many areas, including drug delivery, tissue engineering since they can perform a quick reversible transformation between liquid and gel under temperature stimulation [[1]](#r1). In search for a polymer that can form an undetectable film on skin surface, Pluronic F-127 was the right candidate, because of its lower critical solution temperature that is around the body temperature (37°C) [[2]](#r2).

Furthermore, during the search for carriers of “decoy proteins”, the possibility to use a polymeric material arose, in parallel to the biological one. Nitrilotriacetic acid (NTA) with nickel ions are commonly used in as polymers to purify His-tagged proteins. The NTA has four metal-chelating sites that allow it to hold a stable bonding with metal ions [[3]](#r3). Because of the relatively high affinity and specificity of binding between His-tag and Ni-poly NTA, this polymer was chosen as the polymeric carrier for our designed “decoy proteins” and was named as “Microgel Beads” due to their own physical characteristic and the desired size which is about 6.5μm.

Herein, the Pluronic F-127 based hydrogel was created with a determined gelation time of ~1 minute. Moreover, NTA monomers have been prepared and microscopic images of the Microgel Beads which were composed of these monomers were obtained. Process utilization and the size of the Microgels are limited for now and adjustments must be made to reach the final product.

* Successfully created the target thermo-responsive hydrogel at a concentration of 18% w/v.
* Gelation time of created hydrogel was tested and matched the expectation (~1 min).
* NTA monomers were produced and the amount was relatively large (~11.4gr, 0.23gr was needed per polymerization reaction).
* Microscopic images of the Microgel Beads were obtained.
* The Microgel Beads’ ability to connect to His-tagged proteins was verified using His-tagged-GFP and results were confirmed via microscope and plate reader.

“How are we going to deliver the ‘decoy proteins’?”
This question that has bothered us for a long time since the product was designated to target SARS-CoV-2. With the help of our first public [survey](https://2020.igem.org/Team%3ATechnion-Israel/Human_Practices), and being inspired by the conventional sunscreens, the proposed product was shaped as a gel-type screen product that can form a thin protective layer which holds the “decoy proteins” with their carriers on the skin. After consulting with Prof. Boaz Mizrahi from the faculty of Biotechnology and Food Engineering in the Technion institute, it was found that a thermo-responsive hydrogel made from Pluronic F-127 (Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)) is suitable for the purpose of this project. Thermo-responsive polymers use temperature as their external stimulus to activate solution-gel transition and most of these materials in this family are capable of forming hydrogel at a temperature that is close to body temperature [[1]](#r1). These hydrogels have been used for biomedical purposes, not only because their gelling behavior can be stimulated by temperature, but also due to their ability to swell in situ under physiological conditions [[4]](#r4). These properties have guaranteed that Pluronic F-127 is suitable for the aim of convenient skin administration. This material was from the family of Pluronic copolymers, which also known as PEO-PPO-PEO copolymers [[1]](#r1). Pluronic F-127 was chosen because of its low critical solution temperature (LCST) which is around the body temperature [[2]](#r2), and a previous study has also demonstrated that it is suitable for clinical skin administrations [[5]](#r5). Therefore, the chosen concentration of this hydrogel was 18% w/v because according to the results, this concentration would present a product with gelation time of about one minute, which fits the goal of skin applications [[6]](#r6). Moreover, to further verify the created hydrogel’s gelation time for the sake of ensuring that it would be ideal for this project, the variation of temperature at skin surface was taken into consideration [[6]](#r6). Thus, the gelation time was determined at 32℃ and 37℃ in four replicates.

NTA is frequently used as a chelating ligand for metal ions, and Ni-NTA is frequently used to purify His-tagged proteins because this material can bind the histidine residues selectively and effectively via the immobilized metal ions [[3]](#r3). The bonding between poly-histidine residues and metal ions was characterized as metal-ligand coordinative bonds [[7]](#r7), which can be generally considered as stable and is also recognized as highly selective [[8]](#r8).

In this project, an improved method of preparing the Ni-poly NTA particles was recruited since it enables a higher density of metal ions to be presented on the particles’ surfaces [[9]](#r9). As one of the possible carriers of the “decoy proteins” that targets SARS-CoV-2, Ni-poly NTA matrices were formed and we named them as “Microgel Beads”, due to their spherical characteristic and representing the desired size (Figure 1.1).

First, the NTA monomers, which served as the building blocks of Microgel Beads, were created successfully in a considerable amount. Later, three attempts have been done for fabrication of the Microgel Beads. Although lots of efforts have been invested to optimizing the production of Microgel Beads, the resulting amount of the product was low and after analysis via microscope, it was observed that there was a deviation from the particles’ size (40μm-90μm) and expectation (6.5μm).

![](https://static.igem.org/mediawiki/2020/9/9d/T--Technion-Israel--Gel_1.1.png
)

**Figure 1.1:** Illustration of Microgel Beads linked with Sybodies. B- Illustration of Microgel Beads linked with ACE2.

The main focus of this part of the work includes the fabrication of the hydrogel, determination of the hydrogel’s gelation time, creation of Microgel Beads using NTA monomers and Nickel ions, verification of Microgel Beads’ connection with His-tagged proteins and estimation of the beads’ size. In addition, expression and purification of His-tagged mCherry fused to human ACE2 protein using HEK-293F cells were done, since this fused protein is needed in the proof of concept experiment of Microgel Beads to examine whether the connection with beads would interfere the proteins’ functionalities.

1. **Hydrogel Fabrication and Gelation Time Examination**

   The thermo-responsive hydrogel was created by simply dissolving Pluronic F-127 powder with Double Distilled Water (DDW) (Figure 1.2). Since ACT. is aimed for skin addministration, the chosen concentration of hydrogel was 18% w/v since a previous research has demostrated that at this concentration, it would have a gelation time of approximate one minute at 37℃ [[6]](#r6). The protocol of creating the hydrogel can be found [here](https://2020.igem.org/Team%3ATechnion-Israel/Experiments). Before proceeding to actual gelation time tests, the hydrogel went through a primary transformation test to brifely examine its ability of performing reversible solution-gel transformation. The test was done by transferring a small amount of gel into a glass bottle and placing it in a 37℃ water bath (gel), then putting it on the ice (liquid), as shown in Figure 1.3.

   ![](https://static.igem.org/mediawiki/2020/c/c0/T--Technion-Israel--Gel_1.2.png
   )

   **Figure 1.2:** Illustration of Hydrogel preparation

   ![](https://static.igem.org/mediawiki/2020/9/92/T--Technion-Israel--Gel_1.3.png
   )

   **Figure 1.3:** A- Solidified Hydrogel after warming at 37℃. B- Liquid Hydrogel after cooling on ice.

   Since the hydrogel was designated for external skin application and the skin temperature varies according to the sourroundings, two temperatures, 32℃ and 37℃, were chosen to represent the limit of common skin temperature range. This was done in four replicates to increase the measurments’ accuracy. The detailed protocol could be found [here](https://2020.igem.org/Team%3ATechnion-Israel/Experiments). According to the results (Figure 1.4), the gel’s average gelation time at 32℃ was ~63.5 seconds and for 37℃ was ~40.75 seconds. Although there was a deviation between the measured result and expecataion, such a small difference could be caused by manual error since this measurement was not analytical. In conclusion, it is reasonable to say that the 18% w/v hydrogel’s gelation time was about 1 minute in the common range of skin temperature, which makes it suitable for the purpose of ACT.

   ![](https://static.igem.org/mediawiki/2020/e/e9/T--Technion-Israel--Gel_1.4.png
   )

   **Figure 1.4:** Gelation time of Hydrogel at 32℃ and 37℃

   The created hydrogel was planned to be used in the proof of concept experiments of Bacillus subtilis spores and the whole final product. For the verification of spores, the main idea was to figure out whether the spores that displayed the “decoy proteins” on their surfaces would germinate when they were mixed with the hydrogel at different temperatures. Regarding the examination with the Microgel Beads together, the key goal was to examinate whether the hydrogel would interupt Microgel’s ability to connect with His-tagged proteins. Unfortunately, the later part of experiment wasn’t done because of the time and material limitations.
2. **Microgel Fabrication**

   As mentioned in the introduction, the “Microgel Beads” are polymer of NTA that are linked with Nickel ions. Such a material was chosen because of its ability to perform selective binding with proteins that have His-tags with a high affinity. Detailed procedures of preparing the Microgel Beads could be found [here](https://2020.igem.org/Team%3ATechnion-Israel/Experiments) and these steps can be generally summarized into three parts: **NTA monomer fabrication, polymerization and nickel attachment**. During the time in the lab, two attempts have been made for creating the NTA monomers (Figure 1.5) and three attempts were done for making the final Microgel Beads using these monomers.

   ![](https://static.igem.org/mediawiki/2020/2/28/T--Technion-Israel--Gel_1.5.png
   )

   **Figure 1.5:** A- NTA Product from 1st attempt. B- NTA Product from 2nd attempt.

   As shown in the photos above, the monomers were created successfully, and a relatively large amount of NTA monomers were obtained (~11.4gr) compared to the amount needed for each round of polymerization (0.23gr). However, the problem was mainly related to the steps after obtaining NTA monomers. In total, the Microgel Beads were prepared three times: the first time with the 1st attempt of monomers while the second and third used monomers from the 2nd attempt. Although the amount of produced Microgel from the first preparation was fairly large (Figure 1.6), the color was close to green and led to the suspicion that the majority of the powders in this tube were nickel salts instead of the desired gel particles. Besides, one of the important steps of creating NTA monomers, which was dripping the mixture of acryloyl chloride and toluene into N,N-bis(carboxymethyl)-L-lysine NaOH solution (Figure 1.7) was done incorrectly since the acryloyl chloride toluene mixture was poured into the beaker instead of adding it one drop at a time. Therefore, the NTA monomers of the 1st attempt (Figure 1.5A) and the first Microgel product were abandoned (Figure 1.6).

   ![](https://static.igem.org/mediawiki/2020/d/d4/T--Technion-Israel--Gel_1.6.png
   )

   **Figure 1.6:** : 1st Microgel product

   ![](https://static.igem.org/mediawiki/2020/e/e8/T--Technion-Israel--aug3fig1.png
   )

   **Figure 1.7:** : Dripping system used in the NTA monomers’ preparation

   In the second trial of Microgel Beads creation, the process was improved by changing the power of sonication from 40% to 20%. This change was made because after centrifugation, some of the suspected gel particles were trapped at the interface of water phase and oil phase (Figure 1.8). After discussing with Prof. Boaz Mizrahi, it was found that the sizes of some particles were too small, which meant the sonication power was too strong. Regardless, the amount of resulting product was too little (~0.2mg) (Figure 1.9). To solve the yield problem, in the third attempt, the sonication procedure was replaced by aggressive stirring. Unfortunately, the product yield was even lower.

   ![](https://static.igem.org/mediawiki/2020/5/5b/T--Technion-Israel--Gel_1.8.png
   )

   **Figure 1.8:** : Particle Trapped at Interface after Centrifugation

   ![](https://static.igem.org/mediawiki/2020/b/b2/T--Technion-Israel--Gel_1.9.png
   )

   **Figure 1.9:** : 2nd Microgel product (~0.2mg)

   Before proceeding to the next step, the created Microgel Beads from the 2nd and 3rd attempt were taken for microscopic analysis to estimate their sizes. Figure 1.10 shows that the Microgel Beads are round, and the one which was circled with white dotted line was a “broken” bead. This “broken” bead led us to believe that the particles were polymers instead of water or air bubbles. To make sure that the particles are three dimensional, another image was taken via Z-Stack (Figure 1.11). Although nice images were obtained using samples from the 2nd Microgel products, no proper image was taken for the product from the 3rd attempt because the sizes of particles varied and some of them were oversized for the microscope. After analyzing all the microscopic photos of particles from 2nd Microgel product, the particles’ sizes were estimated to be in the 40μm-90μm range. According to literature [[10]](#r10), the expected size of particles was around 6.5μm. The reason of such a deviation could be caused by the differences in the protocols, since the power of sonication used in the 2nd attempt was 20% instead of 40% that was suggested by Prof. Boaz Mizrahi. Another possible reason is that the sizes of particles increased due to swelling since this polymer can swell in aqueous solutions.

   ![](https://static.igem.org/mediawiki/2020/a/ad/T--Technion-Israel--Gel_1.10.png
   )

   **Figure 1.10:** : Microscopic image of Microgel Beads (Product from the 2nd Attempt, circled in white: a “broken” bead)

   ![](https://static.igem.org/mediawiki/2020/a/a3/T--Technion-Israel--Gel_1.11.png
   )

   **Figure 1.11:** : Height image of Microgel Beads via Z-Stack

   To conclude this part of the work, the preparation of NTA monomers was successful and the high yield resulted in several trials of polymerization experiments, though extra attention is required at the dripping step. However, our main struggle is improving the yield of final Microgel Beads and restraining their sizes in the desired range.

   Our future plans for the Microgel Beads could be divided into three sections: verification of their connection with His-tagged GFP (detailed in the next section), verification of their connection with fluorescence marked hACE2 and verification of their connection to fluorescence-marked hACE2 after mixing with hydrogel. In addition, only the 2nd Microgel Beads were used in further experiments since their sizes were close to the desired size range.
3. **Verification of Microgel Beads via Enhanced Green Fluorescence Protein (eGFP)**

   Since Microgel Beads were meant to be the carrier of “decoy proteins” which have His-tags, it is crucial to examine their ability to selectively bind His-tagged proteins. As mentioned in the previous part, three experiments were planned for this verification but only the first one was performed due to time limit, and the results of it would be presented in this section.

   This experiment was conducted according to the [protocols](https://2020.igem.org/Team%3ATechnion-Israel/Experiments) and could be concluded as followed: setting GFP blank, incubating the Microgel Beads with GFP solutions, and obtaining results using the plate reader and microscope. As written in the protocol, the Microgel Beads were planned to be tested with GFP solutions at two concentrations (33.33ng/μl, 16.67 ng/μl) since the size of particles were slightly oversized compared to the expectation. Nevertheless, the product was tested with only one concentration (33.33ng/μl) because there was only a tiny amount of Microgel available. The tested samples were taken from the Microgel Beads of 2nd attempt because of their relatively large amount and close to proper sizes.

   The microscopic analysis has given us two parts of results, including a rough quantification of connected GFP and some images. Firstly, the green fluorescence intensities of GFP+Microgel samples and pure Microgel negative controls were compared and presented below (Figure 1.12A). To get the value of intensity, an example is shown in Figure 1.12B. Based on the graph (Figure 1.12A), after incubating with GFP, the Microgel’s fluorescence intensity has increased approximately two times, which proves that the beads have connected to the GFP successfully. However, it is obvious that such a measuring method is not analytical and objective enough. Besides, there is also a relatively large amount of GFP trapped inside the polymer particles since there are pores on these spheres’ surfaces. Therefore, we measured the GFP fluorescence by a plate reader. Besides this brief estimation (Figure 1.12), some images were also taken using the microscopy for visual comparison (Figure 1.13). By comparing the images of the Microgel w/o GFP in Figure 1.13, it could be easily told that there is a significant difference between the fluorescence intensities of GFP incubated samples and the control, which meant that the Microgel Beads were capable of binding His-tagged GFP.

   ![](https://static.igem.org/mediawiki/2020/2/28/T--Technion-Israel--Gel_1.12.png
   )

   **Figure 1.12:** A- Comparison of green fluorescence intensity of Microgel Beads incubated with and without GFP via optical microscopy. B- Example of determining the green fluorescence intensity via microscopy, the particle’s fluorescence intensity was represented by the peak value of the graph.

   ![](https://static.igem.org/mediawiki/2020/2/22/T--Technion-Israel--Gel_1.13.png
   )

   **Figure 1.13:** A, B- Microscopic images of Microgel Beads (negative control) taken at 490nm light and were standarized to an intensity of 5\*104. C, D- Microscopic images of Microgel Beads+GFP (490nm light, standarized to an intensity of 5\*104.

   The plate reader was used to verify the Microgel Beads’ ability to bind His-tagged proteins in a more analytical way. Since directly measuring the fluorescence of a dense sample of Microgel+GFP might result in low accuracy, we chose to use an indirect method. This indirect measurement was aimed to use the difference in fluorescence intensities of pure GFP solution and supernatant of Microgel+GFP which contains unbound GFP to represent the amount of connected GFP. According to the results (Figure 1.14), the average value of green fluorescence in the supernatant (Post-treatment) was almost half of the intensity of the original GFP solution (Pre-treatment), which could led to the conclusion that the Microgel Beads created in the lab were able to bind the His-tagged proteins as expected.

   ![](https://static.igem.org/mediawiki/2020/7/7c/T--Technion-Israel--Gel_1.14.png
   )

   **Figure 1.14:** Comparison of green fluorescence intensities between GFP solution (Pre-treatment) and supernatant (Post-treatment) that contains unbound GFP (460nm excitation and 510nm emission)

   After proving the Microgel’s ability to connect with His-tagged proteins, our future plan regarding this part involves combining the created Microgel Beads with fluorescent marked human ACE2 protein to figure out whether the connected proteins’ functionalities would be altered. Then, the Microgel would be connected with the designed “decoy proteins” to construct the final product.
4. **Side Task: hACE2-mCherry-tdPP7-His Protein Expression in HEK-293F Cells**

   Production of fluorescent marked human ACE2 protein were necessary since this material couldn’t be obtained commercially and was needed for one of the proof of concept experiments of the Microgel Beads. The used plasmid that encodes human ACE2 protein with fluorescent marker and His-tag hACE2-mCherry-tdPP7-His (**Target**, Figure 1.15A) and a second plasmid, hACE2-tdPP7-His (Control, Figure 1.15B), was used as a control for the experiment. This **Control** was used because this plasmid has been used for ACE2 protein expression in HEK-293F cells and the proteins were also purified successfully in the lab. And the **Target** is what we actually wanted to obtain. Both plasmids were designed and cloned by Dr. Sarah Goldberg, a Research Assistant from Prof. Roee Amit’s lab in the Faculty of Biotechnology and Food Engineering in the Technion.

   ![](https://static.igem.org/mediawiki/2020/2/21/T--Technion-Israel--Gel_1.15.png
   )

   **Figure 1.15:** A- Gene map of hACE2-mCherry-tdPP7-His (**Target**). B- Gene map of hACE2-tdPP7-His (**Control**).

   The chosen cells for transfection were the HEK Freestyle 293F cells, and the protocol for cell culture could be found [here](https://2020.igem.org/Team%3ATechnion-Israel/Experiments). The whole process includes cell culture, transfection and protein purification. This was done with the help of Dr. Sarah Goldberg and Or Willinger from Prof. Roee Amit’s lab. To check whether the cells were transfected properly, samples were taken for plate reader and Fluorescence-activated cell sorting (FACS) at three time points: one, two and five days after transfection. Results were presented as followed (Figure 1.16). As shown in Figure 1.16A, the mCherry fluorescence intensity has increased significantly at Day 2, which implied that the transfection process has succeeded. However, since the FACS results could not provide useful information regarding amount of fluorescent protein that were secreted into the medium, it is necessary to include plate reader analysis of the medium. This step is important because eventually, the proteins would be purified from the medium. According to the result in Figure 1.16B, the fluorescence intensity of transfected cells’ supernatant, which represents the cell medium, has also increased dramatically at Day 2. Therefore, by combing the results from FACS and plate reader, it could be concluded that the cells were transfected successfully and have started to express the plasmid and explode the proteins to medium two days post-transfection.

   ![](https://static.igem.org/mediawiki/2020/5/53/T--Technion-Israel--Gel_1.16.png
   )

   **Figure 1.16:** Figure 1.16: A- FACS Results at 1, 2, and 5 Days Post Cell Transfection (Percentage of Live & Single Cells was based on total cell count of 104 while percentage of mCherry Expressing was based on the number of cells that were alive and single.). B- Plate Reader Results at 1, 2, and 5 Days Post Cell Transfection (560nm excitation and 612nm emission; Blank: FreeStyle Medium; **Target**: hACE2-mCherry-tdPP7-His.).

   Five days post-transfection, the proteins were purified from the transfected cells’ growth medium for both the Target (hACE-mCherry-tdPP7-His) and Control (hACE2-tdPP7-His). To increase purification yield, this process was split into two parts using different methods: His-affinity agarose (Figure 1.17) and MagneHis Protein Purification System (Figure 1.18). Firstly, samples were taken for concentration measurement (Table 1.1 and Table 1.2) using Nanodrop spectrophotometer in order to select the elution solution that has the highest protein concentration. According to the Nanodrop’s results, the His-affinity agarose’s purification efficiency was higher than its counterpart and in general, the products’ protein concentrations were low. To ensure that the purified proteins of the Target set were with fluorescence as desired, samples were taken for plate reader and the elution buffer was used as control. As shown in the figure (Figure 1.19), compared to the control which was the elution buffer, the elution samples from His-affinity agarose purification method had a significant fluorescence, which indicates the presence of desired proteins. By combining the above-mentioned results, from products purified by His-affinity agarose, the third elution product (E3) of Target and second elution product (E2) of Control were chosen to be loaded in the SDS-page with all the products from MagneHis Protein Purification System as well. After analyzing the images of SDS-page gel (Figure 1.20, Figure 1.21), negative result was found for the elution product of MagneHis Protein Purification System while for its counterpart, bands showed up for both Control and Target, and the sizes were close to expectation.

   To sum up this part, both proteins for the Target and Control were successfully purified from the growth medium of transfected cells although the concentrations were low.

   ![](https://static.igem.org/mediawiki/2020/d/d3/T--Technion-Israel--Gel_1.17.png
   )

   **Figure 1.17:** Protein Purification via His-affinity Agarose

   ![](https://static.igem.org/mediawiki/2020/2/2a/T--Technion-Israel--Gel_1.18.png
   )

   **Figure 1.18:** Protein Purification via MagneHis Protein Purification System

   Table 1.1: Concentrations of Purified Proteins Measured by Nanodrop spectrophotometer (His-affinity Agarose, calibrated by elution buffer, the values of samples that were used for further processes are underlined)

   |  |  |  |  |  |  |
   | --- | --- | --- | --- | --- | --- |
   | **Name** | **Eluted Product (E1) Concentration (mg/ml)** | **Eluted Product (E2) Concentration (mg/ml)** | **Eluted Product (E3) Concentration (mg/ml)** | **Eluted Product (E4) Concentration (mg/ml)** | **Eluted Product (E5) Concentration (mg/ml)** |
   | **Target** | 0.259 | 1.026 | 1.161 | 1.110 | 1.069 |
   | **Control** | 0.615 | 1.379 | 1.339 | 1.218 | 1.117 |
   | **BSA 1:10** | 1.406 | | | | |

   Table 1.2: Concentrations of Purified Proteins Measured by Nanodrop spectrophotometer (MagneHis Protein Purification System, calibrated by elution buffer)

   |  |  |
   | --- | --- |
   | **Name** | **Concentration of Eluted Product (mg/ml)** |
   | **Target** | 0.117 |
   | **Control** | 0.076 |
   | **BSA 1:100** | 0.118 |

   ![](https://static.igem.org/mediawiki/2020/a/af/T--Technion-Israel--Gel_1.19.png
   )

   **Figure 1.19:** Plate reader results of samples post-purification

   ![](https://static.igem.org/mediawiki/2020/5/52/T--Technion-Israel--Gel_1.20.png
   )

   **Figure 1.20:** SDS-Page result of purified proteins from His-affinity agarose (Target: hACE2-mCherry-tdPP7-His; Control: hACE2-tdPP7-His; Protein Standard: BSA 1:10; Protein Ladder: PM2600 ExcelBand)

   ![](https://static.igem.org/mediawiki/2020/4/4c/T--Technion-Israel--Gel_1.21.png
   )

   **Figure 1.21:** SDS-Page result of purified proteins from MagneHis Protein Purification System (Target: hACE2-mCherry-tdPP7-His; Control: hACE2-tdPP7-His; Protein Standard: BSA 1:100; Protein Ladder: PM2600 ExcelBand)

   Due to the low concentrations of the purified proteins, a concentration step was needed. Prior to the actual concentration, a dialysis (Figure 1.22) was required since the buffers used in the purification contained imidazole which would lead to unavailable His-tags. As a result of the SDS-page analysis, only the third elution product from the His-affinity agarose purification was used. The protein concentrations were again measured via Nanodrop (Table 1.3). Post-concentration samples’ concentrations were re-measured after the concentration process (Table 1.4). Unfortunately, although the concentrations of Control and the BSA were lifted successfully, the concentration of Target has failed.

   ![](https://static.igem.org/mediawiki/2020/9/95/T--Technion-Israel--Gel_1.22.png
   )

   **Figure 1.22:** Dialysis of Purified Proteins

   Table 1.3: Concentrations of Purified Proteins Measured by Nanodrop (Post-dialysis, calibrated by dialysis buffer)

   |  |  |
   | --- | --- |
   | **Name** | **Protein Concentration (mg/ml)** |
   | **Target** | 0.179 |
   | **Target’ \*** | 0.015 |
   | **Control** | 0.246 |
   | **BSA 1:100** | 0.005 |

   (\* The sample of Target was split into two since the volume of sample increased significantly after the dialysis. These two sets of samples were combined again during the concentration.)

   Table 1.4: Concentrations of Purified Proteins Measured by Nanodrop (Post-concentration via Amicon, calibrated by dialysis buffer)

   |  |  |
   | --- | --- |
   | **Name** | **Protein Concentration (mg/ml)** |
   | **Target** | 0.108 |
   | **Control** | 1.117 |
   | **BSA 1:100** | 0.013 |

   As a summary to all the work in this part, two plasmids were transfected into HEK-293 cells and the proteins were successfully purified. However, since the proteins’ concentrations were low and the concentration using Amicon did not work well, the products could not be used in the proof of concept experiments for Microgel Beads. One of the possible solutions for obtaining the Target proteins in a higher concentration might be growing more transfected cells to increase the initial protein concentration prior to the purification.

# *Bacillus subtilis* Spore

[Overview](#nav-bacoverview)
[Key Achievements](#nav-backey)
[Introduction](#nav-bacintro)
[Aims](#nav-bacaims)
[Results](#nav-bacresults)

Due to accumulated knowledge of many studies over the years, and the fact that its genome can be easily manipulated, *B. subtilis* bacteria has become one of the most widely used strains in the world of synthetic biology. Moreover, unlike its common counterpart, *E. coli*, *B. subtilis* is considered "Generally Recognized as Safe" (GRAS) and is used even in the food industry.

In the search for a carrier that will display the “decoy proteins” on its surface, one of the most attractive options was to display them on a membrane of living *B. subtilis* bacteria. In that way, the living bacteria would become the front-line producers of those “decoy proteins” and one of the dominant components in the final product against the viruses. While searching for methods to express heterologous proteins on the membranal surface of a vegetative *B. subtilis*, we came across a special technology that involves the use of *B. subtilis* spores. *B. subtilis* spore surface display (BSSD) technology uses the fusion of spore coat protein (or Cot) with heterologous proteins for displaying them on the spore’s coat with high activity and stability.

Here, we present a method for the expression of our heterologous “decoy proteins” on the surface of the *B. subtilis* PY79 spores using BSSD technology. By fusing the spores coat protein (CotC or CotG) to “decoy protein” we will be able to decrease the binding of the viruses to the human cells, thus reduce the chance of infection. During our lab work, we were able to induce sporulation on the *B. subtilis* PY79 strain, as well as design, construct and integrate pBS1C \_SYB15 and pBS1C \_SYB68 plasmids to the *B. subtilis* genome. The integration of pBS1C \_SYB68 into the B. subtilis PY79's genome was successfully verified by Colony-PCR. Germination of the *B. subtilis* PY79 spores in the hydrogel was tested as well and it was found that our hydrogel is a less convenient environment for *B. subtilis* PY79 spores’ germination compared to water.

* Sporulation of *B. subtilis* PY79
* pBS1C \_SYB15 and pBS1C \_SYB68 plasmids construction
* pBS1C \_SYB15 and pBS1C \_SYB68 plasmids integration into the amyE locus of the *B. subtilis*.
* Incorporating of *B. subtilis* in thermo-responsive hydrogel

*Bacillus subtilis*, a gram-positive bacteria that can be found in different environments, has been the model bacteria for many iGEM groups and researchers in the field of synthetic biology over the years. The development of the synthetic biological toolbox in recent years has made the B. subtilis one of the most productive hosts for enzymes and biochemical products alongside *Escherichia coli* (*E. coli*), which is another common host in the world of synthetic biology [[10]](#r10). The industrial need for the ongoing production of highly utilized recombinant proteins has increased over the years and made *B. subtilis* one of the most widely used bacteria thanks to extensive knowledge and ease of genetic manipulation.

Under significant extreme conditions, such as nutrients depletion in the environment, many vegetative gram-positive bacteria, including B. subtilis, can perform sporulation as described in Fig. 2.1 In this form, the bacteria can survive for a long time under extreme conditions. During this process, an asymmetrical division of genetic materials takes place from a mother cell into smaller daughter cells called pre-spores. At the end of this process, the pre-spore cells become endospores and are released into the surrounding medium. The pre-spore organized into three main layers: an amorphous undercoat, a lamellar layer, and a striated electron-dense outer coat. In parallel with the later formation, a proteinaceous layer forms around the surface of the outer spore coat [[11]](#r11).

![](https://static.igem.org/mediawiki/2020/2/20/T--Technion-Israel--B.sub_2.1.png)

**Fig. 2.1:** Illustration of the sporulation process

The outer coat layer of the spore is a source of great interest as a tool for research and application in many fields such as biotechnology, synthetic biology, and molecular biology. The reason for this is the ability to use several dozen proteins that make up the outer coat of the spore (Cot proteins) as an anchor to other heterologous proteins and display them on the outer surface of the spore [[12]](#r12). For example, Negri et al. were able to express fragments of Clostridium difficile (C. diff) flagellar cap protein (FliD protein) on the surface of B. subtilis spores using many Cot proteins, such as CotC and CotG, to produce a FliD-based vaccine against C. diff [[13]](#r13). Another example came from the 2016 iGEM group from Freiburg, which worked on the introduction of functional glutathione S-transferase (GST) and an epitope-specific nanobody for targeted drug delivery [[14]](#r14). The technology used by most researchers is called "Bacillus subtilis spore surface display" (BSSD) which involves a fusion of the target protein to one of the Cot proteins, transforming the relevant plasmid into the B. subtilis and encouraging sporulation. Verma et al. [[15]](#r15) showed a functional expression of one of our chosen “decoy proteins” (Angiotensin-converting enzyme 2 (ACE2)) in Lactobacillus paracasei. Based on the extensive work that is mentioned above, we decided to combine the ideas to create a B. subtilis spore that expresses the “decoy proteins”, which can bind to the virus's spikes, as described in Fig. 2.2 These spores will be an integral component in our final hydrogel-based product, meant to decrease virus' infectivity.

![](https://static.igem.org/mediawiki/2020/0/09/T--Technion-Israel--B.sub_2.2.png)

**Figure 2.2:** Illustration of the binding between the virus’s spike protein with “decoy protein” that will be expressed on the spore's surface of *B. subtilis* PY79.

* Design the final plasmid that includes the gene encoding the coat protein fused to the gene encoding one of the "decoy proteins".
* Transformation and integration of the fusion protein gene into the B. subtilis PY79 genome.
* Induction of sporulation on the *B. subtilis PY79*.
* Testing for the expression of the protein complex.
* Germination test in the hydrogel environment.
* Test the ability of the Cot-decoy protein complex to bind to the virus's spikes.

**Plasmid Construction**

Initially, six different sequences were designed to be incorporated into the genome of *B. subtilis* as describes in Fig. 2.3 Inspired by the articles mentioned above10 [[11]](#r11) [[12]](#r12) [[13]](#r13) [[14]](#r14) [[15]](#r15), a 33 bases linker was attached between the Cot protein genes and the gene of interest. The decision to use the pBS1C\_LacZ as a backbone was based on a study of Radeck et al. in which this vector was specified as capable of undergoing integration into the Amy-E locus (encoding for α-amylase) of *B. subtilis*.

![](https://static.igem.org/mediawiki/2020/5/56/T--Technion-Israel--B.sub_2.3.png)

**Fig. 2.3:** Designed plasmids for integration into the *B. subtilis* PY79's genome: pBS1C\_CotC\_Syb15, pBS1C\_CotC\_Syb68, pBS1C\_CotC\_ACE2, pBS1C\_CotG\_Syb15, pBS1C\_CotG\_Syb68, pBS1C\_CotG\_ACE2.

The first cloning step involved linearization of the vector pBS1C\_LacZ, removal of the LacZ-encoded sequence, and insertion of corresponding edges to the G-block sequences encoding the “decoy proteins” via PCR reaction and Gibson Assembly (Fig. 2.4)

![](https://static.igem.org/mediawiki/2020/7/70/T--Technion-Israel--B.sub_2.4.png)

**Fig 2.4:** First cloning steps – Revers PCR and Gibson Assembly.

After each plasmid transformation into *E.coli* Top10, 2 different tests were performed to verify the insertion of the sequences. The first one was a colony-PCR test, in which two different primers were used- one that overlaps with the backbone (pBS1C) and the other that overlaps with the sequence encoding for each one of the decoy proteins (ACE2 / Sybody15/ Sybody68) separately. Promising clones were then sent for another verification by Sanger sequencing.

After a few attempts, we were able to get both pBS1C\_Sybody15 and pBS1C\_Sybody68 as seen in Fig. 2.5 & 2.6 with a 100% match to the planned sequences. Regarding the ACE2 sequence, several attempts have been made with changed parameters to insert it into the pBS1C backbone using the Gibson assembly method, but no positive result has been obtained. In this case, a second option was planned, which included the use of restriction enzymes, but so far, we have not been able to perform it, due to lack of time. At this stage of the experiment, we decided to proceed only with the pBS1C\_Sybody15 and pBS1C\_Sybody68 plasmids.

![](https://static.igem.org/mediawiki/2020/4/43/T--Technion-Israel--B.sub_2.5.png)

**Fig. 2.5:** 1K NEB Ladder. Black arrows represent bands in the desired sizes. Expected results: 2’.1-2’.8, pBS1C\_Syb15 ~ 600 bp. Negative control ~ no bands.

![](https://static.igem.org/mediawiki/2020/3/39/T--Technion-Israel--B.sub_2.6.png)

**Fig. 2.6:** 1K NEB Ladder. Black arrows represent bands in the desired sizes. Expected results: 3.1-3.4, 3’.5-3’.8: pBS1C\_Syb68 ~ 600 bp. Negative control ~ no bands.

The pBS1C backbone we worked with also contained the mRFP encoding sequence that we removed in a subsequent step, in which we used pre-designed specific restriction sites to insert the sequence encoding the Cot proteins as seen in Fig. 2.7.

![](https://static.igem.org/mediawiki/2020/b/bd/T--Technion-Israel--B.sub_2.7.png)

**Fig. 2.7:** Cot protein insertion and mRFP removal.

Unfortunately, we were unable to insert the designed sequence encoding the coat protein even after several restriction attempts under different conditions. That could be due to low insert/backbone ratio, low inserts concentration (Cot proteins), as a result of relatively low recovery from low-melting agarose gel. Due to a lack of time, we were unable to test all parameters.

**Integration test**

Using an integrative vector in bacillus strains is common and intended to prevent instability of recombinant plasmids as a result of structural rearrangement [[16]](#r16). One of the methods to make a vector integrative is to use a gene encoding to α-amylase (amyE locus). In this method, the desired gene can be integrated within amyE locus by utilizing the natural mechanism of homologous recombination, by flanking the gene of interest on both sides in two halves of the amyE gene designated amyE-front and amyE-back [[17]](#r17) as seen in Fig. 2.8.

![](https://static.igem.org/mediawiki/2020/8/8d/T--Technion-Israel--B.sub_2.8.png)

**Fig. 2.8:** Integration to AmyE locus

Here, we used the described method to integrate pBS1C\_Syb68/15 plasmid into the genome of *B. subtilis*. A colony-PCR test was performed after plasmid transformation and integration. The results in Fig. 2.9 show the success of pBS1C\_Syb68 integration in several colonies.

![](https://static.igem.org/mediawiki/2020/3/36/T--Technion-Israel--B.sub_2.9.png)

**Fig. 2.9:** Log Ladder. Black arrows represent bands in the desired sizes. Expected results: 1-17: PY79\_Syb68 - ~600 bp.

Genomic sequencing of the bacillus, which was pre-designed for the final plasmid (pBS1C\_CotC/G\_ACE2/Sb#15/Sb#68), is another option for testing the integration for further verification.

**Sporulation**

Sporulation is a process that gram-positive species can undergo, including *B. subtilis* bacteria, when they are facing harsh environmental conditions, such as the depletion of nutrient sources. The coat of *B. subtilis* spores composed of more than 20 polypeptides, which confers resistance to lysozyme and too harsh surrounding environments. During sporulation, the bacteria form a rigid shell that maintains the ability to survive under environmental stress [[18]](#r18)[[19]](#r19).

Encouraging sporulation is an integral part of our idea. Since the "decoy proteins" can only be expressed when the bacteria undergo sporulation as described in Fig. 2.10, an adjustment of the working conditions is required to make sure that the majority of the population will be in the form of spores, rather than vegetative bacteria. Therefore, as a preliminary step, a protocol corresponding to the B. subtilis 168 WT was tested, and adjustments were made to adapt it to our chosen strain - PY79.

![](https://static.igem.org/mediawiki/2020/b/b2/T--Technion-Israel--B.sub_2.10.png)

**Fig. 2.10:** Illustration of B. subtilis spore surface display of ACE2 using CotC/CotG protein.

The *B. subtilis* PY79 strain is characterized by a knockout of two genes, which are responsible for the production of proteases – proteins that impair other protein bindings to the outer spore coat. These bacteria were diluted and incubated in a Difco Sporulation Medium (DSM) that stimulates sporulation [[20]](#r20). After several adjustments that included increasing the number of washing steps, we were able to reach ~90% spores out of the general population Fig. 2.11.

![](https://static.igem.org/mediawiki/2020/1/18/T--Technion-Israel--B.sub_2.11.png)

**Fig. 2.11:** *B. subtilis* PY79 spores under a microscope. Spores are shown as white spheres.

**Integration of the Bacillus in the hydrogel**

Although spores can survive for a long time in their dormant state, once the conditions in the environment become more favorable for the growth of the vegetative bacteria, the spores will germinate and become living and functioning bacteria [[21]](#r21). In that scenario, the decoy proteins expressed on the surface of the spore will no longer display and the effectiveness of the product will be significantly reduced.

Many components and conditions trigger germination. For example, the presence of nutrients in the living environment such as L-alanine, L-valine, and L-asparagine, high pressure of several thousand atmospheres, etc.

In this part of the project, the stability of the spores, and the germination rate were examined given the aqueous environment and hydrogel one. The effect of skin temperature (~37°C) and refrigerator storage temperature (4°C) on the stability of the spores were also examined. As a control, samples incubated in water were examined, since high water content is a significant factor in the swelling of the spore and the completion of the germination process.

The first test performed is based on the turbidity in O.D600nm. Spores exhibit light refraction that can be detected using a spectrophotometer set to O.D600nm [[22]](#r22). During germination, the spore changes its physical shape resulting in lower O.D values. As can be seen in Fig. 2.12, even after 1-hour incubation no significant decrease in the spores’ amount was observed in different environments and temperatures, which emphasizes the stability of the spores in our product.

![](https://static.igem.org/mediawiki/2020/f/fc/T--Technion-Israel--B.sub_2.12.png)

**Fig. 2.12:** The change in turbidity (O.D600 values) relative to initial measurement over time

To further examine the germination process, a pyridine-2,6-dicarboxylic acid (DPA) release test was performed using O.D420nm turbidity test. The core region of the spore is rich in that spore-specific molecule (DPA) and poor in water [[22]](#r22). During germination, the same molecules are released into the environment and can serve as a tool for testing the germination rate. In Fig. 2.13, you can see the difference in concentration between the aqueous environment and the hydrogel environment after about an hour. While the DPA concentration reached ~ 250 g/mL in the aqueous environment, its equivalent in the gel showed concentrations that were ~10 times smaller. Therefore, it can be concluded that the hydrogel environment does not constitute a good basis for the germination process and that the integration of the spores in the gel raises the possibility of obtaining a stable product.

![](https://static.igem.org/mediawiki/2020/4/41/T--Technion-Israel--B.sub_2.13.png)

**Fig. 2.13:** DPA concentration in both medium (water and hydrogel) at different temperatures

The structure of endospores makes it difficult for chemicals to penetrate, making them difficult to stain. Some dyes penetrate the spore using steam. Malachite green is one of the dyes that stains endospores in green, as can be seen under the microscope [[23]](#r23). The greenish color does not come out of the stained spores, even after rinsing with water. In contrast, it is easy to remove the green Malachite dye in vegetative cells. For vegetative bacteria, Mercurochrome is frequently used, which stains the cells in a red-purple color [[24]](#r24). By using those dyes, we were able to tangibly see all the results described under the microscope. Fig. 2.14 shows the significant differences in the concentration of vegetative bacteria in water and gel after one hour, when more vegetative bacteria were observed in water. These results are consistent with the results of the DPA test described above.

![](https://static.igem.org/mediawiki/2020/6/68/T--Technion-Israel--B.sub_2.14.png)

**Fig. 2.14:** Spores and vegetative cell assessment in water and hydrogel. **(A)** Spores in water, time zero; **(B)** Spores in water after 1-hour incubation; **(C)** Spores in Hydrogel, time zero; **(D)** Spores in Hydrogel after 1-hour incubation. Spores were stained with Malachite green (greenish spheres) and vegetative *B. subtilis* PY79 cells with Mercurochrome (red-purple rod shapes).

**Future plans**

During the construction of the plasmids, we encountered difficulties in reaching the final constructs. The first hurdle was the construction of the pBS1C\_ACE2 plasmid. After several unsuccessful attempts using Gibson assembly, it was decided to proceed only with the pBS1C\_Syb15/68, as they managed to pass the initial stage of cloning. To ligate the ACE2 sequence, a contingency work plan was planned in advance for further work. This plan involves the use of restriction enzymes instead of Gibson assembly to attach the ACE2 to the pBS1C backbone.

The final plasmid assembly including fusion of the decoy proteins with the sequence encoding the coat proteins was designed using restriction enzymes.

The expression of the proteins on the spores was designed to be tested by marking the c-Myc with a fluorescent anti-Myc followed by FACS measurement.

In the final stages, the affinity of the “decoy-proteins” for the spike proteins was designed to be tested using affinity tests such as Bio-layer interferometry (BLI) real-time test or Surface Plasmon Resonance (SPR) [[25]](#r25).

# Mutated ACE2

[Overview](#nav-aceoverview)
[Key Achievements](#nav-acekey)
[Introduction](#nav-aceintro)
[Aims](#nav-aceaims)
[Results](#nav-aceresults)

Angiotensin-converting enzyme 2 (ACE2) is an enzyme found in cell membranes of cells in the lungs, arteries, heart, kidneys, and intestines. its role is to catalyze the hydrolysis of angiotensin II into angiotensin (1-7) and therefore lower blood pressure. In addition to its natural role in the body, ACE2 is a cellular receptor for SARS-CoV-2 (and other coronaviruses). The infection by SARS-CoV-2 happens through the connection between the ACE2 extracellular peptidase domain (PD) and the SARS-CoV-2 spike proteins’ receptor-binding domain (RBD) [[26]](#r26).

The goal of this sub-project is to produce one of the “decoy proteins”, an improved ACE2. The protein that we aim to produce will play a role in the product as "the bait". The main purpose of producing an improved ACE2 is to enable an interaction with higher affinity between the protein and the virus's spike proteins compared to the native one. This will be achieved by changing different amino acids (AA) that are related to the spike - ACE2 binding to achieve a higher affinity connection. In this way, we can increase the chances of capturing the virus and prevent infection.

Here, we managed to produce linear fragments out of p416-FEC-nCore1 plasmid. Those fragments contain 20-40 overlap base-pairs that enable us to use them in "Any Gene Any Plasmid" (AGAP) cloning method [[27]](#r27) [[28]](#r28). In addition, transformation was conducted with the linear fragments and a designed gBlock that contains a mutated ACE2 sequence into W303 yeasts strain. We observed growth of yeast on the reaction plate, but due to the contamination that was found on the plate, the experiment was terminated. Unfortunately, due to the lockdown, we did not achieve our goals, but in the future, we aim to proceed by repeating the procedures and take special caution regarding a sterile work environment.

* Producing "R-16 cut" fragment by PCR reaction.
* Producing F-9 fragment with additional His-tag by PCR reaction.
* Transformation of 3 linear fragments to W303 yeasts strain.
* Adding His-tag to p416-FEC-nCore1 plasmid.
* Transformation of p4160-FEC-mCore1 with His tag plasmid to top10 *E. coli*.

In our project, we tried to find a variant of human ACE2 protein that has higher affinity to the spike protein compared to the W/T. In order to achieve this goal, we created a library of 93,311 options of mutant ACE2 proteins based on four different studies [[29]](#r29) [[30]](#r30) [[31]](#r31) [[32]](#r32). Efforts have been invested to identify amino acids (AA) in the ACE2 sequence that are related to its binding to the SARS-CoV-2 S protein.

16 AA were identified under that criteria:
N330, K353, G354, D355, F356, R357, A396, A25, T27, K31, Y41, Q42, L79, M82, Y83, P84.

![](https://static.igem.org/mediawiki/2020/7/7b/T--Technion-Israel--ACE2_3.1.png
)

**Figure 3.1:** The identified AA (red) one ACE2.

To create the library, we replaced those AA with other AA that maintain their bio-chemical characteristics (Aliphatic, Aromatic, Acidic, Basic, Hydroxylic, Sulfur containing, Amidic). Those changes gave us total of 97,202 variants.

As part of the efforts to find the most suitable variant, we planned to narrow down the size of the library using the ROSETTA bioinformatics modeling software [[33]](#r33). Due to the lack of time, we started the lab work with 3 initial variants, in parallel to the modeling work.

The three variants we worked with are:

1. X1 isoform, which is the most common ACE2 isoform in human lungs [[14]](#r14), as our positive control.
2. Variant V1 as our library’s representative, which is taken from literature [[13]](#r13). This variant was shown to have high affinity to the spike protein.

   |  |  |
   | --- | --- |
   |  | Mutations deriving from the X1 isoform |
   | V1 | H47A, T105Q, Q338P, A399L |
3. As the negative control, an ACE2 variant which was shown to be non-binding to the spike protein, according to literature.

   |  |  |
   | --- | --- |
   |  | Mutations deriving from the X1 isoform |
   | V-neg | Q89N, L92E, T105S |

After creating the final variant library, we aim to screen the library by a protein interactions model and then transform the sequences into yeast by heat shock. Finally, we plan to analyze the resulting proteins and optimize our design to the best. Results are obtained via SDS-page and FACS analysis.

* Purifying ACE2 protein with Nickel column.
* Purifying YFP protein with Nickel column.
* Analyzing the ACE2 size and perform affinity test for the mutant ACE2 protein.

Our lab work started with cloning a plasmid backbone that will contain the ACE2 variants. We extracted an existing plasmid (p416-FEC-nCore1, ~6000 bp) out of *Saccharomyces cerevisiae* W303 strain that we received from Prof. Roee Amit's lab. This backbone contains a yeCitrine fluorescent protein gene that will be replaced with the ACE2 variants later.

To allow extraction of the ACE2 protein from yeast, a His-tag sequence was added by performing a PCR reaction. A restriction map was created to verify the plasmid sequence, after several failures in PCR reactions.

![](https://static.igem.org/mediawiki/2020/6/6b/T--Technion-Israel--ACE2_3.2.png
)

**Figure 3.2:** 1 Kb Ladder. Black arrows represent bands in the desire size. PCR expected results - ~6 Kb for reaction and no bands on the control. 1 Cut restriction expected results - 6 Kb. 2 Cut restriction expected results - 5 Kb + 1 Kb. Control for restriction – Circular plasmid.

Once we verified the restriction map of the plasmid, we realized the issue might be the primers. Therefore, we used a different polymerase that is designed to work on difficult sequences - PrimeSTAR GXL polymerase (GXL).

Using the GXL polymerase we prepared a circular p4160-FEC-mCore1 plasmid with His-tag via PCR reaction, PNK and ligation. The circular plasmid was transformed to E. coli top10 to produce a yeCitrine protein fused to a His-tag.

![](https://static.igem.org/mediawiki/2020/5/55/T--Technion-Israel--ACE2_3.3.png
)

**Figure 3.3:** Transformation of the circular plasmid to E. coli top10 as shuttle vector.

We attempted to verify our cloning using Colony PCR. Unfortunately, the results were not as expected – 700 bp instead of 3400 bp. Thus, we decided to send the colonies for sequencing. The sequencing showed that during ligation of the blunt end PCR product, a few base-pairs were dropped from the plasmid which lead to the unexpected size. We realized that a different strategy was required.

However, due to the lack of time, we decided to put this part of the project on hold and continue to other parts.

Alongside the yeCitrine purification, we started working on "Any Gene Any Plasmid" (AGAP) cloning method. This method enables us to produce the desired plasmid by transforming linear fragments that contain 20-40 bp overlaps into yeast, which will be closed into a circular plasmid by in vivo homologous recombination [[11]](#r11)[[12]](#r12).

![](https://static.igem.org/mediawiki/2020/b/b5/T--Technion-Israel--ACE2_3.4.png
)

**Figure 3.4:** Illustration of AGAP homologous recombination method.

We produced the linear fragments for the AGAP method by performing several reactions. The 1st fragment "R-16 cut" was amplified from p416-FEC-nCore1 plasmid using PCR reaction followed by restriction (yeCitrine protein was cut out). The 2nd fragment "F-9" was also amplified from p416-FEC-nCore1 plasmid. And the 3rd fragment was ordered as gBlock.

Results of the PCR reactions are exhibited in pictures below.

![](https://static.igem.org/mediawiki/2020/9/99/T--Technion-Israel--ACE2_3.5.png
)

**Figure 3.5:** Log Ladder. Black arrows represent bands in the desired sizes. Expected results: F9 – 4300 bp; R16 – 1600 bp; Negative Controls – no bands.

![](https://static.igem.org/mediawiki/2020/3/36/T--Technion-Israel--ACE2_3.6.png
)

**Figure 3.6:** Log Ladder. Black arrows represent bands in the desired sizes. Expected results: X1, V1, Vneg G-Blocks – 2600 bp; Negative Controls – no bands.

The AGAP method requires a large amount of product (~1 µg) from each fragment. Therefore, many PCR reactions were needed to allow the R-16 cut fragments to reach this amount.

After getting sufficient amounts, we transformed the fragments into W303 type YA619 yeast strain. While yeast grew on the plate, mold grew on it as well, meaning we could not use the yeast colonies. At this point, we could not repeat the experiment because of the lockdown in our country.

Our future plans are to perform another transformation of the fragments into yeast, which will express the desired variant. Then, we aim to extract the proteins and purify them with Nickel column. Afterwards, we will verify the size of the protein extracted with SDS-page analysis and measure the affinity to the spike protein by Fluorescence-activated cell sorting (FACS) using a fluorescently labeled spike protein and an antibody targeting the human ACE2.

# Sybodies

[Overview](#nav-syboverview)
[Key Achievements](#nav-sybkey)
[Introduction](#nav-sybintro)
[Aims](#nav-sybaims)
[Results](#nav-sybresults)

Nanobodies are the variable domain of camelid heavy chain antibodies [[34]](#r34). Despite their small size, nanobodies exhibit full capacity to bind proteins selectively and were chosen due to their good properties and ability to simultaneously associate with the RBD [[35]](#r35).

In this part, the results that we got through creating the Sybodies are presented. The Sybodies are destined to be attached to the Microgel Beads as binders for the SARS-CoV-2’s spike proteins. The creation of the Sybodies was divided into three parts: cloning the desired plasmid containing one of the Sybody’s sequences, over express it in designated over expression bacteria, and extraction of the proteins from the cells. During the time we had in the lab, we successfully created over-expression plasmid containing the Sybodies’ sequence with additional His-tag. The sequence of the ligated plasmid was verified via colony PCR and Sequencing. Additionally, the first proof of concept for Sybody’s expression and extraction has achieved via SDS-PAGE and Western Blot.

* Cloning a ligated plasmid into TOP10 *E.coli* and verifying its sequence.
* Expression and Extraction of the Sybodies as proteins from KRX *E.coli* cells.

Most antibodies are composed of two heavy chains and two light chains while both chains contribute to the two identical antigen-binding sites. In addition to these conventional antibodies, camelids and sharks also produce antibodies composed only the heavy chains [[18]](#r18). These peculiar heavy chain antibodies contain only a single variable domain and two constant domains. The single variable domain is called nanobody or single domain antibody [[36]](#r36). Although their sizes were small, nanobodies are capable to bind proteins selectively and with high affinity [[37]](#r37) with full capacity. The differences between the three types of antibodies can be seen in the figure below.

![](https://static.igem.org/mediawiki/2020/4/44/T--Technion-Israel--sybodies_alpaca.png)

**Figure 4.1:** Antibodies, Nanobodies and Sybodies

Antibodies are widely used for the detection and manipulation of proteins in diverse research fields. However, the number of validated antibodies is limited and there is an unmet need for binding proteins tailored to fulfill desired technical and biological requirements [[38]](#r38). Creation of synthetic nanobody can solve this problem. Sybodies, which stand for synthetic nanobodies, are easy to produce, stable, soluble in aqueous solutions and can perform reversible refolding without the need for additional improvements. All those properties are ideal for a practical binder, especially for bacterial expression [[19]](#r19). Various researches that used synthetic nanobody for detection can be found [[12]](#r12) [[40]](#r40) [[41]](#r41) as well as a variety of protocols for creating them [[22]](#r22) [[42]](#r42) [[43]](#r43) [[44]](#r44). One significant example of synthetic nanobody’s novel application is the cancer treatments. Chimeric antigen receptor-modified T cells (CAR T-cells) are highly researched and used in various clinical trials [[45]](#r45) [[46]](#r46) [[47]](#r47) [[48]](#r48).

Sybodies were chosen as “decoy proteins” for the SARS-CoV-2 due to the advantages listed above. During the search on Sybodies, we came across the work of Dr. Justin D. Walter et al, in which 3 Sybody libraries targeting the receptor-binding domain (RBD) of SARS-CoV-2 were created and tested. The sequences for Sybody number 15 (Syb15) and Sybody number 68 (Syb68) were chosen for our project. Those sequences are two of the top six Sybodies’ sequences with the best properties tested in this article, such as kinetic parameters for interactions between Sybodies and spike proteins of SARS-CoV-2. Moreover, those two specific Sybodies were chosen because of their ability to simultaneously associate with the RBD [[25]](#r25). Using both proteins together in our product can, potentially, increase the overall avidity to the spike proteins and by that increase the efficiency of the product. We have consulted with Dr. Walter regarding this choice and he supported it.

In our project, the Sybodies are used to target the RBD of the SARS-CoV-2 spike proteins. Those Sybodies contain additional His tag that allows to bind them to the Microgel Beads and will be suspended in the Hydrogel later. Together, all three parts complete the ACT. product.

One of the advantages of Sybodies is that they can be produced easily in bacteria once their sequence is determined. In this project, the production of the Sybodies is composed of few steps. First, the Sybodies’ sequences and the pET9D backbone were restricted and ligated together. The ligated plasmid was transformed into TOP10 E.coli for sequence verification and amplification. Then, the amplified plasmid was transformed into KRX competent cells to overexpress the proteins. The cell content was extracted and the proteins themselves were purified. After that, a verification for the extracted proteins’ size and structure was done. The final step planned for this part was verification of the Sybodies’ functionality. This experiment was not preformed due to a lack of time.

The main goal of this part of the project is to produce functional Sybody proteins, that will be connected to Microgel Beads and serve as binders for the SARS-CoV-2 spike proteins. The production of the Sybodies was divided into two stages (cloning and expression), after which there would be another step of functionality test for the proteins. All the process is described in the schema below, in Figure 4.2.

![](https://static.igem.org/mediawiki/2020/b/ba/T--Technion-Israel--sybodies_scheme.png)

**Figure 4.2:** Sybodies creation process

**Cloning a ligated plasmid:**

The first step in the process of creating the desired proteins is to create a whole plasmid containing both the plasmid backbone and one of the Sybodies’ sequences.

This step began with amplifying the pET-9D backbone and inserting into it a Sybody sequence with additional His-tag. The insertion was done by restriction and ligation. This ligated plasmid was transformed into TOP10 E.coli in order to amplify it and its sequence was verified by colony PCR and Sanger sequencing. In Figure 4.3 & 4.4, the gels of the colony PCR are shown, for both Sybodies.
In the end of this step we had two verified plasmids containing each Sybody’s sequence.

![](https://static.igem.org/mediawiki/2020/9/9e/T--Technion-Israel--Syb_4.4.png
)

**Figure 4.3:** Colony PCR gel for Sybody 15 (2-Log ladder. Black arrows represent bands in the desired sizes. Expected results: 1-19: pET9d\_syb15 – 555bp).

![](https://static.igem.org/mediawiki/2020/6/6d/T--Technion-Israel--Syb_4.5.png
)

**Figure 4.4:** Colony PCR gel for Sybody 68 (2-Log ladder. Black arrows represent bands in the desired sizes. Expected results: 1-18: pET9d\_syb168 – 773bp)

**Expressing the Sybodies in KRX cells and purification:**

The second step in the process is creating the Sybodies as proteins.

This part began with overexpression of the ligated plasmid in KRX competent cells, to produce a large amount of Sybody proteins. The transformation into KRX competent cells was done by heat shock. Then, the bacteria were grown until 0.8-1.0 OD was reached. Afterward, the overexpression was induced by adding the inducer rhamnose into the cells’ growth medium. Extraction of the cell content was done by Homogenizer and the purification of the Sybody proteins was done using a Nickel column. In this stage, purification Nickel-bound beads from two different companies were used (Cube Biotech and Promega) to compare their efficiency and as duplicates. Eventually, to verify the proteins’ presence in the purified solution, SDS-PAGE and Western blot were performed. Pictures of the gels from both the SDS-PAGE and Western blot are shown below, in Figure 4.5 & 4.8.

![](https://static.igem.org/mediawiki/2020/a/a1/T--Technion-Israel--syb_4.5.jpeg
)

**Figure 4.5:** SDS-PAGE for the extract of KRX cells producing Sybody 68 (Excel band PX2600 marker. Black arrows represent bands in the desired sizes. Expected results: Sybody 68 – 14.26 kDa). E- Elution, F- Flow, W- Wash

![](https://static.igem.org/mediawiki/2020/7/70/T--Technion-Israel--Syb_4.8.png
)

**Figure 4.6:** SDS-PAGE for the extract of KRX cells producing Sybody 15 (Excel band PM2600 marker. Black arrows represent bands in the desired sizes. Expected results: Sybody15 – 13.12 kDa)

![](https://static.igem.org/mediawiki/2020/5/50/T--Technion-Israel--Syb_4.9.png
)

**Figure 4.7:** SDS-PAGE for the extract of control KRX cells (Excel band PM2600 marker).

![](https://static.igem.org/mediawiki/2020/0/0d/T--Technion-Israel--Syb_4.10.png
)

**Figure 4.8:** Western Blot with anti his-tag antibody (PageRuler ladder. Black arrows represent bands in the desired sizes. Expected results: Sybody 15 – 13.12 kDa; Sybody 68 – 14.26 kDa).

**Future plans:**

The last step in this part of the project is to prove the Sybodies’ functionality: their ability to capture the SARS-CoV-2 virus. This will be done by inducing and measuring a reaction between the Sybodies and the spike proteins, as shown in Figure 4.11. This was planned to be achieved with SARS-CoV-2 Spike Protein-Coated 96 wells Plates via ELISA procedure. In this procedure the produced proteins are reacted with the SARS-CoV-2 spike protein bound to the commercial plate. The amount of protein reacted with the plate measured by absorbance of light. Unfortunately, because of the COVID19 situation and the lockdowns in Israel, we did not manage to do this test.

After completing the Sybodies’ testing, verification of the interaction between the Sybodies and the SARS-CoV-2’s spike protein on the surface of the Microgel Beads will be done by the Microgel group.

**References**

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10. Liu Y, Liu L, Li J, Du G, Chen J. Synthetic Biology Toolbox and Chassis Development in Bacillus subtilis. Trends Biotechnol. 2019;37(5):548-562. doi:10.1016/j.tibtech.2018.10.005
11. Henriques AO, Moran CP. Structure, assembly, and function of the spore surface layers. Annu Rev Microbiol. 2007;61:555-588. doi:10.1146/annurev.micro.61.080706.093224
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13. Negri A, Potocki W, Iwanicki A, Obuchowski M, Hinc K. Expression and display of Clostridium difficile protein FliD on the surface of Bacillus subtilis spores. J Med Microbiol. 2013;62:1379-1385. doi:10.1099/jmm.0.057372-0
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