iGEM 2020 — Team SDU-Denmark

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# Team:SDU-Denmark/Notebook

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Videos

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[Final Presentation](https://2020.igem.org/Team%3ASDU-Denmark/Presentaion)

"An experiment is worth nothing if its procedure is not written down"

# Notebook for laboratory work

In the lab we strived to note everything we did each day. This helped to eep us structured and was a great place to assamble vital information.

For showing who was in the lab which days following abbreviations is used:
AMK: Anne Mette Kristensen
AR: Aicha Robgo
FF: Flora Fuglsang
FT: Frederik Tolberg
JL: Jakob Larsen
KDL: Kristine de Leon
NJ: Nikolaj Juul
RHA: Rikke Haugbølle Andersen

\*BE AWARE the notebook includes a few tables that can be hard to see if you visit this page from a mobile. If you wish to see these tables fully please switch to a tablet or computer sized screen.

INTRODUCTION

While we waited for our ordered DNA-sequences for Cas-proteins, we did a lot og introduction exercises in the lab to make sure we knew the protocols when our parts arrived

Week 25 (15th-21st of June)

**Monday**
 *Present: FF, FT, JL and NJ*
We learned how to make LB medium, LB agar and TSB buffer used for transformation.

 We made 500 mL medium and 500 mL agar. Furthermore stock solutions of CaCl2 and MgCl2 were diluted.

 An over-night (ON) culture of E. coli was set over.

  **Tuesday**
 *Present: FF, FT, JL and NJ*
Creation of competent cell culture (CCC) by CaCl2 protocol.

 New Top10 stock taken from -80°C freezer and streaked onto petri dishes are placed in 37°C incubator ON.

 **Wednesday**
 *Present: FF, FT, JL and NJ*
Transformation by CaCl2 into CCC from yesterday.
Plasmid transformed into CCC, contains Lac promoter and ampicillin resistance. Derived from 5H, plate 4 of the biobrick kit from 2017. DNA is resuspended into 10 µL milliQ water.
Transformation protocol followed. However, as Amp plates were not made, 20 µL Amp was added to the plates before transformed cells were added. Laboratory supervisor noted this as a “Gangster method”.
Cultures are plated onto plates as such:

|  | LB plate | LB plate w. ampicillin |
| --- | --- | --- |
| CCC + water | 50 µL | 50 µL |
| CCC + plasmid | 50 µL | 50 µL |

Incubated at 37°C ON.
Top10 colonies from yesterday transferred into tube with 2mL LB medium and put into fridge in stead of incubator, by mistake.

 **Thursday**
 *Present: FF, FT, JL and NJ*
Top10 colonies from 16/06 are transferred to 2 mL LB and cooled until 14:00, where they are transferred to incubation ON.
100 mL 50% glycerol mixed and autoclaved, ready for use in freezestocks.
Amp plates created. 14g LB+agar broth + 400 mL milliQ water.
• Autoclaved. When temperature is down to 40°C, 400 µL (100γ) Amp is added.
• Poured into plates and left ON on work bench.

 **Friday**
 *Present: FF, FT, JL and NJ*
TSB transformation performed from protocol.
• TSB buffer always kept on ice.
• ON cultures from yesterday were transferred to 3x 1.5 mL Eppendorf tubes.
• Protocol for TSB transformation was followed.
• Plasmid DNA concentration was measured on the Nanodrop spectrophotometer.
• 4µL plasmid DNA was added.
After transformation was done, cells were plated as such:

|  | LB plate | LB plate w. ampicillin |
| --- | --- | --- |
| CCC + water | 100 µL | 100 µL |
| CCC + plasmid without lac promoter | 100 µL | 100 µL, 250 µL |
 CCC + plasmid without lac promoter | 100 µL | 100 µL, 250 µL |

Freezestock (-80°C) of Top10 culture was created using 700 µL culture and 300 µL 50% glycerol.

 **Saturday**
 *Present: FF and NJ*
TSB transformation results were checked
• Control: colonies. Cells survived.
• Amp plates: No colonies. Transformation unsuccessful and therefore no Amp resistance. Possibly because cells were not in mid-exponential phase.
ON culture made for Top10: 2 mL LB medium and one Top10 colony.

  **Sunday**
 *Present: AMK and AR*
TSB-transformation into Top10 cells. Cells were plated as follows:

|  | LB plate | LB plate w. ampicillin |
| --- | --- | --- |
| CCC + water | 100 µL | 100 µL |
| CCC + plasmid without lac promoter | 100 µL | 100 µL, 250 µL |
 CCC + plasmid with lac promoter | 100 µL | 100 µL, 250 µL |

Week 26 (22nd-28th of June)

**Monday**
 *Present: AMK, KDL and RHA*
Results from transformation was checked The following scheme shows the results:

|  | LB plate | LB plate w. ampicillin |
| --- | --- | --- |
| CCC + water | Growth | No growth |
| CCC + plasmid without lac promoter | Growth | No growth |
 CCC + plasmid with lac promoter | Growth | Growth |

As the transformation worked for the plasmid with lac promoter, ON cultures were made from this transformation.

  **Tuesday**
 *Present: AMK, AR, KDL and RHA*
Plasmid isolation was done from ON culture. Protocol from GenElute plasmid Miniprep kit was followed.

  **Wednesday**
 *Present: AMK, KDL and RHA*
Plasmid digestion:
Protocol for DNA digestion using FastDigest enzymes was followed.
From this, DNA purification was done by following the Illustra GFX PCR DNA and Gel Band Purification Kit.
ON culture was made from transformed colony.

  **Thursday**
 *Present: AMK, KDL, RHA*
Plasmid isolation:
• GenElute Miniprep was followed
• 2x2 mL Top10 with plasmid containing GFP gene and cml resistance.
PCR was done with no result. New primer dilutions were made.

 **Friday**
 *Present: AMK, KDL and RHA*
Plasmid cutting was done again, as PCR from yesterday did not show the expected results.
Cutting was done on pSB1A2, carrying Lac promoter. This was cut with PstI and SpeI.
pSB1C3 carrying GFP was cut using XbaI and PstI.
No FastAP was added, though it should have been, to prevent re-ligation.
Colony PCR was performed with newly diluted primers (10µM).
Following this, the pSB1A2 plasmid was purified using the Illustra GFX PCR DNA and Gel Band purification kit, and pSB1C3 was run on an agarose gel to separate cut plasmids from uncut.
The band of interest was cut out from the gel and purified in miniprep kit for TAE and TBE agarose gels.
![](https://static.igem.org/mediawiki/2020/9/98/T--SDU-Denmark--GelPicture26-7.jpeg)
![Gel that eperated caut and uncut plasmid](https://static.igem.org/mediawiki/2020/f/f1/T--SDU-Denmark--Gel260620_3.png)

Week 27 (29th of June - 5th of July)

**Monday**
 *Present: AMK, RHA and KDL*
Culture with pSB1A2 (part 27) was streaked onto plate and incubated ON at 37°C.

  **Tuesday**
 *Present: None*
Lab people at virtual exam. No progress.

  **Wednesday**
 *Present: NJ, KDL, AMK and RHA*
Plasmid purification of pSB1A2. Protocol for plasmid purification is followed.
• Plasmid cutting by 2 different enzymes was carried out, total volume: 70µL
• This was run on agarose gel, next to 5 µL #402 DNA ladder.
Ligation of 20 µL total volume was carried out, ligating pSB1A2 and GFP in the ratio 10:20, 10:50 and 10:0, with numbers indicating amount of substance in fmol.

  **Thursday**
 *Present: AMK, NJ, FF, KDL, AR*
Top10 put to grow to mid-exponential. Growing to mid-exponential failed, as cells were dead. New ON put over.
TSB is made for transformation.
40 LB+agar plates madeGrowing to mid-exponential failed, as cells were dead. New ON put over.
• Grown ON culture used for TSB transformation, following protocol for transformation.
Transformations done:

| Transformations | Name on petridish |
| --- | --- |
| Top10 + water | C1 |
| Top10 + uncut pSB1A2 | C2 |
| Top10 + cut pSB1A2 | C3 |
| Top10 + ligation 10:20 | T1 |
| Top10 + ligation 10:10 | T2 |
| Top10 + ligation 10:0 | T3 |

**Friday**
 *Present: AMK, FF, FT, JL and RHA*
Results from the transformation from yesterday: Controls were as expected. Uncut gave 6208 colonies, while cut plasmid gave 45 colonies. Ligations, 10:0, 10:20, and 10:50 gave 9, 0 and 12 colonies, respectively.
Colony PCR was performed on transformations from yesterday, following protocol. All 12 colonies in 10:50 were used.
The PCR showed GFP at 1000 bp0, indicating positive results in the transformation in all, except for 2 samples. One of which was not loaded correctly into gel. The positive colonies were cleanly streaked onto plates.
CaCl2 competent cells were made, with remaining cells used for glycerol stocks.
![Gel showing colony PCR](https://static.igem.org/mediawiki/2020/b/ba/T--SDU-Denmark--Gel030720.png)

  **Saturday**
 *Present: FF, NJ, AR and RHA*
ON culture of cleanly streaked plates from yesterday was made. One culture did not express GFP after all, as indicated by its lack of green color. This was not made ON culture of.
Top10 with GFP induced was streaked onto Amp-plates and incubated ON.
Further colony PCR was performed and run on gel after this. However, no bands were visible.
![Agarosegel without visible bands from samples](https://static.igem.org/mediawiki/2020/3/33/T--SDU-Denmark--Gel040720.png)

 **Sunday**
 *Present: AR, FF, KDL, RHA and AMK*
While waiting to get our own sequences we performed transformation of plasmid [BBa\_J45014](http://parts.igem.org/Part%3ABBa_J45014) from 2019 into CCC. This part have a his-tag which make it posiible to reherse protein purification.
We did another plasmid purification from cells expressing and not expressing GFP, following protocol from miniprep kit.
• Cut by MfeI, PstI and EcoRI. One sample cutting by EcoRI and PstI, and one with only MfeI, as MfeI recognizes Lac-promoter in the plasmid.
• Protocol for FD restriction enzymes is followed.
• This was loaded into a gel, showing GFP expression with 3 extra unidentified, showing no GFP expression.
Colony PCR was repeated from Saturday. This showed GFP expression in the cells.
![Gel of DNA digestion with enzymes](https://static.igem.org/mediawiki/2020/5/59/T--SDU-Denmark--Gel050720.png) ![Colony PCR](https://static.igem.org/mediawiki/2020/6/60/T--SDU-Denmark--Gel050720_2.png)

Week 28 (6th-12th of July)

**Monday**
 *Present: AMK, KDL and RHA*
Transformation of [BBa\_J45014](http://parts.igem.org/Part%3ABBa_J45014) (his-tag protein) from 2017.
• Competent cells taken from freezestock, with CaCl2 protocol being followed afterwards.
• DNA from iGEM plates was resuspended in water and taken out of well.
• Plating of the transformation was done as such:

|  |  |  |
| --- | --- | --- |
| **Plate 1**   LB Top10 + 1 µL water   100 µL sample | **Plate 4**   LB + CML  Top10 + [BBa\_J45014](http://parts.igem.org/Part%3ABBa_J45014)   250 µL sample | **Plate 7**   LB + Amp  Top10 + pSB1A2   100 µL sample |
| **Plate 2**   LB + Amp Top10 + 1 µL water   100 µL sample | **Plate 5**   LB + CML  Top10 + L19 2014   100 µL sample | **Plate 8**   LB + Amp  Top10 + pSB1A2   250 µL sample |
| **Plate 3**   LB + CML Top10 + [BBa\_J45014](http://parts.igem.org/Part%3ABBa_J45014)   100 µL sample | **Plate 6**   LB + CML  Top10 + L19 2014   250 µL sample | **Plate 9**   LB + CML  Top10 + 1 µL water   100 µL sample |

**Tuesday**
 *Present: FF, FT and NJ*
LB medium and agar plates made.
• 800 mL LB medium
• 3x400 mL agar with cml.
CaCl2 competent cells made following protocol.

 **Wednesday**
 *Present: AR, FT, JL and RHA*
Transformation of competent cells from yesterday, using [BBa\_J45014](http://parts.igem.org/Part%3ABBa_J45014) from 2017

|  | LB | LB+amp |
| --- | --- | --- |
| Top10 + water | 50 µL | 50 µL |
| Top10 + [BBa\_J45014](http://parts.igem.org/Part%3ABBa_J45014) | 50 µL | 50 µL, 150 µL, 350 µL |

**Thursday**
 *Present: none*
No progress made

 **Friday**
 *Present: AMK, FT, JL and KDL*
As earlier attempts have failed, we repeat transformation of His-tag plasmid into CCC.

 **Saturday**
 *Present: AMK, FF, KDL and RHA*
No introduction lab exercises was done - See assembling of CAS
![](https://static.igem.org/mediawiki/2020/3/39/T--SDU-Denmark--Gel110720_2.png)

 **Sunday**
 *Present: AR, FF, KDL, JL and NJ*
As earlier attempts have failed, we repeat transformation of His-tag plasmid into CCC. Lac containing plasmid was also tranformated as a control.

|  | LB | LB + Amp | LB + Cml |
| --- | --- | --- | --- |
| Water | 50 µL | 50 µL | 50 µL |
| His-tag plasmid | 50 µL |  | 50 µL, 150 µL |
| Lac plasmid | 50 µL | 50 µL, 150 µL |  |

Week 29 (13th-19th of July)

**Monday**
 *Present: AR, KDL, FT and RHA*
Plasmid purification done by following GenElute protocol.
• pSB1A2 purified from Top10, which had been growing ON.
• When gel had been run on this, no plasmid was seen in the gel, indicating that no His-tag was present.
![Gel only showing ladder](https://static.igem.org/mediawiki/2020/c/c1/T--SDU-Denmark--Gel130720.png)

 **Tuesday**
 *Present: AR, FT, KDL and NJ*
Test of, whether His-tag is a plasmid or a linear piece of DNA is tested on gel.
• Purify His-tag and insertion into plasmid, followed by test on gel.
• This showed, that the PCR product annealed at 63°C was optimal.
• There were performed PCR on the same His-Tag plasmid from different iGEM distribution kits (2016, 2017 and 2019). And only the one from 2016 gave a band. A vague band was also seen from the control.
![aarose gel](https://static.igem.org/mediawiki/2020/5/56/T--SDU-Denmark--Gel140720_2.png)

 **Wednesday**
 *Present: AR, JL and NJ*
ON put over of an E. coli strain (ER2566) from Dr. Sergi Torres Puig.

 **Thursday**
 *Present: AR, JL and NJ*
Protein induction, as guided by Dr. Sergi Torres Puig:
• 10 mL LB medium, 10 Kanamycin (50γ) 100 µL ER2566 culture with plasmid, containing a protein with 6x His-tag.
• These were grown to an OD600 of 0.547.
• 10 µL IPTG was added, with a control having no IPTG induction.
• Every hour, 1 mL is taken out to and spun down, and frozen for protein expression.
Pellets are resuspended in 100 µL PBS and is boiled for 10 minutes at 98°C.

 **Friday**
 *Present: AR and NJ*
ON culture of ER2566 are set over for incubation at 37°C for protein induction tomorrow.
• 4x 250 mL flasks with 50 mL LB, 0.5 mL ER2566 culture, 50 µL 50γ Km.

 **Saturday**
 *Present: AR and NJ*
4x 50 mL set for inoculation in 2x 1L flasks of 198 mL LB + Km50 and 2 mL culture.
• OD600 at 2.5 hours is 0.578, whereafter cultures are spun. Supernatant is removed and pellets are cooled until Sunday 19/07.

 **Sunday**
 *Present: AR and NJ*
Samples from yesterday are resuspended in 2x 200 mL LB + 2x 200 µL Km50 + 500 µL IPTG in 1L Erlenmeyer flasks.
• Two hours growth at 37°C.
• Spun down, supernatant removed and samples places in freezer. 2 flasks induced with IPTG, 2 without.
LB medium produced.

ASSEMBLING OF CAS12 AND CAS13 DNA SEQUENCES

Week 28 (6th-12th of July)

**Friday**
 *Present: AMK, AR, FT and NJ*
Cas13a part 2 has arrived from IDT.
PCR amplification of Cas13a part 2, and a serial dilution was performed to get working stocks of different concentrations.
• Dilution from 100 ng/µL to 20 ng/µL, 2 µL Cas13a-2, 8 µL water.
• Dilution from 20 ng/µL to 10 ng/µL, 4 µL Cas13a-2, 4 µL water.
• Dilution from 10 ng/µL to 5 ng/µL, 4 µL Cas13a-2, 4 µL water.
• Dilution from 5 ng/µL to 1 ng/µL, 1 µL Cas13a-2, 4 µL water.
• PCR mix was made, inserting the Cas13a-2 gene fragment.
• Settings for PCR was made in Excel sheet with specific calculations for primers and template.
• Gel purification showed no results. Neither in controls, nor in actual attempts. most likely no EtBr was added to the agarose gel

 **Saturday**
 *Present: AMK, AR, NJ and FT*
PCR for purification, followed protocol by Ampliqon.
• 1, 5, 10 and 20 ng template were added to the PCR tubes along with tubes, where 2 µL 25 mM MgCl2 was added. This was done alongside a negative control with water and a positive control with pSB1A2.
Run on a gel for 35 minutes did not show anything other than the positive control. Possibly due to primers for the Cas13a-gene. New primers were designed and ordered.

 **Sunday**
 *Present: AR, FF, KDL, JL and NJ*
See introduction chapter. No progress made in assembling Cas proteins

Week 29 (13th-19th of July)

No progress made in assembling Cas proteins, as we still wait for arrivals from IDT.
See introduction for what we did this week.

Week 30 (20th-26th of July)

**Monday**
 *Present: AMK, AR, FF, FT, JL, KDL, NJ and RHA*
Drylab. We used the day to research in biomarkers.

 **Tuesday**
 *Present:*
Parts for Cas13a for IDT arrived and PCR is performed on these.
3 PCR tubes of 50 µL, 25 µL Taq Polymerase Master mix was added.
Negative result after run on gel.

 **Wednesday**
 *Present: none*
We held an internal social meet-up.

 **Thursday**
 *Present: none*
We held an internal social meet-up.

 **Friday**
 *Present: none*
We were not in the lab as we hosted the Danish meet-up.

 **Saturday**
 *Present: none*
We were not in the lab as we hosted the Danish meet-up.

 **Sunday**
 *Present: none*
We were not in the lab as we hosted the Danish meet-up.

Week 31 (27th of July - 2nd of August)

**Monday**
 *Present: AR, NJ*
PCR amplification of Cas13a parts:
• Primers are diluted to a concentration of 10 µM.
• Cas13a parts, 1, 2, and 3 are diluted to 10 ng/µL.
• Taq-polymerase mastermix is used at 25 µL.
• Primers for parts:
• Cas13a-1: Prefix-F + Cas13a-1R
• Cas13a-2: Cas13a-2F + Cas13a-2R
• Cas13a-3: Cas13a-3F + Suffix-R
These were loaded into PCR tubes at a volume of 50 µL and PCR were performed .

 **Tuesday**
 *Present: AR, NJ*
A gel was run on PCR products from yesterday, where these were purified from the gel, using the miniprep kit, for gel purification.
• Protocol was followed, though only for parts 1 and 3.
•Part 2 only showed a band around 500 bp, 1500 bp short of the expected result.
PCR run for part 2, only showed primer-dimer.
Yet another PCR run with temperature gradient and extension period of 2 minutes, only showed a band around 400-500 bp.
![bands from all Cas13 DNA sequences received](https://static.igem.org/mediawiki/2020/5/52/T--SDU-Denmark--Gel280720_2.png)

 **Wednesday**
 *Present: NJ*
Amplification of Cas13a-2:
As IDT wrote, that Part 2 might not contain the right sequence, this was tested using the part 2-primers as well as Prefix-F and Suffix-R.
Samples:
1. Prefix-F + Suffix-R2: Cas13a-2F + Cas13a-2R3: Prefix-F + Cas13a-2R4: Control (no DNA).
• Extension at 72oC, 2:30 minutes.
• Still only bands at 500 bp, as shown on gel.

Another PCR run was made, using temperature gradient in annealing time, was made.
• Mainly bands at 500 bp, as seen on gel, though bands at 2000 bp was visible, yet faint.
• Jens, one of our supervisors, talked of using another polymerase than TAQ from Ampliqon, this being Phusion polymerase.
![Bands with a lenght of approximately 300bp](https://static.igem.org/mediawiki/2020/7/76/T--SDU-Denmark--290720_2.png) ![Bands with a lenght of approximately 300bp](https://static.igem.org/mediawiki/2020/e/e0/T--SDU-Denmark--290720_3.png)

 **Thursday**
 *Present: AR, NJ*
SOP from 2017, on using Phusion DNA polymerase is used and followed.
• Temperature gradient and different buffers, GC, and HF are used.
• Protocol demanded sacrifice to the PCR gods, followed by doing the PCR dance. This was not carried out, as the resident laboratory assistant deemed this a health risk.
• (Editor’s note: This may explain the following 3 weeks of unsuccessful PCR runs.)
• HF buffer showed positive results, with temperature gradient having a minor effect on product amount.
![Bands with length of 2000bp](https://static.igem.org/mediawiki/2020/d/d9/T--SDU-Denmark--300720_1.png)

 **Friday**
 *Present: Ar, NJ, JL*
Ligation of all Cas13a parts:

1. Part 1, Part 2, Part 3, HF buffer, Prefix-F, Suffix-R
2. Same as 1.
3. Part 1, Part 2, HF buffer, Prefix-F, Cas13a-2R.
4. Part 2, Part 3, HF buffer, Cas13a-2F, Suffix-R.
5. Part 1, Part 2, Part 3, GC buffer, Prefix-F, Suffix-R
6. Same as 5.
7. Part 1, Part 2, GC buffer, Prefix-F, Cas13a-2R.
8. Part 2, Part 3, GC buffer, Cas13a-2F, Suffix-R.
9. Control

• Annealing temperature, 55°C for 30 sec.
• Extension period, 2:30 minutes, 72°C.
Another PCR run was made with the samples, though with TAQ polymerase.
• Extension period, 4:30 minutes, 72°C.
Neither TAQ nor Phusion showed any significant results, as multiple bands were visible in each well. This prompted a further look into part concentrations 01/08, so 1:1 relations can be reached.
![Bands with length of 750bp](https://static.igem.org/mediawiki/2020/c/cd/T--SDU-Denmark--310720.png) ![Bands with length of 750bp](https://static.igem.org/mediawiki/2020/8/81/T--SDU-Denmark--310720_2.png)

 **Saturday**
 *Present: JL, RA*
PCR ran yesterday, with gel run today showed bands for 1+2 and 2+3 in both GC and HF buffer.
• These were purified from gel and put in anther PCR run to add on the final part, with 1+2 missing 3 and 2+3 missing 1.
• 2 bands on the gel named “6” and “8” showed a faint, yet present band of 4000 bp, the length of the Cas13a gene. These both stemmed from reactions with GC buffer.
![Gel with bands at many lengths](https://static.igem.org/mediawiki/2020/f/fe/T--SDU-Denmark--010820.png) ![Bands with length of 750bp](https://static.igem.org/mediawiki/2020/f/f6/T--SDU-Denmark--010820_2.png)

 **Sunday**
 *Present: none*

Week 32 (3rd-9th of August)

**Monday**
 *Present: JL, NJ, RA*
“6” and “8” was purified from gel using the miniprep kit for gels.
• Protocol was followed.
A new attempt at PCR was done, now testing for different primer concentrations:
“6” and “8” were also attempted to be amplified, in case these were actually the assembled Cas13a gene.

| Tube | Parts | Buffer | Primer relations, Forward:Reverse |
| --- | --- | --- | --- |
| 1 | (1+2)+(2+3) | HF | 1:1 |
| 2 | (1+2)+3 | HF | 1:3 |
| 3 | (2+3)+1 | HF | 1:3 |
| 4 | (1+2)+(2+3) | GC | 1:10 |
| 5 | (1+2)+3 | GC | 1:16 |
| 6 | (1+2)+3 | GC | 1:3 |
| 7 | (1+2+3) “6” | GC | 1:3 |
| 1 | (1+2+3) “8” | GC | 1:3 |

Annealing temperature: 54°C, Extension: 3:30 minutes, 30 cycles.

 **Tuesday**
 *Present: NJ*
Gel of PCR from yesterday did not show any positive results, even “6” and “8” showed barely any discernable bands.
• A PCR, attempting to ligate (1+2) and (2+3) is made for both GC and HF buffer with an annealing temperature of 54°C-59°C.
• Likewise, 2 more PCR’s are made, using Phusion, though without adding additional MgCl2, with annealing temperature of 53-63°C.
• Another PCR attempts to use the High-Fidelity polymerase from Ampliqon again, with an annealing temperature of 53-63°C.
![Agarose gel](https://static.igem.org/mediawiki/2020/b/b0/T--SDU-Denmark--040820.png)

 **Wednesday**
 *Present: JL, NJ, RHA & AMK, FF, KD*
Gel run on PCR runs from yesterday.
• With #402 ladder used, only the final PCR made on 04/08 showed any significant bands.
• Though the gel showed multiple bands, two samples made of (2+3)+1 showed bands around 4000 bp.
• This was cut out from the gel and purified using the miniprep kit for gels.
A PCR run was made to replicate the two possibly successful PCR runs.
• PCR with Phusion polymerase was followed, without the addition of MgCl2, and with GC buffer.
• Annealing temperatures were: 53, 53.7, 56.5 and 59°C.
Gelmix for sgRNA purification was prepared.

1. TBE buffer 10x50 mL
2. 40% acrylamide100 mL
3. 2% bis-acrylamide40 mL
4. 7M urea210 g
5. Water to a total volume of 500 mL

2M ammonium acetate was also made.
![Agarose gel showing bands that possibly is Cas13](https://static.igem.org/mediawiki/2020/a/a6/T--SDU-Denmark--050820.png) ![Agarose gel without discernable bands](https://static.igem.org/mediawiki/2020/2/23/T--SDU-Denmark--050820_2.png)

 **Thursday**
 *Present: AMK, FT & AR, NJ*
Purification of Ascpf1 plasmid was done using the miniprep kit.
Gel was also run on the PCR products from yesterday, showing high contamination in the products.
• As uncertainty in the purification of Cas13a-2 arose, a PCR run on Cas13a-2 was done, using Phusion, additional MgCl2 and HF buffer, with annealing temp. at 54°C and 59°C, as well as ext. time of 1:30 minutes.
• Three bands were still visible here, though the bands were not spread out anymore.
• A band around 2000 bp was cut out and purified from the miniprep kit.
![Agarose gel showing bands at 3000bp](https://static.igem.org/mediawiki/2020/f/f6/T--SDU-Denmark--060820_1.png)
![Agarose gel showing bands at 3000bp](https://static.igem.org/mediawiki/2020/b/b7/T--SDU-Denmark--060820_2.png)
![Agarose gel showing bands at 1500bp and 2000bp](https://static.igem.org/mediawiki/2020/f/f6/T--SDU-Denmark--060820_3.png)

 **Friday**
 *Present: AMK, NJ*
PCR was done on parts 1, new 2, and 3.
• Phusion, MgCl2, Ann. Temp: 54°C-59°C, 2 minutes. Ext time: 2 min.
• part 3 was also amplified, as new part 3 was missing. Phusion, MgCl2, Ann. Temp: 54°C, Ext time: 30 sec.

| HF | GC |
| --- | --- |
| 1+2 Ann. Temp: 54°C, 56°C, 59°C | 1+2 Ann. Temp: 54°C, 56°C, 59°C |
| 3 Ann. Temp: 54°C | 3 Ann. Temp: 54°C |

Plasmid for Cas12 transformation is purified.

 **Saturday**
 *Present: none*

 **Sunday**
 *Present: none*

Week 33 (10th-16th of August)

**Monday**
 *Present: NJ, FT*
Cas13a-1 amplified by PCR
New part3 is purified from gel by miniprep kit.
New part2 and new part 3 were put together in PCR.

|  |  |
| --- | --- |
| 2+3, GC, 54°C, Ext time: 1:30 min. | 2+3, GC, 56°C, Ext time: 1:30 min. |
| 2+3, HF, 54°C, Ext time: 1:30 min. | 2+3, HF, 56°C, Ext time: 1:30 min. |
| 1, GC, 54°C, Ext time: 1:00 min. | 1, GC, 56°C, Ext time: 1:00 min |
| 1, HF, 54°C, Ext time: 1:00 min. | 1, HF, 56°C, Ext time: 1:00 min. |

Transformation of Cas12 into plasmid purified 07/08.
• Done with CaCl2, where CCC made 09/08.
Plates done:

|  |  |  |
| --- | --- | --- |
| LB   50 µL   Cas12 | LB+amp   50 µL   Cas12 | LB+amp   100 µL   Cas12 |
| LB+amp   250 µL   cas12 | LB+amp   100 µL   lac | LB+amp   250 µL   lac |

**Tuesday**
 *Present: AR, NJ*
Gel run for PCR from yesterday.
• Part 1 was purified from the gel by the Miniprep kit.
• Part 2+3 was too weak to use, though a band at 2700 bp was visible.
New working stock of all 3 parts were made, after consulting Simon Rose on the working parameters of Phusion. A color code for the parts was also made.

Part 1: 0.5 ng/µL

Part 2: 0.3 ng/µL

Part 3: 1 ng/µL

• This was done, as to only add 1 µL of each template into a 20 µL reaction.
• 6 different reactions were made, with following:

|  |  |  |
| --- | --- | --- |
| 1+2, HF | 2+3, HF | 1+2+3, HF |
| 1+2, GC | 2+3, GC | 1+2+3, GC |

• Denaturation temperature: 98°C, 15 sec.
• Ext. 2:10
• Ann. Temp. 55, 57, 59, 61, 63°C
A gel was run on this, and the same pattern of contamination seen in 1+2 was also seen in 1+2+3, indicating contamination stemming from the cleanliness of part 1. Even so, a small band at 2700 bp was visible in 2+3, indicating a successful ligation through the overhangs put onto part 2 by Cas13a-2R.
![Agarose gel attempt to assemble Cas](https://static.igem.org/mediawiki/2020/d/df/T--SDU-Denmark--110820_1.png)
![Gel with PCR at 55 and 57 degrees celcius to assemble cas13](https://static.igem.org/mediawiki/2020/2/2d/T--SDU-Denmark--110820_2.png)
![Gel with PCR at 59 and 61 degrees celcius to assemble cas13](https://static.igem.org/mediawiki/2020/d/d9/T--SDU-Denmark--110820_3.png)
![Gel with PCR at 63 degrees celcius to assemble cas13](https://static.igem.org/mediawiki/2020/3/3a/T--SDU-Denmark--110820_4.png)

 **Wednesday**
 *Present: NJ*
As primers may be contaminated, new working stocks of 10 µM were made.
Concentrations of templates were measured. This was run on a gel, again showing the same pattern of unspecificity in part 1, as seen in all gels from yesterday. The band at 1400 bp, corresponding to part 1 was purified and used for the following, once a working stock had been made.
A PCR for 1+2, with new primer working stocks was done.

|  |  |  |
| --- | --- | --- |
| 1+2, HF | -Template, HF | -Primer, HF |
| 1+2, GC | 2-Template, GC | -Primer, GC |

Denat temp. 98°C Ann. Temp.: 59oCExt time: 1:3030 cycles
Gel of this only showed the same product as Part 1 in gel made for the individual parts. The gel without template showed more PCR product than 1+2, the reason of which, is unknown.
• This same PCR is done tomorrow with the new part 1.
![Agarose gel](https://static.igem.org/mediawiki/2020/a/a0/T--SDU-Denmark--120820_1.png)
![Faild gel from cas 13](https://static.igem.org/mediawiki/2020/6/6a/T--SDU-Denmark--120820_2.png)

 **Thursday**
 *Present: NJ*
Working stock is made from new Part 1 to 0.5 ng/µL.
Simon Rose discovered more bands in the gel of the individual parts and recommended a new PCR on these was necessary.
• 2x5 10 µL reactions:Denat: 98°C, 35x cycles,Ext. 1:10 min., Ann. Temp: 55, 57, 59, 61, 63°C.
• Gel shows no products that are viable. Contamination is suspected. New PCR with new buffers, dNTPs, dilutions of template and primers is carried out with the same conditions for the PCR run as the previous.
![Agarose gel from gradient PCR](https://static.igem.org/mediawiki/2020/7/71/T--SDU-Denmark--130820_1.png)

 **Friday**
 *Present: FT, NJ*
PCR done for parts 1, 2, and 3.
• 3x5 10 µL reactions showed positive results. These were purified using the miniprep kit.
PCR done to amplify part of the Cas13a-1 part for insertion onto Cas12a given by PhyLife.
• Cas13a-1F and Ascpf-1-pre-reverse was used to amplify this.
• Phusion protocol was carried out and aliquoted into 5 tubes with Ann. Temp. of 55-63°C.
![Agarose gel bands at 600bp](https://static.igem.org/mediawiki/2020/4/4f/T--SDU-Denmark--140820_1.png)
![Agarose gel bands at 200bp](https://static.igem.org/mediawiki/2020/f/f1/T--SDU-Denmark--gel140820_2.png)

 **Saturday**
 *Present: none*

 **Sunday**
 *Present: none*

Week 34 (17st-23rd of August)

**Monday**
 *Present: FT, NJ*
During the weekend, part 3 was purified and PCR was run for amplifying parts 1, and 2.
• 2x5 50 µL, Denat: 98°C, 15 sec., Ann. Temp: 55-63°C, Ext temp.: 1:10 min., 35x cycles.
Gel run was run, but pictures were unclear. As these were clearer under UV light, products were still cut out of the gel and purified.
Part 1+2 were put together through PCR: 5x HF, 5x GC.
• Denat: 98°C, 15 sec., Ann. Temp: 55-63°C, Ext temp.: 1:10 min., 35x cycles.
The promoter region for Cas12a was loaded onto gel, purified, and run on another gel, as the first gel had been 2 days old, at the time of cutting.
• A clear band at 75bp was visible and showed the presence of the promoter region, ready to be cut out.
Cas12a was also purified, using the Miniprep kit.

 **Tuesday**
 *Present: FT, NJ*
Cas13a: Products from yesterday’s PCR were run on gel to see if these could be separated further.
• This showed part 1 and part2 being separated even further.
Cas12a: Gel was run on the Cas12a-part 1 purification done yesterday. This was purified and put into freezer.
![Agarose gel bands at 200bp](https://static.igem.org/mediawiki/2020/f/f1/T--SDU-Denmark--gel140820_2.png)
![Agarose gel bands at 200bp](https://static.igem.org/mediawiki/2020/4/4d/T--SDU-Denmark--gel180820_1.png)
![Agarose gel bands at 1500bp and 2000bp](https://static.igem.org/mediawiki/2020/8/88/T--SDU-Denmark--gel180820_2.png)

 **Wednesday**
 *Present: NJ*
All parts were made into new working stocks. And part2+3 were put together using a new Phusion master mix with HF buffer. Run as followed:
• Denat: 98°C, 15 sec., Ann. Temp: 57-61°C, Ext temp.: 1:30 min., 35x cycles
![Agarose gel with faint to no bands](https://static.igem.org/mediawiki/2020/e/ea/T--SDU-Denmark--gel190820_1.png)
![Failed gel with giant smeares](https://static.igem.org/mediawiki/2020/5/5e/T--SDU-Denmark--gel190820_2.png)

 **Thursday**
 *Present: NJ*
PCR from yesterday is run on a gel, which turned out to be completely useless. This may be due to unclean combs. The same PCR is run again.
• The PCR was not done in vain. Lines for part 2 and part 3 are seen on the gel.
• These were purified on the miniprep kit.
PCR was then done for putting together part 2+3.
• This was done, using the same conditions as PCR from 19/08.
• Only part 3 was shown amplified.
PCR with smaller concentrations of part 3 is made.
0,3 ng/µL, noted as ”I”0,06 ng/µL, noted as ”V”0,03 ng/µL, noted as ”X”.
Using Cas13a-3R primer and Suffix-R, as it was shown to also bind in the prefix-region of the gene fraction.
• Denat: 98°C, Ann temp: 58°C, Ext time: 1:00 minutes, 35x cycles

|  |  |  |  |
| --- | --- | --- | --- |
| Suf-R, I | Suf-R, V | Suf-R, X | Suf-R, No template |
| Cas-3R, I | Cas-3R, V | Cas-3R, X | Cas-3R, No template |

![Agarose gel](https://static.igem.org/mediawiki/2020/c/c8/T--SDU-Denmark--gel200820_1.png)
![bands at 600bp](https://static.igem.org/mediawiki/2020/e/e0/T--SDU-Denmark--gel200820_2.png)

 **Friday**
 *Present: NJ*
Gel run on PCR from yesterday, showed a clear part 3 being amplified, when adding Cas13a-3R.
A new PCR is run, using Cas13a-1F instead of Prefix-F as forward primer.
• Denat: 98°C, Ann temp: 58°C, Ext time: 1:00 minutes, 35x cycles.

|  |  |  |
| --- | --- | --- |
| Pre-F, No template | Pre-F (Noted on gel as SufR by mistake) | Cas-1F |

Gel run showed a far greater part 1 product, by using Cas13a-1F.
• This was cut out and purified from gel.
![Gel with bands representing cas13a](https://static.igem.org/mediawiki/2020/7/77/T--SDU-Denmark--gel210820_1.png)

 **Saturday**
 *Present: none*

 **Sunday**
 *Present: none*

Week 35 (24th-30th) of August

**Monday**
 *Present: FT, KD, NJ*
Cas12 group:
PCR was done for Cas12 in two reactions of 25 µL.
• Gel was run, which did not show any amplification at 4000 bp.
• This be an issue with primers or the Phusion Mastermix, which will be further tested tomorrow.

Cas13a group:
PCR done to put together the new part 1 and d part (2+3):
3x10 µL reactions, Denat: 98oC, 15 sec., Ann temp: 57, 59, 61oC, Ext time 2:30 min., 35x cycles.
![Gel with bands from 1500bp and downwards](https://static.igem.org/mediawiki/2020/b/bf/T--SDU-Denmark--gel240820_1.png)
![Unsuccesful gel](https://static.igem.org/mediawiki/2020/8/85/T--SDU-Denmark--gel240820_2.png)

 **Tuesday**
 *Present: NJ, FF, KD, FT*
Cas13a group:
Gel run on PCR from yesterday.
• Three clear lines at 4000 bp show successful ligation of the Cas13a gene.
The same reaction is done with the same conditions to produce as much of gene as possible.
• 2x50 µL reactions, Ann temp.: 58°C.
Gel run for 45 minutes.
• Cas13a is clearly shown again.
• The left line is crooked, though this may be due to someone pushing the gel, during the run.
Cas13a was cut out of gel.

Cas12 group:
PCR done with annealing temperature gradient of 55-65°C.
• Reaction are taken from the two purified plasmids, Ascpf1.1, and Ascpf 1.2, creating a total of 10 reactions.
Gel run showed nothing successful, so primers are checked again.
• Template was checked to see if it was even present. It was.
![Gel with bands at 4000bp indicating cas13](https://static.igem.org/mediawiki/2020/8/84/T--SDU-Denmark--gel250820_1.png)
![Gel with bands at 6000bp](https://static.igem.org/mediawiki/2020/9/93/T--SDU-Denmark--gel250820_2.png)

 **Wednesday**
 *Present: FF, KD, FT, NJ, RA*
Cas12a group:
PCR is done, with annealing temperature gradient at 65-72°C and template as Ascpf 1.1.
• 4 reactions are run.
Gel run showed no product. Picture of gel was not taken.

Cas13a group:
Cas13a was purified from gel. Preparations for plasmid cutting and ligation for tomorrow was done.

sgRNA group:
Protocol for in vitro transcription of sgRNA was initiated.

 **Thursday**
 *Present: NJ*
DNA digestion by FastDigest enzymes:
• Cas13a was cut by enzymes XbaI, and SpeI (BcuI).
• Backbone pSB1A2 was cut by SpeI (BcuI), one sample with alkaline phosphatase (AP) and one without.
• Protocol was followed, though digestion took 1 hour to maximize cutting.
Cas13a was purified using the Miniprep kit for enzymatic reactions.
Backbone was run on gel, cut out and purified.
Ligation of Cas13a and backbone was done after protocol:
Relations noted as “Backbone:Cas13a”.

| No AP | With AP |
| --- | --- |
| 1:0 | 1:0 |
| 1:1 | 1:1 |
| 2:1 | 2:1 |

Put over at 16°C overnight.

 **Friday**
 *Present: NJ, FT*
Cas13a group:
ON culture Top10 created for transformation tomorrow.

sgRNA group:
Protocol followed for in vitro transcription.
• When using gel, a 13% acrylamide gel was used instead of 8%.
• Bands on gel marked with marker and cut out of gel.
• Precipitation was done, following protocol, though with an extra 10-minute spin after each wash.
Concentration of RNAs:

| sgRNA | Target |  |
| --- | --- | --- |
| 23.6 ng/µL | 21.1 ng/µL | T |
| 21.25 ng/µL | 34.25 ng/µL | ERG |
| 43.1 ng/µL | 14.5 ng/µL | G |

**Saturday**
 *Present: RA*
Cas13a group:
TSB Transformation into Top10 with ligations performed 27/08 was done, following protocol.
• Concentrations on template was measured:

| Ligation | Concentration |
| --- | --- |
| 1:0 AP | 1378.6 ng/µL |
| 1:0 No AP | 1628.01 ng/µL |
| 1:1 AP | 1500.9 ng/µL |
| 1:1 No AP | 1303.09 ng/µL |
| 2:1 AP | 1107.44 ng/µL |
| 2:1 No AP | 1411.06 ng/µL |

• 4 µL template was added to each transformation.
• Control with water was also made.

 **Sunday**
 *Present: NJ, RA*
Cas13a group:
Transformation yesterday was unsuccessful.

Week 36 (31st of August - 6th of September)

**Monday**
 *Present: FF, AMK, KD*
Cas12a group:
PCR is done, using new primers:
9x 100 µL reactions, Denat.: 98°C, Ann temp: 54.5, 58.9, 62.5°C, Ext time: 2:10 minutes.

|  |  |
| --- | --- |
| 3x version 1 reverse primer | Diluted 10x |
| 3x version 2 reverse primer | Diluted 10x |
| 3x version 3 reverse primer | Not diluted |

• Version 1 and version 2 showed Cas12a at 4000 bp. These were cut from gel and stored in freezer.
![Gel with bands at 4000bp indicating cas12](https://static.igem.org/mediawiki/2020/0/05/T--SDU-Denmark--gel310820_1.png)

 **Tuesday**
 *Present: KDL, AMK, FF*
Cas12 group:
DNA purified from gel in miniprep kit.
• Protocol followed.
Concentrations ranged from 9.7 to 20.1 ng/µL.
PCR is run to put on the promoter region from Cas13a onto Cas12a.
• Cas12a: ≈4200 bpPromoter region: ≈275 bpOptimal relation in PCR is 1:20.
Dilution made according to recommendations from Phusion.
• Promoter: 2 ng/µLCas12 v 1.3: 0.1 ng/µLCas12 v 2.3: 0.1 ng/µL.
• 3x 25 µL reactions were done, with annealing temperature of 62°C.
• Gel was run on the PCR, showing no ligation.
![Agarose gel](https://static.igem.org/mediawiki/2020/f/f7/T--SDU-Denmark--gel010920_1.png)

 **Wednesday**
 *Present: KDL, FF*
Cas12 group:
The same PCR was run and run on gel, as the gel yesterday may have been defective.
• Pieces were cut out and purified from miniprep kit.
![Agarose gel](https://static.igem.org/mediawiki/2020/3/3e/T--SDU-Denmark--gel020920_1.png)

 **Thursday**
 *Present: AMK, FT, KDL*
Cas12 group:
Part from 2016, plate 3, 5D is used. This contains part [BBa\_B1006](http://parts.igem.org/Part%3ABBa_B1006)+pSB1C3.
• Plasmid was cut with XbaI, while Cas12a was cut with XbaI and SpeI.
• Protocol was followed, though FastAP was added to both digestion reactions. Cut for 1.5 hours.
These were purified on miniprep kit for enzymatic reactions.
Ligation was done in relations (Backbone: insert), 10:0, and 10:100.
• Protocol was followed.
 **Friday**
 *Present: AMK, KDL, FT*
Agar plates with cml were made.
Using ON culture from yesterday, 2 cml plates were plated with 50 µL culture, as the backbone carries cml resistance.
Another ON culture was made from a 1:100 dilution of this ON culture.

 **Saturday**
 *Present: FF, KDL*
Cas12 group:
CaCl2 CCC is made.
• During growth to mid-exp. phase, cells were grown to OD450=0.625, which is too high.
• CCC were left to incubate ON on 4°C.
Cas12a + promoter v 2.3p was amplified on PCR and run on gel.
![Agarose gel](https://static.igem.org/mediawiki/2020/3/3e/T--SDU-Denmark--gel020920_1.png)
New CCC were made with CaCl2 protocol.

 **Sunday**
 *Present: none*

Week 37 (7th-13th of September)

**Monday**
 *Present: AMK, NJ, FF, FT*
Cas13a group:
Top10 culture from Simon Rose shown not to be Amp resistant and set into incubation until mid-exponential phase.
• TSB transformation was not done, as backbone attempted to be used, was already cut with SpeI and PstI.
• New digestion was performed on backbone with XbaI.

Cas12 group:
CaCl2 transformation done for ligations 10:0, and 10:100. Controls with water “transformed” into Top10 was also done.
Transformation was done for ligations.

|  |  |  |
| --- | --- | --- |
| 50 µL 10:100, LB | 50 µL 10:100, Amp | 400 µL 10:100, cml |
| 50 µL 10:100, cml | 150 µL 10:100, cml | 250 µL 10:100, cml |
| 250 µL 10:0, cml | 50 µL 10:0, LB | 50 µL 10:0, Amp |
| 250 µL water, cml | 50 µL water, LB | 150 µL water, cml |

**Tuesday**
 *Present: FF, FT, KDL, JL, AR, NJ*
Cas12 group:
Transformations from yesterday were looked at.
• Controls look good, but transformation was unsuccessful, as both control plasmids with Amp resistance is not shown to grow.
New ligation of Cas12a+promoter into backbone is made.
The rest of the ligation from yesterday is run on gel after digestion by restriction enzymes, to see if any ligation has taken place.
• Ligations are cut after FastDigest protocol.
![Agarose gel](https://static.igem.org/mediawiki/2020/b/bb/T--SDU-Denmark--gel080920_1.png)
• No bands are visible, indicating an unsuccessful ligation.

Cas13a group:
Ligation of Cas13a and pSB1A2.
• Since all concentrations of backbone and Cas13a were approx. 5 fmol/µL, the unit in ligation relations equates 10 fmol or 2 µL. Relations in ligation: Backbone:Cas13a.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Ligations | 1:0 | 1:1 | 2:1 | 3:1 | 5:1 | 1:4 |

**Wednesday**
 *Present: FF, AMK*
Cas12 group:
Gel run on PCR from yesterday. Clear bands at 4000 bp indicate Cas12a+promoter.
![Agarose gel with clear bands at 4000bp](https://static.igem.org/mediawiki/2020/d/d9/T--SDU-Denmark--gel090920_1.png)

• cas12 without promoter was not shown here, so another gel with Cas12+promoter and Cas12 v1.2 was run.
![Agarose gel of cas with and without promoter region](https://static.igem.org/mediawiki/2020/d/d1/T--SDU-Denmark--gel090920_2.png)

Picture was unclear, so new PCR of Cas12+promoter and Cas12 is done.
• Cas12+promoter (Cas12P) is done in two reactions with either Prefix-F or Cas13a-1F as forward primer.

 **Thursday**
 *Present: FF, FT, KDL, NJ, RA, AR, JL*
Cas12 group:
![Agarose gel of cas12](https://static.igem.org/mediawiki/2020/9/98/T--SDU-Denmark--gel100920_2.png)

Gel run on PCR from yesterday shows longer bands for Cas12P, with both PreF and Cas13-1F as forward primer.
• Cas12P v2.3 is used as primary part from this point on.
PCR to make extra Cas12P v2.3 is done, so more can be used for transformations.
• 6x 25 µL reactions, Ann temp: 62.5°C.
CaCl2 CCC made in 5x falcon tubes.
Genes inserted: “2017, 5D” (BB), Water, Lac promoter with Lac resistance.
• Transformations done:

|  |  |  |  |
| --- | --- | --- | --- |
| 50 µL, LB, BB | 50 µL, cml, BB | 200 µL, cml, BB | 500 µL, cml, BB |
| 50 µL, LB, Water | 300 µL, Amp, Water | 250 µL, cml, Water |  |
| 300 µL, Amp, Lac | 250 µL, cml, Lac |  |  |

Cas13a group:
TSB transformation for ligation inserted into Top10 culture is performed.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| 50 µL, C, LB | 50 µL, 1:1, No AP, Amp | 50 µL, 2:1, No AP, Amp | 50 µL, 3:1, No AP, Amp | 50 µL, 5:1, No AP, Amp | 50 µL, 1:4, No AP, Amp |
| 50 µL, C, Amp | 100 µL, 1.1, No AP, Amp | 100 µL, 2:1, No AP, Amp | 100 µL, 3:1, No AP, Amp | 100 µL, 5:1, No AP, Amp | 100 µL, 1:4, No AP, Amp |
| 50 µL, 1:0 No AP, Amp |  | 200 µL, 2:1, No AP, Amp | 200 µL, 3:1, No AP, Amp | 200 µL, 5:1, No AP, Amp | 200 µL, 1:4, No AP, Amp |
| 50 µL, 1:0 AP, Amp | 50 µL, 1:1, AP, Amp | 50 µL, 2:1, AP, Amp | 50 µL, 3:1, AP, Amp | 50 µL, 5:1, AP, Amp | 50 µL, 1:4, AP, Amp |
|  | 100 µL, 1:1, AP, Amp | 100 µL, 2:1, AP, Amp | 100 µL, 3:1, AP, Amp | 100 µL, 5:1, AP, Amp | 100 µL, 1:4, AP, Amp |
|  |  | 200 µL, 2:1, AP, Amp | 200 µL, 3:1, AP, Amp | 200 µL, 5:1, AP, Amp | 200 µL, 1:4, AP, Amp |

**Friday**
 *Present: FF, AMK, KDL*
Cas12 group:
PCR from yesterday is run on a 0.8% gel.
![Agarose gel of cas12](https://static.igem.org/mediawiki/2020/4/47/T--SDU-Denmark--gel110920_1.png)

Bands at 4000 bp indicate successful PCR.
• Bands are cut out and amplified in Miniprep kit for gels.
Transformation yesterday unsuccessful. Backbone seems to not be working. New backbones are found in old iGEM-racks.
• Possible candidates: [BBa\_B1005](http://parts.igem.org/Part%3ABBa_B1005) (6A), [BBa\_B1003](http://parts.igem.org/Part%3ABBa_B1003) (4O) and [BBa\_B1006](http://parts.igem.org/Part%3ABBa_B1006) (6C).
Transformation is done on these and plated onto cml plates, as all parts carried cml resistance.

 **Saturday**
 *Present: AMK*
Cas12 group:
ON culture done of transformations from yesterday, while colonies of these were also plated.

Cas 13 group:
PCR was run:
![Agarose gel of cas13](https://static.igem.org/mediawiki/2020/4/4b/T--SDU-Denmark--gel120920_1.png) ![Agarose gel of faint band of cas13](https://static.igem.org/mediawiki/2020/5/56/T--SDU-Denmark--gel120920_2.png)

 **Sunday**
 *Present: AMK, KDL, FF, FT*
Nothing grown in 4O, so, but all (4O, 6A and 6C) are set for plasmid isolation.
• Protocol is followed.
These are set for digestion by XbaI.
• Protocol followed.
• Cut 1.5 hours.
Cas12a is also cut, though with XbaI and SpeI.
Protocol followed.
Digestions run on gel.
![Agarose gel of cas12](https://static.igem.org/mediawiki/2020/2/2a/T--SDU-Denmark--gel130920_1.png) ![Agarose gel backbone, 2000bp](https://static.igem.org/mediawiki/2020/e/e8/T--SDU-Denmark--gel130920_2.png)
Bands of Cas12a and plasmids are cut out and purified, though 6C was dropped by mistake.
Ligations of these were done of Backbone:Cas12a; 1:0, 1:5, 1:7.7

Week 38 (14th-20th of September)

**Monday**
 *Present: KDL, AMK, RA, JL, NJ*
Cas12 group:
Transformations done for ligations from yesterday.
• Done on CCC, protocol followed.
• Transformations:

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Water, LB | Water, cml | Lac, cml | Lac, Amp | 1:0, cml | 1:5, cml | 1:7.7, cml |

• Incubated ON

Cas13a group:
After transformation, colonies were present on 1:4 plates.
• ON culture of these was made 12/09.
• Plasmid purification was done for these colonies and put into gel, both cut and uncut by XbaI.
![colony PCR of cas13 transformants](https://static.igem.org/mediawiki/2020/f/f0/T--SDU-Denmark--gel140920_1.png)

• No Cas13a insertion is shown. This has probably been due to relegation of the plasmid.
Colony-PCR was run on these colonies, and found a weak band at 4000 bp.
Plasmid purification of this colony showed no Cas13a, only backbone.
(INSERT GEL)

 **Tuesday**
 *Present: NJ, JL, AR*
Cas13a group:
Part from 2019, pSB1A2 ROOII is cut by XbaI and SpeI.
• Ligations are made on new backbone and Cas13a.
• Ligations, Plasmid:Cas13a: 1:0, 1:1, 2:1, 5:1, 1:5
Set on 16°C ON.
ON culture made for Top10 with 6C and 6C.

 **Wednesday**
 *Present: FF, AMK, NJ, RA, JL, AR*
Cas12a group:
Plasmids purified from ON culture.
To ensure no relegation, backbone is cut with both XbaI and SpeI for 10 min. and run on gel.
Mixes of each was done: 1:5 1, 1:5 2, 1:7\_1, 1:7\_2
These were run on gel.
![Gel showing failed transformation](https://static.igem.org/mediawiki/2020/3/3e/T--SDU-Denmark--gel160920_1.png)
Only backbone is visible, so more transformants are set ON. 10 from 1:7.7 and 8 from 1:5.

Cas13a group:
Plasmid purification for Top10-6C cultures, which is then cut with XbaI+AP.
• Cas13a is cut by XbaI and SpeI.
Ligations made: 1:0, 1:1, 2:1, 3:1, 5:1, 1:5, Uncut.
More Cas13a is also made, as we were running low. It was done from the same PCR settings as 25/08.
Transformation of ligations from yesterday is made.
![Gel showing Cas12](https://static.igem.org/mediawiki/2020/0/03/T--SDU-Denmark--gel160920_5.png)

 **Thursday**
 *Present: F, AMK, KDL, NJ, RA, JL, AR*
Cas13a group:
Cas13a from gel and purified by miniprep kit.

Cas12 group:
ER2566 E. coli have been growing ON and have been made competent by CaCl2.
Colony PCR on ON clean-streaked plates was performed.
• Protocol for TAQ-polymerase was performed, with annealing temp. at 61.5°C.
![Gel on transformation](https://static.igem.org/mediawiki/2020/7/76/T--SDU-Denmark--gel170920_1.png) ![Gel on transformation](https://static.igem.org/mediawiki/2020/1/1f/T--SDU-Denmark--gel170920_2.png)
• No Cas12a was ligated into the backbone, as no bands at 6000 bp were visible.

Cas13a group:
Transformation of Top10 from yesterday showed no growth on Amp-plates.
TSB transformation is done from ligations from yesterday with the new “6C” backbone.

 **Friday**
 *Present: AMK*
INSERT TEXT HERECas12a group:
Transformation from yesterday did not work, so new transformation is done on new CCC.
• Ligations used: 1:0, 1:1, 2:1, 3:1, 5:1, 1:2, uncut backbone.
• 8 µL plasmid added.
Because of suspicion of relegation in both backbone and Cas genes, a new primer with EcoRI-site is attempted to be added.
• PCR is done to add the EcoRI site.
• Gel is run on PCR product, showing almost only primer.
![Gel showing primerdimer](https://static.igem.org/mediawiki/2020/5/53/T--SDU-Denmark--gel190920_2.png)

Cas13a group:
The same EcoRI site is attempted to be added by PCR, yielding the same result as for the Cas12a group.
![Gel showing primer dimer](https://static.igem.org/mediawiki/2020/9/90/T--SDU-Denmark--gel190920_1.png)

 **Saturday**
 *Present: none*

 **Sunday**
 *Present: none*

Week 39 (21st-27th of September)

**Monday**
 *Present: KDL, NJ, RA, JL*
Cas12a group:
EcoRI is once again attempted top be added to Cas12a by PCR.
• EcoRI primer is diluted to working stock, 10 µM.
• Cas12+promoter, promoter region (EcoRI-primer), promoter region (Cas1F-primer)
• 9x 20 µL reactions: Denat.: 98°C, 15 sec., Ann temp. 55-65°C, 30 sec., Ext time: 2:30 min, 35x cycles.
• PCR left ON in machine on infinite hold.

Cas13a group:
PCR was also done in the same way as the Cas12a group, with Cas13a and Cas13 part 1.
• Ann temp. gradient of 58-63°C.
Gel run on PCR products show very weak bands at 1400 bp for part 1 and an even weaker band at 4000 bp for Cas13a.
![Gel swith strong primer dimer, and weak cas13 band](https://static.igem.org/mediawiki/2020/3/30/T--SDU-Denmark--gel210920_1.png)
• A large amount of primer may indicate a primer concentration being too high.
A new PCR is done, with the same conditions, but lower EcoRI concentration.
![Cas 13 PCR](https://static.igem.org/mediawiki/2020/e/e2/T--SDU-Denmark--gel210920_2.png)
• Cas13a with EcoRI site was cut out of gel and purified from miniprep kit for gels.

 **Tuesday**
 *Present: AMK, NJ*
Cas12a group:
PCR from yesterday was run on gel.
• No positive results.
![Cas 13 PCR](https://static.igem.org/mediawiki/2020/7/76/T--SDU-Denmark--gel220920_2.png)

Cas13a group:
Gel pieces purified by miniprep.
Backbone “6C” and Cas13a was cut by restriction enzymes:
• Cas13a: EcoRI + SpeIBackbone: EcoRI + XbaI
Gel run for backbone, both cut and uncut.
![Cas 13 PCR](https://static.igem.org/mediawiki/2020/2/26/T--SDU-Denmark--gel220920_1.png)

Cut backbone was cut from gel and purified from miniprep kit.
Cas13a was purified by miniprep kit for enzymatic reactions.
Ligations were done:
• Uncut, 1:0, 1:1, 2:1, 3:1, 1:2, 1:3.
• Set ON at 16°C.

 **Wednesday**
 *Present: AMK, NJ, RA, KDL*
Cas13a group: br>
ON culture of Top10 is made, both plated and in tubes.
Top10 with 6C plasmid is also grown, so plasmid purification can take place tomorrow.
Transformation tomorrow as well.

Cas12a group:
As the last PCR runs have been unsuccessful, new promoter region from Cas13a is made, now with the EcoRI-site added.
• New cas12a is also amplified from the Ascpf1-plasmid.
PCR products run on gel.
• Promoter region was amplified, but Cas12a failed to be amplified.
![](https://static.igem.org/mediawiki/2020/7/7d/T--SDU-Denmark--gel230920_1.png)

Purification from gel of promoter region.
Two gels in fridge say “Clean Cas12”, from 2/9. These were also purified along with Cas12+promoter.
• These were all stored in freezer for later use.

 **Thursday**
 *Present: AMK, KDL, FT, RA, FF*
PCR done on purification made yesterday to put together Cas12 and promoter region.
• Confusion as to, what reverse primer had been used earlier, made it so that multiple PCRs were done.
• Reactions
• At 61.1°C

|  |  |  |
| --- | --- | --- |
| Clean Cas12 1.3 + promoter | Primer Rv1 | EcoRI-F |
| Clean Cas12 2.3 + promoter | Primer Rv1 | EcoRI-F |
| Clean Cas12 1.3 + promoter | Primer Rv2 | EcoRI-F |
| Clean Cas12 2.3 + promoter | Primer Rv2 | EcoRI-F |
| Clean Cas12 1.3 + promoter | Primer Rv3 | EcoRI-F |
| Clean Cas12 2.3 + promoter | Primer Rv3 | EcoRI-F |
| Water | Primer Rv1 | EcoRI-F |

• At 58.8°C

|  |  |  |
| --- | --- | --- |
| Clean Cas12 1.3 + promoter | Primer Rv1 | Ascpf-F |
| Clean Cas12 2.3 + promoter | Primer Rv1 | Ascpf-F |
| Clean Cas12 1.3 + promoter | Primer Rv2 | Ascpf-F |
| Clean Cas12 2.3 + promoter | Primer Rv2 | Ascpf-F |
| Clean Cas12 1.3 + promoter | Primer Rv3 | Ascpf-F |
| Clean Cas12 2.3 + promoter | Primer Rv3 | Ascpf-F |
| Water | Primer Rv1 | Ascpf-F |

• At 55.7°C, the same as 61.1oC, though without water control.
PCR products were run on gel.
![Gel of cas12 with promoter region](https://static.igem.org/mediawiki/2020/e/e5/T--SDU-Denmark--gel240920_1.png)

• Pieces of Cas12+promoter with EcoRI-site were cut from gel and purified.
6C backbone was cut by restriction enzymes, EcoRI and SpeI, with AP being added 30 minutes into the cutting.
Ligations were made in relations: Backbone:Cas12a: 10:0, 10:20, 10:50, 10:130
• Placed on 16°C ON.

Cas13a group:
Purification of 6C form Top10 and cutting of 6C backbone by EcoRI and SpeI.
• Run on gel
![Gel with band showing Earlier biobrick](https://static.igem.org/mediawiki/2020/3/3c/T--SDU-Denmark--gel240920_2.png)

• Cut out of gel and purified in miniprep kit.

 **Friday**
 *Present: AMK, KDL*
Cas12a group:
TSB transformation on ligations from yesterday.
• Lack of LB plates caused that only 1 volume per ligation was plated.

|  |  |  |  |
| --- | --- | --- | --- |
| 50 µL, LB, water | 100 µL, cml, uncut | 100 µL, cml, 10:20 | 100 µL, cml, 10:50 |
| 50 µL, cml, water | 100 µL, cml, 10:0 | 50 µL, cml, 10:50 | 100 µL, cml, 10:130 |

Cas13a group:
TSB transformation of ligation from 23/09.
• Lack of LB plates caused that only 1 volume per ligation was plated.

|  |  |  |  |
| --- | --- | --- | --- |
| 100 µL, LB, water | 100 µL, cml, uncut | 100 µL, cml, 10:20 | 100 µL, cml, 10:50 |
| 100 µL, cml, water | 100 µL, cml, 10:0 | 50 µL, cml, 10:50 | 100 µL, cml, 10:130 |

**Saturday**
 *Present: AM, RA*
Cas12a group:
Transformations from yesterday did not work.
• Cells were competent, but no ligations were taken up.
PCR was done on the ligations, to see, if ligation had even worked.
• These were run on gel, which showed no ligation had taken place.
![Smeared gel](https://static.igem.org/mediawiki/2020/0/0e/T--SDU-Denmark--gel260920_1.png)
New LB medium and cml plates were made.

Cas13a group:
PCR was also done for ligations, as previous transformation had not worked, though cells were competent.
• No ligation had taken place.
New ligation was made: 1:0, 1:1, 2:1, 3:1, 1:2, 1:3, 1:5, uncut.

 **Sunday**
 *Present: RA*
Cas13a group:
Protocol followed for CaCl2 transformation.

|  |  |  |  |
| --- | --- | --- | --- |
| 100 µL, LB, water | 100 µL, cml, water | 100 µL, cml, 1:0 | 100 µL, cml, Uncut |
| 100 µL, cml, 1:1 | 100 µL, cml, 1:2 | 100 µL, cml, 1:3 | 100 µL, cml, 2:1 |
| 100 µL, cml, 3:1 | 200 µL, cml, 1:1 | 200 µL, cml, 1:2 | 200 µL, cml, 1:3 |
| 200 µL, cml, 2:1 | 200 µL, cml, 3:1 |  |  |

Week 40 (28th of September - 4th of October)

**Monday**
 *Present: FF*
No progress in assembling Cas, see Flowstrip work

 **Tuesday**
 *Present: FF, FT*
No progress in assembling Cas, see Flowstrip work

 **Wednesday**
 *Present: AMK, NJ*

 **Thursday**
 *Present:*
Cas12 group:
PCR was conducted with the new primers that arrived yesterday. PCR mixes were made for Cas12, Cas12 promoter region and Cas12+promoter region, along with a water control that contained the EcoRI 2F and Rv2 primers.
![Gel with bands showing Cas12](https://static.igem.org/mediawiki/2020/e/e0/T--SDU-Denmark--gel011020_1.png) ![Gel with bands showing the promoter region](https://static.igem.org/mediawiki/2020/7/77/T--SDU-Denmark--gel011020_2.png)

20 µl of Cas12+promoter region (Ecov2) was digested with EcoRI and SpeI and afterwards were purified. The concentration was measured to 6,9 ng/µl. Digested 6C backbone was ligated to the purified Cas12+promoter region.
![Image of equation](https://static.igem.org/mediawiki/2020/0/01/T--SDU-Denmark--EquationW40.png)
From the equation above, it is known that from 10 fmol, 4,1 µl of Cas12 is to be used.

|  | Total volume | Cas12 | 6C backbone | Ligation buffer | Ligase | H2O |
| --- | --- | --- | --- | --- | --- | --- |
| 10:0 | 20 µl | - | 2.1 µl | 2 µl | 1 µl | 15.4 µl |
| 10:10 | 20 µl | 4.1 µl | 2.1 µl | 2 µl | 1 µl | 11.3 µl |
| 10:20 | 20 µl | 8.2 µl | 2.1 µl | 2 µl | 1 µl | 7.2 µl |
| 10:50 | 30 µl | 21 µl | 2.1 µl | 3 µl | 1.5 µl | 3.15 µl |
| 20:10 | 20 µl | 4.1 µl | 4.2 µl | 2 µl | 1 µl | 9.2 µl |
| 30:10 | 20 µl | 4.1 µl | 4.2 µl | 2 µl | 1 µl | 9.2 µl |
| Undi- gested 6C | 20 µl | - | 1 µl | 2 µl | 1 µl | 16.5 µl |

A new PCR with purified Cas12 and promoter region from earlier today were ran to be ligated together.

Cas13 group:
PCR for Cas13a was done to add EcoRI+6 extra nucleotides on Cas13a. 2 PCR tubes with 50 µl were made to run on the PCR and these were run on a gel.
Under gel purification, a column was accidentally thrown away when it should not have been. Therefore only 1 tube was continued with.
Digestion was done with EcoRI and SpeI and 6C backbone cut with EcoRI and XbaI were ligated using the following ratios: 1:0, 1:1, 2:1, 3:1, 1:2 and undigested 6C backbone.

 **Friday**
 *Present:*
INSERT TEXT HERE
 **Saturday**
 *Present:*
INSERT TEXT HERE
 **Sunday**
 *Present: AMK, KDL, NJ*
Cas12 group:
CaCl2 transformation was done for the Cas12+promv2 that was purified yesterday. 3,5 µl ligation mixture was added to 100 µl competent cells and the protocol for CaCl2 transformation was followed.

Cas13a group:
CaCl2 transformation using competent cells that were made on 03/10 was done. 5 µl of the different ligation mixtures were added to 200 µl competent cells. The protocol was followed.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| 50 µl, Amp, TV | 50 µl, Cml, TUS | 50 µl, Cml, T1:1 | 50 µl, Cml, T2:1 | 50 µl, Cml, T3:1 | 50 µl, Cml, T1:2 |
| 50 µl, Amp, TV | 50 µl, Cml, T1:0 | 100 µL, Cml, T2:1 | 100 µl, Cml, T2:1 | 100 µl, Cml, T3:1 | 100 µl, Cml, T1:2 |
|  |  |  | 200 µl, Cml, T2:1 | 200 µl, Cml, T3:1 | 200 µl, Cml, T1:2 |

Week 41 (5th-11th of October)

**Monday**
 *Present: RHA, AMK*
Colony PCR was done on many colonies from the successful transformation that happened from yesterday. Cas13a had one of the colonies with Cas13a in and that was left to incubate. Cas12 did another colony CPS, since there was a mix up with the primers initially.
![Colony PCR, succesfull for cas13](https://static.igem.org/mediawiki/2020/9/94/T--SDU-Denmark--gel051020_2.png)

 **Tuesday**
 *Present: RHA, NJ, AMK*
Top10 and ER2566 cells were left to grow in the heating cabinet in order to make competent ER cells. The completed competent cells were left in the refrigerator.
Overnight cultures of Cas13a from Top10 cells were left to incubate. 1 colony was taken and added to 4 tubes containing 5 ml LB, 5 µl Cml.
Pictures of the gels from the Cas12 colony PCR can be found here:
![Colony PCR, succesfull for cas12](https://static.igem.org/mediawiki/2020/f/f4/T--SDU-Denmark--gel01020_1.png) ![Colony PCR, succesfull for cas12 close-up](https://static.igem.org/mediawiki/2020/f/f0/T--SDU-Denmark--gel06020_2.png)

 **Wednesday**
 *Present: RHA*
Plasmid purification was made out of 20 ml Cas13a+Top10 culture. Concentrations that were measured all lay above 60 ng/µl.
ER2566 was left to grow in the incubator for TSB transformation tomorrow.

 **Thursday**
 *Present: RHA*
TSB competent cells were made after the protocol.

 **Friday**
 *Present: KDL, FF, NJ*
Using the ON cultures made from the colonies that grew from our transformation yesterday, Cas12 and Cas13a plasmids were purified. The protocol for plasmid purification was followed and concentrations were measured to the following:

* Cas12-1: 50.47 ng/µl
* Cas12-2: 44.85 ng/µl
* Cas13-1: 178.74 ng/µl
* Cas13-2: 75.09 ng/µl

Glycerol stocks of all the ON cultures of Cas12 and Cas13a were made by adding 700 µl ON culture to 300 µl 50% glycerol in freeze stock tubes.
The plasmids were digested with EcoRI and PstI where the protocol was followed.
A gel was run for the digested plasmids and were loaded as follows:
402–Cas12-1–Cas12-2–Cas13-2
Some other blue dye was accidentally added to Cas13-1, so this was disregarded.
![Colony PCR, succesfull for cas12](https://static.igem.org/mediawiki/2020/d/de/T--SDU-Denmark--gel091020_2.png)
It seems that from the photo, Cas13 is present. We cannot say this with full certainty since controls were not added.

 **Saturday**
 *Present: FF, KDL*
The same procedure as yesterday is done, but with the ON culture that was made yesterday. The plasmids were purified after the protocol and glycerol stocks were made out of these. The concentrations were measured to be:

* Cas12-3: 82.46 ng/µl
* Cas12-4: 87.84 ng/µl
* Cas13-5: 79.70 ng/µl
* Cas13-3: 82.19 ng/µl
* Cas13-4: 89.75 ng/µl

The following was used to make the digestion mixture:

|  |  |
| --- | --- |
| Total volume | 20 µl |
| 10x buffer | 2 µl |
| DNA | 12 µl (1000 ng/~80 ng/µl) |
| PstI | 1 µl |
| FastAP | 1 µl |
| EcoRI | 1 µl |
| H2O | 3 µl |

The Cas13 from yesterday that is known to work was used as a control, including 6C undigested plasmid. They were loaded on a gel as follows:
Cas12-3–Cas12-4–Cas12-5–Cas13-3–Cas13-4–Cas13 control–6C uskåret.
![CBands with succesfull cas 13 (and cas12)](https://static.igem.org/mediawiki/2020/f/f8/T--SDU-Denmark--gel101020_2.png)
The LB plates whre transformations of Cas12 and Cas13 were made yesterday. The control looked a little strange, but there were some colonies on Cas13-2 (200 µl) and Cas12-2 (200µl). We made ON cultures from 1 colony and added it to an inoculation tube with 5 ml LB and 5 µl Cml. The tubes were left I the heating cabinet (37℃) to grow.

 **Sunday**
 *Present:*
INSERT TEXT HERE

Week 42 (12th-18th of October)

**Monday**
 *Present: KDL*
Cas12 and Cas13a are purified once again by following the protocol. The concentrations were measured to be:

* Cas12-6: 71,35 ng/µl
* Cas12-7: 100 ng/µl

The following table illustrates what comprises the digestion mixture.

|  | Cas12-6 | Cas13-5 | H2O | Digested Cas12-6 with EcoRI | Digested Cas12-6 with PstI |
| --- | --- | --- | --- | --- | --- |
| Total volume | 20 µl | 20 µl | 20 µl | 20 µl | 20 µl |
| 10x buffer | 2 µl | 2 µl | 2 µl | 2 µl | 2 µl |
| DNA | 14 µl (1000 ng/~71 ng/µl) | 10 µl (1000 ng/~100 ng/µl) | 14 µl | 14 µl | 14 µl |
| PstI | 1 µl | 1 µl | 1 µl | - | 1 µl |
| FastAP | 1 µl | 1 µl | 1 µl | 1 µl | - |
| EcoRI | 1 µl | 1 µl | 1 µl | 1 µl | 1 µl |
| H2O | 1 µl | 1 µl | 1 µl | 1 µl | 1 µl |

These were left to digest for 1 hour at 37℃ and were ran on a 1% agarose gel. The gel was loaded as follows:
402–Cas12-6–Cas12(E)–Cas12(P)–Cas13-5– H2O –Undigested Cas12–Undigested 6C.
![Bands shows succesfull cas 12](https://static.igem.org/mediawiki/2020/f/f2/T--SDU-Denmark--gel121020_1.png)
After checking the SnapGene file for Cas12, it was found that PstI cuts on 5 different sites on our Cas12 sequence. This means that we have Cas12.
200 µl of the remaining ON cultures were plated on Cml plates and were left to grow overnight at 37℃.

 **Tuesday**
 *Present:*
 **Wednesday**
 *Present: KDL, AMK*
Cas12 group:
We received our codon optimized Cas12 from Genscript and decided to run a PCR reaction on it in order to have more of it on stock.
According to the instructions sent by Genscript, the plasmid had to first be centrifuged at 6000 g for 1 min at 4℃. Then 20 µl sterilized water was addded to dissolve the DNA. The mixture was then vortexed slightly.
Seeing that the sequences primers were “VF2” and “AsCpf1\_R,” working stocks were made by adding 10 µl primer and 90 µl H2O to a new Eppendorf tube.
The following was used for the PCR mix and a water control was also used:

|  |  |
| --- | --- |
| Total volume | 25 µl |
| VF2 | 1 µl |
| AsCpf1\_R | 1 µl |
| Template | 1 µl |
| H2O | 9.5 µl |

The temperature was set to 60℃ and the annealing time at 2:20 min. This ran for about 2.5 hours.
Apart from this, Top10 cells were grown to make competent cells. This was done in order to transform the Cas12 gene to the iGEM backbone. The cells never grew to mid exponential so we are trying again tomorrow.

 **Thursday**
 *Present: KDL*
Cas12 group:
The PCR machine was left on the whole night, so the sample from yesterday had to be run through gel electrophoresis. A 1% gel was moulded and the PCR samples were loaded.
The optimized Cas12 sequence is found on a PUC57 vector, which has a size of 2710 bp. The size of the sequence is 4259 np, so in total, we would be looking at a band with a size of about 7000 bp.
![CBands with succesfull cas 13 (and cas12)](https://static.igem.org/mediawiki/2020/4/48/T--SDU-Denmark--gel151020_2.png)

The results did not come out as expected, since none of the bands are lying on the right places. We then decided to transform the plasmid directly to Top10 cells instead of trying again with PCR.

 **Friday**
 *Present: KDL*
Cas12 group:
Using the competent cells that were made yesterday, Cas12 was transformed to Top10 cells. 3 µl of the optimized Cas12 and 100 µl competent cells were used. Fir the controls, 3 µl H2O and 0,5 µl LAC was used. The protocol for CaCl2 transformation was followed and the samples were plated out as follows:

|  |  |  |
| --- | --- | --- |
| 250 µl, Amp, Optimized Cas12 | 100 µl, Amp, H2O | 100 µl, Amp, LAC |
| 150 µl, Amp, Optimized Cas12 | 100 µl, H2O | 100 µl, cml, LAC |
| 50 µl, Amp, Optimized Cas12 |  |  |

The plates were left to grow ON at 37℃.

 **Saturday**
 *Present: KDL*
It was decided on that we would not continue with working on the codon optimized Cas12 and that we would just try to focus our efforts on deriving a proof of concept for the Cas12 we already have.

 **Sunday**
 *Present:*
INSERT TEXT HERE

Week 43 (19th-25th)

**Monday**
 *Present:*
INSERT TEXT HERE

 **Tuesday**
 *Present:*
INSERT TEXT HERE
 **Wednesday**
 *Present:*
INSERT TEXT HERE
 **Thursday**
 *Present:*
INSERT TEXT HERE
 **Friday**
 *Present:*
INSERT TEXT HERE
 **Saturday**
 *Present:*
INSERT TEXT HERE
 **Sunday**
 *Present:*
INSERT TEXT HERE

PROTEIN PURIFICATION

Week 40 (28th of September-4thd of October)

**Friday**
 *Present: AMK, RHA, JL, FT*
ER2566 containg plasmid with Papa\_gene, was incubated at 37C with vigorous shaking ON. This done to practice protein purification, due to a lack of time.

 **Saturday**
 *Present: JL, FT*
ER2566 with Papa gene was harvested when OD600=0,6 the protocol:  [“protein purification with His-tag”](https://2020.igem.org/Team%3ASDU-Denmark/Experiments%20)  was followed. We stopped at step after sonication due to the needed for sleep.

 **Sunday**
 *Present: AMK, KDL, NJ, JL, FT*
The sonicated ER 2566 papa was incubated with Ni-agarose then washed, pre-eluted, and eluted.

Week 41 (5th-11th of October)

**Monday - Friday**
No progress in Protein purification.

 **Saturday**
 *Present: FF, KDL, JL, FT*
ER2566 with plasmid containing Cas13a was grown up to OD600=0,7. Then induced with IPTG (retrospect to low finale concentration of IPTG) the followed the steps described in the protocol  [“protein purification with His-tag”](https://2020.igem.org/Team%3ASDU-Denmark/Experiments%20)
We put the sonicated lysates at –20℃.

 **Sunday**
 *Present:*
The sonicated lysates for Cas13a was washed, pre-elution and eluted in fractions.

Week 42 (12th-18th of October)

**Monday**
 *Present: KDL, JL, FT*
The sonicated lysates for Cas13a was washed, pre-elution and eluted in fractions.

 **Tuesday**
 *Present: JL, FT*
The elution fractions of protein from Cas13a was runed on SDS-page gel. The result of gel was displayed with “instant blue” and showed clear bands at the first and second elution fraction. There were also artifacts in a clearly pattern in both elution fractions. (in retrospect higher concentration of Imidazole in the pre-elution step could solve the problem, additively a step more of pre-elution could be added)

 **Wednesday**
 *Present:*

 **Thursday**
 *Present: KDL, JL, FT*
ER2566 with plasmids containing Cas13a and Cas12a was grown up to OD600=0,7. Then induced with IPTG (finale concentration 1mM) the followed the steps described in the protocol  [“protein purification with His-tag”](https://2020.igem.org/Team%3ASDU-Denmark/Experiments%20)
We put the sonicated lysates at –20.
 **Friday**
 *Present: KDL, JL, FT*
The sonicated lysates for Cas13a and Cas12a was incubated with Ni-agarose, then flushed with washbuffer, pre-pre-elution (Imidazole conc: 50mM), pre-elution (Imidazole conc: 75mM) and eluted in fractions.

Then an SDS-page gel for each Cas protein was made, to evoke bands instant blue was used. The result of gels. Both gels had clear bands in the correct place for Cas13a and Cas12a. There were clear bands in elution fractions 2-6 for cas12a without noticeable artifacts Cas13a had also clear bands in the elution fractions 2-6, but we saw still the same artifacts as described at the first Cas13a protein purification. This time it was not as clear.

 **Saturday**
 *Present: KDL, JL, FT*
Bradford assay was conducted on the elution fractions to estimate protein concentrations.
After Sumo-protease assay was conducted to cleave of SUMO-6xhistag. This was verified on an SDS-page gel. The cleaving was only successful on Cas13a

 **Sunday**
 *Present:*
INSERT TEXT HERE

Week 43 (19th-25th of October)

**Monday**
 *Present:*
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 **Tuesday**
 *Present:*
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 **Wednesday**
 *Present:*
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 **Thursday**
 *Present:*
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 **Friday**
 *Present:*
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 **Saturday**
 *Present:*
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 **Sunday**
 *Present:*
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RNASE AND FLOWSTRIP EXPERIMENTS

Week 40 (28th of September - 4th of October)

**Monday**
 *Present: FF*
RNA reporter group:
Flowstrip site recommends 0.02-2 pmol to work for negative controls.
• Changing concentrations is looked at for the test: 100 pmol, 10 pmol, 1 pmol, 0.1 pmol, 0.01 pmol RNA reporter is added into 100 µL assay buffer. Results are seen after 3 minutes, though result sheet is gone.

 **Tuesday**
 *Present: FF, FT*
Flowstrip group:
Inhibition of RNases.
Test to see if Protease K inhibits RNases in saliva and urine.
• 1 µL Protease K is added to 100 µL urine, 100 µL saliva and 100 µL water.
• Left ON at RT.
• 20 µL Inhibitor is added to each sample and flowstrips, RNA reporter + buffer is added.
![Flowstrips from experiments](https://static.igem.org/mediawiki/2020/1/11/T--SDU-Denmark--Flowstrip290920.png)
RNases in urine is inhibited, but not in saliva. More urine and saliva are added and shows stronger signals.
• Protease is also inhibited afterwards, so Cas can work.
• Positive controls, only with urine and saliva show a clear positive line, but also a faint control-line, which has not been observed before. RNA reporter may have been needed to linger in the samples before flowstrip was inserted.
• A sample with and without assay buffer a made, which seams to have an impact.

 **Wednesday**
 *Present: FF, Ft*
We tested if we could inhibit RNAse in saliva. It worked with 4 and 8 protease K after ON incubation at 55.
We also saw inhibition after only 3 hours of incubation at 55, though the inhibition was smaller.
![Flowstrips from experiments](https://static.igem.org/mediawiki/2020/8/82/T--SDU-Denmark--Flowstrip300920_1.png) ![Flowstrips from experiments](https://static.igem.org/mediawiki/2020/b/bb/T--SDU-Denmark--Flowstrip300920_2.png)

 **Thursday**
 *Present: AMK, KDL, FT, RA, FF*
RNase digestion:
5-day old tubes with Protease K, RNase alert was added and shown not to fluoresce after 50 minutes.
• Protease K inhibitor was added to 0.5 mM PMSF concetration.
• Added new urine and saliva to the tubes for 1 hour to see if Protease K activity stops.
• 5 µL RNase alert is added, leaving it for +30 minutes.

Flowstrip test:
Flowstrips are tested with positive and negative controls
• Positive control has a single in the top, while the negative has two, though the lower is strongest.
• Different concentrations of RNA reporter was used. Two lines on all samples, show that negative control not having a line in the top is not dependent on RNA reporter concentration.
![Flowstrips from experiments](https://static.igem.org/mediawiki/2020/7/7f/T--SDU-Denmark--Flowstrip011020_1.png)

 **Friday**
 *Present:*
See assembling of cas proteins

 **Saturday**
 *Present:*
See assembling of cas proteins

 **Sunday**
 *Present:*
See assembling of cas proteins

Week 41 (5th-11th) of October

**Monday - Sunday**
 *Present:*
Focus was on other aspects of our project

Week 42 (12th-18th of October)

**Monday**
 *Present:*
No progress in this area

 **Tuesday**
 *Present: RHA, FF*
**POC group:**
The purified Cas13a protein was obtained from the protein purification crew, and an attempt at making proof of concept on flow strips was made. For the reactions, Cas13a was diluted in Protein SB to a concentration of 63.3 ng/µl and mixed with 1 µl of 10 ng/ µl sgRNA.

Storage buffer was made from:

* 2.5 ml Tris HCl (pH = 7.5)
* 6 mL NaCl (5 M)
* 2.5 µl Glycerol
* 100 µl DTT (1M)
* 100 µl DTT (1M)

And was filtered through a 0.22 µm 50 ml vacuum filter.

Proof of concept was not successful, as all flow strips presented with two lines instead of one, making it impossible to distinguish between whether they were negative or positive. Therefore, it was suggested that the concentration of target RNA be varied to obtain optimal readout.

 **Wednesday**
 *Present:AMK, FF, RHA*
**POC group:**
As suggested yesterday, the amount of target RNA was varied in a new attempt at proof of concept on flow strips. The following concentrations were used: 1x, 10x, 100x and 1000x.

The flow strips still presented with two lines.

 **Thursday**
 *Present: KDL, JL, FT, FF, RHA*
**POC group:**
It was decided that the system should be tested using RNase alert, to determine whether the protein was activated in the presence of sgRNA and target, as it was discovered that a half positive could present as two lines on flow strips. RNase alert showed that after 20 min incubation, no RNase activity could be measured. However, after ON incubation, a noticeable difference between negative controls and the samples could be observed, and it was therefore chosen to do more experiments where the concentration of sgRNA was varied, to test if the incubation time could be decreased. No difference was observed between the samples.

 **Friday**
 *Present: KDL, JL, FT, FF, RHA*
 **POC group:**
All the equipment for RNase Alert had been used up, and a new method to detect POC therefore had to be chosen. Here, it was discovered that 2% agarose gels should be able to show RNase activity, if random RNA was added to each sample as a marker of RNase activity. In the presence of RNases, the random RNA would be degraded and visible as a smeared band on the gel.

 **Saturday**
 *Present: KDL, JL, FT*
 **POC group:**
POC was attempted on a 2% agarose gel. This gel showed that the random RNA had been degraded in the samples, including negative controls with Cas13a alone or with Cas13a + sgRNA. Therfore, it was hypothesised that Cas13a might have intrinsic activity on its own without sgRNA and target RNA bound.

 **Sunday**
 *Present:*
No progress in this area

Week 43 (19th-25th of October)

**Monday**
 *Present:*
No pregres in this area

 **Tuesday**
 *Present: KDL, FT, FF, RHA*
Cas12a group:
Purified Cas12 is to be run on a gel to see if it is active and is cleaving RNAs. We followed the protocol that was being used for the Cas13a POC.
The following were added to RNAse-free tubes where samples were made for 3 reactions = total volume of 60 µl.

|  |  |
| --- | --- |
| Cas13a | 3.5 µl |
| Target | 3.75 µl |
| Guide RNA | 0,6 µl or 1,2 µl |
| Cas12a | 13 µl |
| H2O | 39.15 µl – for 1 µM gRNA samples   38.9 µl – for 2 µM gRNA samples   39.75 µl – for no target samples |

The following tubes included the following:

* Water + C13
* 1 µM gRNA + target + Cas12a + Cas13a + H2O
* 2 µM gRNA + target + Cas12a + Cas13a + H2O
* No target 1 µM gRNA + Cas12a + Cas13a + H2O
* DNAse + C13 + H2O

This was done for both T and G SNPs and 20 µl of sample was taken out after 1 hour, 2 hours, and 3 hours.

The results did not turn out as expected, so we think that it could have something to do with the fact that Cas12a works better with a buffer. Also, there does not seem to be an effect in changing the time.
We will try to make the buffer tomorrow and do the whole procedure again.

 **POC group:**
POC was attempted by using a protein without the sumo his-tag cut off, as it was hypothesized that the tag may help to lower the intrinsic activity indicated yesterday. Additionally, varying concentrations of sgRNA was used, and it was seen that higher concentrations of sgRNA led to higher degradation of random RNA. However, as no control holding only Cas13a in MiliQ was included, it could not be determined whether it was actually the presence of target RNA that activated the protein, or the hypostasized intrinsic activity.

 **Wednesday**
 *Present: KDL, FT, FF, RHA*
Cas12a group:
A Cas12a buffer was made from the protocol published for NeBuffer 2.1. Before beginning, it was decided to use Cas13a that is cut open with EcoRI. The following digestion mixture was used:

|  |  |
| --- | --- |
| Total volume | 20 µl |
| 10x fast digest | 2 µl |
| EcoRI | 1 µl |
| Cas13 | 14,7 µl (1000 ng/67,8 ng/µl) |
| FastAP | 1 µl |
| H2O | 1.3 µl |

The sample was left to digest at 37℃ for 1 hour. Afterwards, the sample was boiled at 95℃ to turn the plasmid into single stranded DNA.
The same protocol was used but here we used the buffer in the place of water. The following were added to RNAse-free tubes where samples were made for 2 reactions = total volume of 40 µl:

|  |  |
| --- | --- |
| Cas13/single-stranded Cas13 | 2.5 µl |
| Target | 1 µl |
| Guide RNA | 0.5 µl or 1 µl |
| Cas12a | 8.5 µl |
| H2O | 27.5 µl – for 1 µM gRNA samples   27 µl – for 2 µM gRNA samples   28.5 µl – for no target samples |

The tubes included the following:

* Buffer + Cas13a
* Buffer + digested Cas13a
* Buffer + digested single-stranded Cas13a
* DNAse + buffer + Cas13a
* 1 µM sgRNA + target + Cas12a + Cas13 + Buffer
* 1 µM sgRNA + target + Cas12a + single stranded Cas13a + Buffer
* 2 µM sgRNA + target + Cas12a + Cas13 + Buffer
* 2 µM sgRNA + target + Cas12a + single stranded Cas13a + Buffer
* 2 µM sgRNA + Cas12a + Cas13a + Buffer
* 2 µM sgRNA + Cas12a + single stranded Cas13a + Buffer

This was done for both T and G SNPs and 20 µl of sample was taken out after 1 hour and 2 hours. The samples were then ran on a 2% agarose gel for 30 mins.
INDSÆT GEL BILLEDE
From the results, it seems that Cas12a is active for the G-SNP, as seen from the very weak band present where 2 µM ssC13 is used. The buffer and single-stranded Cas13a showed to be a good idea to use. We will try again tomorrow to get a better picture of the results.

**POC group:**
POC was attempted with the same protein and “best” estimated concentration of sgRNA from yesterday, but all samples containing Cas showed degraded RNA. Therefore, we became suspicious of whether Cas13a had been contaminated with RNases during protein purification.

 **Thursday**
 *Present: KDL, FF, RHA*
The same procedure from yesterday is followed, but now the gel will run on a 1% agarose gel for 1 hour. The process will only be done for the G-SNP and the samples will only incubate for 15 mins and 1 hour.
INDSÆT GEL BILLEDE
The results do not show anything about Cas12a’s activity. We think that it could be due to storage problems for Cas12a, where it should have been stored with a storage buffer. The storage buffer was, however, not added. Thus, the Cas12 could potentially not be active anymore.
Luckily, we have another tube of undigested Cas12a. Tomorrow we will try to cut it open and store it properly and do the procedure again to get a better gel picture.

 **POC group:**
A 2% agarose gel was run with random RNA and different Cas13a proteins from the freezer, to determine whether RNases was present in our protein samples or if Cas13a had intrinsic activity . This showed that some of our protein samples was in fact contaminated with RNases, however, random RNA was not fully degraded in all samples. Therefore, it was decided to attempt POC with one of the proteins which showed no contamination with RNases.

 **Friday**
 *Present: KDL, JL, FF, RHA*
 **POC group:**
POC WAS ACHIEVED

##### igem@sdu.dk [@igem.sdu](https://www.instagram.com/igem.sdu/) [/igem.sdu](https://www.facebook.com/igem.sdu)

[![SDU](https://static.igem.org/mediawiki/2020/9/93/T--SDU-Denmark--SDUlogo.png)](https://www.sdu.dk/en)

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[![SnapGene](https://www.snapgene.com/wordpress/wp-content/uploads/2016/07/SnapGeneLogoLight@2x.png)](https://www.snapgene.com/)

[![Ampliqon](https://ampliqon.com/images/logo.png)](https://ampliqon.com/)

[![Integrated DNA Technologies](https://static.igem.org/mediawiki/2020/5/5a/IDT_Logo_TM_19_RGB_%281%29.png)](https://eu.idtdna.com/pages)

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# Team:SDU-Denmark/Engineering

[Home](https://2020.igem.org/Team%3ASDU-Denmark)

Our Project

[Description](https://2020.igem.org/Team%3ASDU-Denmark/Description)
[Results](https://2020.igem.org/Team%3ASDU-Denmark/Results)
[Proof of Concept](https://2020.igem.org/Team%3ASDU-Denmark/Proof_Of_Concept)
[Contribution](https://2020.igem.org/Team%3ASDU-Denmark/Contribution)
[UN SDGs](https://2020.igem.org/Team%3ASDU-Denmark/Sustainable)
[Poster](https://2020.igem.org/Team%3ASDU-Denmark/Poster)

Our Team

[Overview](https://2020.igem.org/Team%3ASDU-Denmark/Team)
[Partnership](https://2020.igem.org/Team%3ASDU-Denmark/Partnership)
[Attributions](https://2020.igem.org/Team%3ASDU-Denmark/Attributions)
[Collaborations](https://2020.igem.org/Team%3ASDU-Denmark/Collaborations)
[Inclusion](https://2020.igem.org/Team%3ASDU-Denmark/Inclusion)

Laboratory

[Design](https://2020.igem.org/Team%3ASDU-Denmark/Laboratory)
[Engineering](https://2020.igem.org/Team%3ASDU-Denmark/Engineering)
[Parts](https://2020.igem.org/Team%3ASDU-Denmark/Parts)
[Notebook](https://2020.igem.org/Team%3ASDU-Denmark/Notebook)
[Protocols](https://2020.igem.org/Team%3ASDU-Denmark/Experiments)
[Safety](https://2020.igem.org/Team%3ASDU-Denmark/Safety)

Human Practices

[Overview](https://2020.igem.org/Team%3ASDU-Denmark/Human_Practices)
[Ethics](https://2020.igem.org/Team%3ASDU-Denmark/Ethics)
[Interview](https://2020.igem.org/Team%3ASDU-Denmark/Interview)
[Science Communication](https://2020.igem.org/Team%3ASDU-Denmark/Education)
[Gamification](https://2020.igem.org/Team%3ASDU-Denmark/Human_Practices/Gamification)
[Early Detection Game](https://2020.igem.org/Team%3ASDU-Denmark/Human_Practices/Card_game)
[Proposed Implementation](https://2020.igem.org/Team%3ASDU-Denmark/Implementation)

Hardware

[Overview](https://2020.igem.org/Team%3ASDU-Denmark/Hardware)
[Integrated Design](https://2020.igem.org/Team%3ASDU-Denmark/Design)
[Modeling](https://2020.igem.org/Team%3ASDU-Denmark/Model)
[Entrepreneurship](https://2020.igem.org/Team%3ASDU-Denmark/Entrepreneurship)

Videos

[Project Promotion](https://2020.igem.org/Team%3ASDU-Denmark/Promotion)
[Music Video](https://2020.igem.org/Team%3ASDU-Denmark/Excellence)
[Final Presentation](https://2020.igem.org/Team%3ASDU-Denmark/Presentaion)

# Engineering Success

"Nothing resembles the joy of when a gel showing correctly assembled DNA."

To make a functional test, both the Cas13a and Cas12a proteins had to be synthesized which was carried out in *E. coli.* The specific genes for *cas13a* and *cas12a* that we used were derived from *Leptotrichia wadei* and *Acidaminococcus sp.*, respectively. We codon optimized the genes for expression in *E. coli* with IDT’s codon optimizing tool and included features that allowed for the inducible gene expression. Both the *cas13a* ([BBa\_K3602001](http://parts.igem.org/Part%3ABBa_K3602001)) and the *cas12a* ([BBa\_K3602015](http://parts.igem.org/Part%3ABBa_K3602015)) genes contain an IPTG inducible promoter region ([BBa\_R0011](http://parts.igem.org/Part%3ABBa_R0011)), a ribosomal binding site ([BBa\_0034](http://parts.igem.org/Part%3ABBa_0034)), and a terminator ([BBa\_B1006](http://parts.igem.org/Part%3ABBa_B1006)). Additionally, both genes have a 6x-histidine and SUMO-tag (Small Ubiquitin-like modification protein) N-terminally, for subsequent protein purification and TEV cleavage.

![Cas13 IPTG Inducible plasmid](https://static.igem.org/mediawiki/2020/b/b7/T--SDU-Denmark--Cas13_construct.png) ![Cas12 Codon optimized plasmid](https://static.igem.org/mediawiki/2020/d/dd/T--SDU-Denmark--Cas12_construct_3.png)

**Figure 1. An overview of the significant features of our Cas13a (left) and Cas12a (right) biobricks designed with SnapGene.**

Due to its high complexity, IDT was unable to synthesize the entire *cas13a* gene. So, we divided the original Cas13a construct into three parts: Cas13a-1 (approx. 1400 bp), Cas13a-2 (approx. 2000 bp) and Cas13a-3 (approx. 700 bp).

Moreover, we were unable to order the *cas12a* gene from IDT, as shipment of the gene outside of the United States required an export license. The research unit “PhyLife”, from the University of Southern Denmark was, however, working with the gene and kindly offered us a plasmid containing the *cas12a* gene. This gene was not codon optimized for *E. coli* and did not contain the desired promoter region or terminator sequence, see [Parts](https://2020.igem.org/Team%3ASDU-Denmark/Parts).

![Cas assembly overview](https://static.igem.org/mediawiki/2020/9/93/T--SDU-Denmark--CasAssemblyEngineering.png)

**Figure 2. Assembly of Cas proteins by way of primer overhangs.** Above, Cas13a is seen assembled in 3 parts, with a promoter region belonging to Cas13a-1. This was amplified from the gene fragment and assembled onto Cas12a from PhyLife. Primer and gene fragment length not to scale. Created with BioRender.com

We assembled Cas13a using PCR amplification, by designed primers that generated overhang between the separate gene parts. The forward overhang primer for Cas13-1 and the reverse Cas13a-3 overhang primer were designed to include an EcoRI-site and SpeI-site, respectively. These restriction sites allowed for digestion and ligation of the gene into the backbone of choice.

As previously mentioned, the obtained Cas12a did not have our desired promoter region. Instead we designed primers to amplify these features from the Cas13a-1 gene and attaching them to Cas12a with PCR. Finally, we cloned the *cas* genes into pSB1C3, which carries chloramphenicol resistance for selection of successful transformants. The newly synthesized plasmids, encoding Cas13a and Cas12a were transformed into the ER2566 strain of *E. coli*, making the genes suitable for protein expression.

![Gel showing bands that represent correctly assembled cas12 and 13 as well as controls](https://static.igem.org/mediawiki/2020/6/63/T--SDU-Denmark--Cas-assembly.png)

**Figure 3. Agarose gel run with backbone + terminator and gene inserts, Cas13a and Cas12a.**  Bands at 2000 bp indicate linear backbone + terminator, bands at 4000 bp indicate linear Cas13a or Cas12a genes and bands at 6000 indicate linear part backbone + terminator with Cas13a or Cas12a inserted. Lane 1) Undigested backbone + terminator Lane 2) Backbone + terminator digested with FastDigest restriction enzyme EcoRI. Lane 3) Backbone + terminator digested with FastDigest restriction enzymes, EcoRI, and SpeI. Lane 4) Undigested backbone + terminator with Cas13a gene inserted. Lane 5) Backbone + terminator with Cas13a gene inserted, digested with FastDigest restriction enzyme EcoRI. Lane 6) Backbone + terminator with Cas13a gene inserted, digested with FastDigest restriction enzyme EcoRI and SpeI. Lane 7) Undigested backbone + terminator with Cas12a gene inserted. Lane 8) Backbone + terminator with Cas12a gene inserted, digested with FastDigest restriction enzyme EcoRI. Lane 9) Backbone + terminator with Cas13a gene inserted, digested with FastDigest restriction enzyme EcoRI and SpeI.

Once the proteins had been purified the unpurified and purified protein was run SDS-PAGE along with some wash buffers. The SDS-PAGE shows that the Cas proteins are successfully expressed and that the His-tag purification is efficient. For more information on the function of the Cas proteins in complex with sgRNAs can be found on our [Proof of Concept](https://2020.igem.org/Team%3ASDU-Denmark/Proof_Of_Concept) page.

![SDS-gel protein purification of cas12a](https://static.igem.org/mediawiki/2020/b/bb/T--SDU-Denmark--protein_purification_cas12.png)

**Figure 4. Successful protein purification of Cas12a.** The first lane displays a sample of lysed *E. coli*  ‘ER2536 + Cas12a plasmid’ flow-through before induction of IPTG. The second lane displays a sample of lysed *E. coli*  ‘ER2536 + Cas12a plasmid’ flow-through after induction of IPTG. The third lane displays flow-through from wash-buffer. The fourth lane displays pre-pre-elution-buffer flow-through. The fifth lane displays pre-elution buffer flow-through. The sixth lane displays 0,5 mL elution-buffer flow-through. Lanes 7-12 display 1 mL elution-buffer fractions. In the lines with elution fractions, there is a clear band above 140 kDa correlating well with the molecular weight of Cas12a+SUMO-Histag = 156,31 kDa. Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”; Gel: Bis-Tris 4-12%; Running buffer: MOPS.

![SDS-gel protein purification of Cas13a](https://static.igem.org/mediawiki/2020/1/18/T--SDU-Denmark--protein_purification_cas13.png)

**Figure 5. Successful protein purification of Cas13a.** The first lane displays a sample of lysed *E. coli*  ‘ER2536+ Cas13a plasmid’ flow-through before induction with IPTG. The second lane displays a sample of lysed *E. coli*  ‘ER2536 + Cas13a plasmid’ flow-through after induction of IPTG. The third lane displays flow-through from wash-buffer. The four lane displays pre-pre-elution-buffer flow-through. The fifth lane displays pre-elution buffer flow-through. The sixth lane displays 0,5 mL elution-buffer flow-through. Lane 7-12 displays 1 mL elution-buffer fractions.In the lines with elution fractions, there is a clear band above 140 kDa correlating well with the molecular weight of Cas13a+SUMO-Histag = 153,8 kDa. Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”; Gel: Bis-Tris 4-12%; Running buffer: MOPS.

Additional information about the assembled parts and the in vitro transcribed guide RNA can be found in the parts registry. Functionality of the Cas-guide RNA complex is illustrated on the [Proof of Concept](https://2020.igem.org/Team%3ASDU-Denmark/Proof_Of_Concept) page.

##### igem@sdu.dk [@igem.sdu](https://www.instagram.com/igem.sdu/) [/igem.sdu](https://www.facebook.com/igem.sdu)

[![SDU](https://static.igem.org/mediawiki/2020/9/93/T--SDU-Denmark--SDUlogo.png)](https://www.sdu.dk/en)

[![Novo Nordisk](https://static.igem.org/mediawiki/2020/6/6c/T--SDU-Denmark--NovoNordisk.PNG)](https://www.novonordisk.com/)

[![SnapGene](https://www.snapgene.com/wordpress/wp-content/uploads/2016/07/SnapGeneLogoLight@2x.png)](https://www.snapgene.com/)

[![Ampliqon](https://ampliqon.com/images/logo.png)](https://ampliqon.com/)

[![Integrated DNA Technologies](https://static.igem.org/mediawiki/2020/5/5a/IDT_Logo_TM_19_RGB_%281%29.png)](https://eu.idtdna.com/pages)

[![Red Bull](https://resources.redbull.com/logos/redbullcom/v1/redbullcom-logo.svg)](https://www.redbull.com/)

[![Cocio](https://cocio.dk/images/logo.svg)](https://cocio.dk/)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:SDU-Denmark/Experiments

[Home](https://2020.igem.org/Team%3ASDU-Denmark)

Our Project

[Description](https://2020.igem.org/Team%3ASDU-Denmark/Description)
[Results](https://2020.igem.org/Team%3ASDU-Denmark/Results)
[Proof of Concept](https://2020.igem.org/Team%3ASDU-Denmark/Proof_Of_Concept)
[Contribution](https://2020.igem.org/Team%3ASDU-Denmark/Contribution)
[UN SDGs](https://2020.igem.org/Team%3ASDU-Denmark/Sustainable)
[Poster](https://2020.igem.org/Team%3ASDU-Denmark/Poster)

Our Team

[Overview](https://2020.igem.org/Team%3ASDU-Denmark/Team)
[Partnership](https://2020.igem.org/Team%3ASDU-Denmark/Partnership)
[Attributions](https://2020.igem.org/Team%3ASDU-Denmark/Attributions)
[Collaborations](https://2020.igem.org/Team%3ASDU-Denmark/Collaborations)
[Inclusion](https://2020.igem.org/Team%3ASDU-Denmark/Inclusion)

Laboratory

[Design](https://2020.igem.org/Team%3ASDU-Denmark/Laboratory)
[Engineering](https://2020.igem.org/Team%3ASDU-Denmark/Engineering)
[Parts](https://2020.igem.org/Team%3ASDU-Denmark/Parts)
[Notebook](https://2020.igem.org/Team%3ASDU-Denmark/Notebook)
[Protocols](https://2020.igem.org/Team%3ASDU-Denmark/Experiments)
[Safety](https://2020.igem.org/Team%3ASDU-Denmark/Safety)

Human Practices

[Overview](https://2020.igem.org/Team%3ASDU-Denmark/Human_Practices)
[Ethics](https://2020.igem.org/Team%3ASDU-Denmark/Ethics)
[Interview](https://2020.igem.org/Team%3ASDU-Denmark/Interview)
[Science Communication](https://2020.igem.org/Team%3ASDU-Denmark/Education)
[Gamification](https://2020.igem.org/Team%3ASDU-Denmark/Human_Practices/Gamification)
[Early Detection Game](https://2020.igem.org/Team%3ASDU-Denmark/Human_Practices/Card_game)
[Proposed Implementation](https://2020.igem.org/Team%3ASDU-Denmark/Implementation)

Hardware

[Overview](https://2020.igem.org/Team%3ASDU-Denmark/Hardware)
[Integrated Design](https://2020.igem.org/Team%3ASDU-Denmark/Design)
[Modeling](https://2020.igem.org/Team%3ASDU-Denmark/Model)
[Entrepreneurship](https://2020.igem.org/Team%3ASDU-Denmark/Entrepreneurship)

Videos

[Project Promotion](https://2020.igem.org/Team%3ASDU-Denmark/Promotion)
[Music Video](https://2020.igem.org/Team%3ASDU-Denmark/Excellence)
[Final Presentation](https://2020.igem.org/Team%3ASDU-Denmark/Presentaion)

# Experiments and protocols

"All great experiments are build upon a 1000 failed ones."

## Experiments

To get and follow our results through the iGEM experience in the wet lab we used a range of different experiments. A collection of which experiments have been used and for what can be found here:
 **Assembling the DNA sequences of Cas13 and Cas12 and inserting it into an iGEM plasmid:**

1. PCR amplification and PCR overhang ligation
2. DNA digestion and ligation
3. Transformation
4. Colony PCR

 **Protein expression and purification:**

1. Protein expression inducement
2. Protein purification
3. Cleavage of His-tag from protein
4. Bradford assay

 **Protein complex assembly and activation:**

1. Co-incubation
2. Activation of nuclease activity
3. In vitro transcription of target and guide RNA

 **Product readout:**

1. Universal lateral flow dipsticks for the detection of biotin and FAM-labeled RNA-reporters
2. Test of RNase presence in spit and urine
3. RNase degradation in urine and spit
4. Proteinase inhibition

## Protocols

Here you can find all the protocols we have followed in the wet lab during this project. Some of the protocols are for basic laboratory practice as making LB broth and agar as well as agarose gels. These protocols are therefore not mentioned in the experiments section above.

Agar with/without antibiotics

**Procedure:**

1. Dissolve 3,5 g of LB-agar per 100 mL of milli-Q.
2. Autoclave the LB-agar solution.
3. Cool the agar down to room temperature (or approximate <50°C).
If no antibiotics is needed skip step 4.4. Add antibiotic. (e.g. volume of antibiotics needed = (desired volume of antibiotics • total volume) / Concentration of antibiotic).
5. Pour the LB-agar in empty sterile Petri dishes under sterile conditions (close to a flame).
6. Let the agar Petri dishes dry before storing them at 4°C.

LB media

**Procedure:**

1. Suspend 20 g of LB broth in 1 L of distilled water.
2. Autoclave for 15 minutes at 121 °C.

Colony picking and culturing

**Procedure:**

1. Fill 5 ml of LB media into an inoculation tube.
2. Add antibiotic to appropriate concentration.
3. Take an agar plate with appropriate medium and antibiotic.
4. Scrape off a single colony from agar plate and mix into the LB-filled inoculation tube.
5. Vortex medium with colony.
6. Place inoculation tube in incubator set to 37°C and leave for 16 hours.

Preperation of agarose gel

**Reagents:**

* Agarose powder
* 1x TAE buffer
* Ethidium bromide

 **Procedure (example given for 1% agarose gel):**

1. Add 1g agarose powder per 10 mL TAE buffer to an erlenmeyer flask.
2. Swirl the flask around, so no large clumps are left. Cover the flask with (PAPER)
3. Dissolve the agarose in the buffer by heating up in a microwave oven, making sure to take the flask out every 2 minutes, to see if particles are left.
4. Let the flask cool for 2-3 minutes.
5. Add 4 g ethidium bromide per 100 mL gel and swirl the flask, to disperse it thrpughout the flask.
6. Place the flask in a 60 C cabinet until use.

Gel electrophoresis (agarose)

**Reagents:**

* Agarose gel with ethidium bromide
* DNA samples
* Loading dye
* DNA ladder
* (TAE buffer)

 **Procedure:**

1. Tape up an agarose mold, making sure that no leaks are present.
2. Add the relevant comb and pour in the agarose gel.
3. Once the gel has stiffened, carefully take out the comb and remove the tape from the mold.
4. Transfer to an electroforesis table.

CaCl2 transformation

**Making CaCl2 competent cells (E. coli)**

1. Streak cells on LB plates and grow at 37°C O/N.
2. Inoculate 5mL LB media with a single colony and grow at 37°C O/N.
3. Inoculate 20mL LB media with 20µL O/N-culture (1:1000 dilution).
4. Grow cells at 37°C (shaking), until OD450= 0.5 (~3 hours).
5. Transfer cells to 50mL tubes and place cells on ice for >20min.
6. Spin down cells at 4°C for 10min at 3000g (4830rpm).
Cells must remain cold for the rest of the procedure e.g. transport tubes on ice and resuspend on ice.7. Pour off media and resuspend (gently) in 10mL ice-cold 0.1 M MgCl2.
8. Spin down cells at 4°C for 10min at 3000g (4830rpm).
9. Pour off media and resuspend cells (gently) in 2mL ice-cold 0.1M CaCl2. Incubate >1hour on ice, but O/N at 4°C is better
10. Use as they are or add 0.5mL 50% glycerol and transfer 100 µL into Eppendorf tubes placed on dry-ice for long term storage.

**Transformation**

1. Thaw CCC on ice (5-10min).
2. Add DNA\* and incubate 10-30min on ice.
3. Heatshock the cells at 42°C for 90sec. on a heatblock or waterbath
4. Move the cells back to ice for 2min.
5. Add 500µL LB (RT)
6. Incubate at 37°C 60min (preferably horizontal shaking).
7. Plate 100µL and 500 µL on LB-plates w. antibiotics.
8. Grow cells o/n at 37°C.

\*Max 10 uL; plasmids: 1-10ng and ligation ~25ng

TSB transformation

**Procedure:**

 **Preparation of E. coli culture:**

1. Add >5mL Lb-media (depends on finale desired volume of transformation) to a bulb
2. Add specific E. coli strain (possible with a pipette tip) to the bulb
3. Incubate at 37°C and swirling until abobance for OD600 is between 0,3-0,5

 **Preparation of TSB buffer:**

1. Add the following components to a 15 mL falcon tube:

   - PEG 3,350 1 g,

   - DMSO 500 µL,

   - MgCl 2 (1M) 200 µL, (OR MgCl 2 6H 2 O 203 g)

   - Fill LB-media up to 10 mL
2. Mix roughly until reagents are completely dissolved
3. Transfer the solution to a new falcon tube through a sterile filter with a syringe

 **TSB transformation:**

1. Spin 1mL E.coli (at OD600: 0,3-0,5) at 3000g at 4°C, remove the supernatant.
2. Dissolve pellet in 200 μL prepared TSB buffer
3. Add plasmid (>1000 pg/mL), and incubate at ice bath for 30 min
4. Transfer from ice to heating block at 42°C for 45 sec, after set heating block to 37°C
5. Add 1mL fort. LB, and let the sample rest to phenotypical expression at 37°C for 0-2 hrs.
6. Spin 3000g for 5 min
7. Remove 90% of supernatant, dissolve the pellet in the rest supernatant
8. Plate on agar plate with appropriate antibiotic

Protein purification with His-tag

It is important to consider that the expressing of the gene/protein of interest happens in a bacterium which have high expression of protein. e.g. *E.coli* ER2566.

 **Procedure:**

1. Inoculate appropriate amount of LB-medium (depends on desired final yield of protein– for this protocol 200mL is applied) with relevant antibiotic and with O/N. culture.
2. Grow at 37°C with vigorous shaking until end of exponential phase is reached (OD: 0,6-0,75).  *(It is possible to collect 1 mL, for later control and comparison)*
3. Induce with IPTG for 3-4 hours (final concentration of IPTG: 0,5-1 mM).  *(It is possible to collect 1 mL, for later control and comparison).*
4. Harvest the cells by centrifugation at ≈10.000g, 4°C for 10 minutes. Discard the supernatant and resuspend the pellet in 100 mL TMN. Then centrifuge again with same settings as above. Discard the supernatant again and resuspend the pellet in 8 mL TMN-buffer.
5. Sonicate on ice: 5X 30 sec with pause > 30 sec for each. (settings for sonicator: Hold, constant and 2,5).
6. Centrifuge the lysates at 8.000g at 4°C for 10 min. Decent the supernatant to a new tube and repeat the centrifugation step. Adjust the supernatant so it gets the same concentration of ions as the wash buffer by adding the following:

   | Stock solutions | Add | Final conc. |
   | --- | --- | --- |
   | 50% glycerol | 2,8 mL | 10% |
   | 1M NH4Cl | 2,7 mL | 250 mM |
   | 1M Tris-HCl pH8 | 0,12 mL | 20 mM |
   | 1M mg(O Ac) | 0,06 mL | 10 mM |
   | 2M Imidazol | 0,14 mL | 20 mM |
   | 14,3M 2-mercaptoethanol | 6,6 µL | 6 mM |
7. Save 30 µL of the sample and mix with 10µ SDS-PAGE buffer and freeze until SDS-PAGE analysis.

 **Preparation of resin:**

1. Wash 1 mL Ni-NTA agarose with 2X 10 mL water and 2X 10 mL wash buffer. Next steps are on 4°C.
2. Add the liquid and mix gently together with the resin centrifuge 2 min at 2000g then remove buffer.
3. Mix supernatant with 1 mL washed resin and incubate for 1 hour at 4°C on rotating table.
4. Apply the resin with supernatant to a Poly-Prep Chromatography column and collect 1 mL flow-through.
5. Wash the column with 10 mL wash buffer and collect 1 mL flow-through
6. Wash the column with 10 mL pre-elution buffer and collect 1 mL flow-through
7. Elute the protein by adding 0,5 mL elution buffer, collect all flow-through. Then add 7,5 mL elution buffer collect 1 mL flow-through.

 *All the collections of ≈ 1mL can be used in SDS-PAGE.*

 **Table for making the different buffer solutions:**

| Stock solutions | Wash buffer 30 mL | Pre- elution buffer 10 mL. | Elution buffer 8 mL | Final conc. |
| --- | --- | --- | --- | --- |
| Water | 20,99 mL | 6,925 mL | 5,06 mL | - |
| 1M NH4Cl | 1,875 mL | 0,625 mL | 0,5 mL | 250 mM |
| 50% glycerol | 6 mL L | 2 mL | 1,6 mL L | 10% |
| 1M MgOAc | 0,3 mL | 0,1 mL | 0,08 mL | 20 mM |
| 1M Tris-HCl pH8,0 | 0,6 mL | 0,2 mL | 0,16 mL | 20 mM |
| 2M Imidazol | 0,3 mL | 0,15 mL | 0,6 mL | 20/30/150 mM |
| 14,3M 2-mercaptoethanol | 12,6 µL | 4,2 µL | 3,36 µL | 6mM |

DNA purification from gel

Kit used: *GE healthcare/ Cytiva:* illustra™ GFX™ PCR DNA and Gel Band Purification Kit.

 **Procedure:**

1. Add 10µL capture buffer (type 3) per 10 mg agarose gel in a DNase free tube
2. Heat the tube up to 60 °C and mix occasional
3. Add 600µL capture buffer (type 3) to/through assembled GFX-microspin, mix and spin 30s at 16.000g, discard flow through. (possible to load more agarose gel, and repeat step 3)
4. Add 500µL wash buffer (type 1) and spin 30s at 16.000g, discard tube and transfer GFX-microspin to a new DNase free tube.
5. Add 10-50µL Elution buffer (type 4 -or 6) let sit for 60s and spin 30s at 16.000g
6. Discard GFX-microspin
7. Measure the DNA-conc., 230/260, and 260/280
8. Store DNA-solution at -20°C

DNA purification from solution

Kit used: *GE healthcare/ Cytiva:* illustra™ GFX™ PCR DNA and Gel Band Purification Kit.

 **Procedure:**

1. Add 500 µL capture buffer to max. 100 µL sample
2. Transfer capture buffer + sample mix into GFX-microspin column and spin 30s at 16.000g. Discard flow-through.
3. Add 500µL wash buffer (type 1) and spin 30s at 16.000g, discard tube and transfer GFX-microspin column to a new DNase free tube.
4. Add 10-50µL Elution buffer (type 4 -or 6) let sit for 60s and spin 30s at 16.000g
5. Discard GFX-microspin
6. Measure the DNA-conc., 230/260, and 260/280
7. Store DNA-solution at -20°C

Ligation (sticky end)

**Procedure:**

1. Add reagents to ligation mix
2. Leave the ligation mix overnight at 16°C
3. Use ligation solution for transformations

| Reagents | Volume |
| --- | --- |
| 10x T4 DNA ligase buffer | 2 µL |
| T4 DNA ligase (add last!) | 1 µL |
| PCR product (cut) of each brick which is to be ligated – **or** 1 part plasmid and 5 part bricks | 5 µL **or** 10 fmol Plasmid, 0, 10 and 20 fmol PCR product |
| H2O | to reach a total volume of 20µL |

While adding digested brick and digested backbone, optimal ligation happens when molar concentration of backbone and brick is approximately the same. Molar concentration is calculated using:
![Equation for optimal ligation](https://static.igem.org/mediawiki/2020/5/53/T--SDU-Denmark--EquationDNADigestion.png)
Cng/µl: concentration in ng/µl
Cfmol: concentration in fmol/µl
nbp: number of base pairs in the fragment of DNA

DNA digestion with FastDigest restriction enzymes

**Procedure:**

1. Add reagents in the order indicated in the table below.

   | # | Reagent | Plasmid DNA | PCR product | Genomic DNA |
   | --- | --- | --- | --- | --- |
   | 1 | Sterile water | x μl (up to total volume) | x μl (up to total volume) | x μl (up to total volume) |
   | 2 | 10x FastDigest or 10x FastDigest Green Buffer | 2 μl | 3 μl | 5 μl |
   | 3 | DNA | Depends on konc. (~1 μg) | Depends on konc. (up to ~0.2 μg) | Depends on konc. (~5 μg) |
   | 4 | FastDigest enzyme | 1 μl | 1 μl | 5 μl |
   | 5 | FastAP (only when digesting backbone) | 1 μl |  |  |
   |  | Total volume | 20 μl | 30 μl | 50 μl |
2. Mix and spin down
3. Incubate at 37°C in a heat block for at least 1 hour.
4. The enzyme can sometimes be inactivated by heating, this is optional)
5. If the 10x FastDigest Green buffer was used, the mixture can be added directly to the gel without loading buffer.- Run the gel and purify the appropriate band protocols for gel electrophoresis and DNA purification from gel. If yield is necessary for next procedure, the gel piece containing the product can be purified using the GE miniprep kit procedure from enzymatic reactions.

PCR amplification

**Reagents for a 20 µL reaction:**

* MilliQ water to a total volume of 50 µL
* 10 µL 2x High Fidelity Polymerase master mix with HF buffer
* 1 µL 10 mM Forward primer
* 1 µL 10 mM Reverse primer
* template (amount depends on concentration)

 **Procedure:**

1. Pipette the largest volume into the PCR tube, followed by the second largest volume and so on, while keeping all reagents not in use on ice.
2. If multiple PCR reactions are made, create a mastermix of all the reagents, which all the reactions have in common, and alliquot the different reagents to their specific tubes.
3. Place into the PCR machine, with following instructions:

   | Step | Temperature | Time (min.) |
   | --- | --- | --- |
   | Step 1 | 98°C | 3:00 |
   | Step 2 | 98°C | 0:15 |
   | Step 3 | 55°C-65°C | 0:30 |
   | Step 4 | 72°C | 0:30 pr. kilobase template |
   | Step 5 | Repeat step 2-4 | x34 |
   | Step 6 | 72°C | 0:15 |
   | Step 7 | 910°C | infinite hold |

Colony PCR

**PCR set up for 1 sample:**
 *Notes: Smaller volume than recommended by Bioline*
Template: 200 ng **or** Template: 0.5 μl of 50 μl sterile water boiled with some of the bacteria colony or mix directly into the PCR tube with the microwaved colony
Primers (20 pmol): 0.2 μl each
Amplicon TAQ polymarase (Red), 2x: 5 μl
Water (sterile): 4.1 μl
Total: 10 μl

**Procedure:**

1. Add 50 μl sterile water to a 1.5 ml tube – one for each sample.
2. Dip a pipette tip in a single colony and mix into an Eppendorf tube.
3. Place the Eppendorf tube in a heating block set at 96 °C for 5 min.
4. Place the samples on ice.
5. Mix primers, water and Taq as described under PCR set up. Be sure that all the samples are in the bottom of the PCR tubes by spinning on PCR tube rotator
6. Place the tubes inside the PCR machine.
7. Start the appropriate PCR program for Taq or design one yourself.
8. Keep at 4-5 °C until use, if more than 2 days waiting time place in -20°C

 **PCR cycling conditions:**

| Step | Temperature | Time (min.) |
| --- | --- | --- |
| Step 1 | 98°C | 3:00 |
| Step 2 | 98°C | 0:15 |
| Step 3 | 55°C-65°C | 0:30 |
| Step 4 | 72°C | 0:30 pr. kilobase template |
| Step 5 | Repeat step 2-4 | x34 |
| Step 6 | 72°C | 0:15 |
| Step 7 | 910°C | infinite hold |

Freezestock

**Procedure:**

1. Add 250 μL of 50 % glycerol to each sterile freezing tubes.
2. Vortex ON culture of bacteria.
3. Add 750 μL ON culture to each of the 3 freezing tubes with glycerol.
4. Pipette up and down to mix.
5. Check register sheet for number for the cultures and write on the tubes

Coincubation and activation of Cas

**Procedure:**

1. After purification, the dilute the Cas proteins and store in protein storage buffer containing (pr 50 mL) with the following:

* 5 ml Tris HCL (pH 7,5; 0,5 M)
* 6 ml NaCl (5 M)
* 2,5 ml glycerol (50%)
* 100 µl DTT (1 M)
* 36,4 ml mQ water
* This was filtered through 0,22 µm 50 ml vacuum filters.

2. Add an appropriate ratio of Cas and gRNA to a tube and incubate for 10 min at 37°C to ensure complex formation.
3. Add target RNA or the testing sample into the tubes. They are now ready for detection using either flow strips or RNase Alert.

Universal lateral flow dipsticks for the detection of biotin and FAM-labeled RNA-reporters

**Procedure:**

1. 100 µl HybriDetect Assay Buffer is added to each reaction tube
2. 20 µl reaction product is added to the tubes along with 1 µl 10 ng/µl biotin and FAM-labeled RNA-reporter.
3. The samples are incubated 10 min at 37°C to ensure cleavage of all RNA-reporters before introducing the flow dipsticks.
4. Dipsticks are added to each reaction tube, incubated for 5 minutes, and then results are interpreted.

RNase Alert Lab Test Kit v2

**Procedure:**

1. 5 µl RNase Alert Lab Test Buffer is added to each sample in one tube of RNase Alert Substrate v2.
2. 45 µl test solution is added to each tube, vortexed, and mixed.
3. For the negative control 45 µl nuclease free water is added instead for test solution and for the positive control 40 µl nuclease free water and 5 µl RNase A is added.
4. The tubes are incubated 30 min to 1 hour at 37°C.
5. The samples are then looked at under fluorescence light (490/520 nm) or by using a plate fluorometer to see the read-out.

Inactivation of RNAse in urine and spit

**Procedure:**

1. Collect sample fluid.
2. Cell lysis. Add 10 µL lysis solution to 50 V urine.
3. Add proteinase K to a concentration of 400 µg/mL.
4. Vortex and spin down three times.
5. Incubate at 55°C for 3 hours.
6. Add PMSF to a concentration of 1 mM.

Plasmid extraction from O/N cultures (GenElute Miniprep)

The protocol from the GenElute Plasmid Miniprep Kit by Sigma-Aldrich was used throughout the project and can be found [here](https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/pln70bul.pdf).

 Upconcentration of samples

**Procedure:**

1. Turn on the compressor.
2. Put the samples in the Speedy Vac with open lid.
3. Close the lid on the Speedy Vac and press run.
4. Vacuum the Speedy Vac by turning the upper green switch to the mark.
5. When the Speedy Vac has run for 15 min. or until the samples are concentrated enough, the vacuum is closed by turning the upper green switch back to start position so air again enters the Speedy Vac.
6. Turn off the Speedy Vac, when it has stopped entering air.
7. Open the Speedy Vac and take out the samples.
8. Turn off the compressor.

Determination of protein concentration

**Procedure:**

1. Make BSA protein standard dilutions of 2.0 µg/mL, 1.5 µg/mL, 1.0 µg/mL, 0.5 µg/mL, 0.25 µg/mL og 0.0 µg/mL (pure milliQ H2O).
2. Dilute 5x Biorad reagent down to 1x.
3. Make a 5x and 10x dilution of protein sample by transferring 5 µL to 20 and 45 µL milliQ water, respectively
4. Prepare an eppendorf tube pr. BSA standard as well as protein sample and add 1 mL 1x biorad reagent to the tubes.
5. Transfer 5 µL of each BSA standard as well as protein sample to the respective eppendorf tubes and incubate at room temperature in 30 minutes.
6. Calibrate a spectrophotometer with a sample of 1 mL pure Biorad reagent.
7. Measure OD595.
8. Make a standard curve and use this to determine protein concentration.

SUMO protease

**Procedure:**

1. Add the following to a RNase free tube.

* Protein of interest contains SUMO-tag 20 µg
* 10X SUMO Protease Buffer +/- Salt 20 μl
* Water to 190 μl
* UMO Protease 10 µL

2. Incubate ≈ 2 hours at appropriate temperature (Cas12a or Cas13a 30°C).
3. Confirm SUMO-cleaving by SDS-PAGE utilizing a suitable gel

In-vitro transcription

This protocol has 4 different subparts and can be performed over several days.
 **Part 1 - Anneal ssDNA oligo's**

1. Make a 2x Annealing Buffer

* 20 mM Tris, pH 7.5-8.0
* 100 mM NaCl
* 2 mM EDTA

2. Mix the following in a PCR tube:

* 20 µL ssDNA oligo #1 (e.g., T7-Scaffold-ERG)
* 20 µL ssDNA oligo #2 (e.g., T7-Scaffold-ERG\_REV)
* 40 µL 2x Annealing Buffer

3. Put it in the PCR machine with following program:

1. 95 °C for 2 min
2. Cool slowly to 25°C over 45 min.
3. Cool to 4 °C for temporary storage.

4. Centrifuge PCR tubes.

 **Part 2 – T7 Transkription with MEGAscript Kit**

1. Mix the following reagents (for small RNAs) in an 1.5 mL Eppendorf tubes (RNase free)

* 2 µL ATP solution
* 2 µL CTP solution
* 2 µL GTP solution
* 2 µL UTP solution
* 2 µL 10x Reaction Buffer
* 250 ng (0.25 µg) Template
* 2 µL Enzyme mix
* Nuclease-free Water à 20 µL

2. Mix Thoroughly
3. Incubate at 37 °C over-night (around 16 hours )
4. Add 1 µL TURBO DNase, **mix well** and incubate 15 min at 37 °C.
5. Keep on ICE or store at -20 or -80 °C.

 **Part 3 – Run RNA transcripts on a 8 % polyacrylamide gel**

1. Cast polyacrylamide gel, and let it polymerize for at least 45 min.
2. Prepare samples by adding RNA loading dye to your samples (5 µL to a 20 µL sample).
3. When gel is polymerized, prepare the gel electrophoresis system, and pre-run the gel for 20 min at 300 V.
4. Clean up your wells with a syringe before loading your samples.
5. Load your samples, and run the gel for 1 h 30 min.
6. Remove the glass plates and transfer the gel to UV-transparent plastic wrap (cover both sides).

 **Part 4 – Extract RNA from gel and precipitate**

1. Put the wrapped-in gel onto a TLC plate and use a UV lamp to detect your RNA bands.
2. Mark your RNA bands, cut out the gel piece and put it into a RNase-free 1.5 mL Eppendorf tube with safe-lock.
3. Cover the gel pieces with 2 M NH4-Acetate (maximum 500 µL) and incubate over-night at 4 °C with shaking.
4. Transfer the NH4-Acetate+RNA solution to 3x volume of 96 % EtOH + 25 µL 3 M NaAcetate (use a RNase-free 2 mL Eppendorf tube). Put it on ice for at least 1 hour (or ON in -20 °C freezer).
5. Spin down a max speed for 45 min, at 4 °C.
6. Carefully remove supernatant.
7. Wash your RNA pellet by adding 150 µL 70 % EtOH (cold) and invert the tube a couple of times (do not resuspend RNA pellet – RNA pellet may loosen itself from the bottom of the tube. Be careful not to lose it).
8. Remove supernatant, and wash again with 150 µL 96 % EtOH (cold) and invert the tube a couple of times (do not resuspend RNA pellet).
9. Remove supernatant, and air dry the pellets.
10. Add RNase-free miliQ H2O (30-100 µL).
11. Measure RNA concentration with Denovix.
12. Store at -20/-80 °C.

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# Team:SDU-Denmark/Results

[Home](https://2020.igem.org/Team%3ASDU-Denmark)

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[Description](https://2020.igem.org/Team%3ASDU-Denmark/Description)
[Results](https://2020.igem.org/Team%3ASDU-Denmark/Results)
[Proof of Concept](https://2020.igem.org/Team%3ASDU-Denmark/Proof_Of_Concept)
[Contribution](https://2020.igem.org/Team%3ASDU-Denmark/Contribution)
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[Poster](https://2020.igem.org/Team%3ASDU-Denmark/Poster)

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[Overview](https://2020.igem.org/Team%3ASDU-Denmark/Team)
[Partnership](https://2020.igem.org/Team%3ASDU-Denmark/Partnership)
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[Inclusion](https://2020.igem.org/Team%3ASDU-Denmark/Inclusion)

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Human Practices

[Overview](https://2020.igem.org/Team%3ASDU-Denmark/Human_Practices)
[Ethics](https://2020.igem.org/Team%3ASDU-Denmark/Ethics)
[Interview](https://2020.igem.org/Team%3ASDU-Denmark/Interview)
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[Gamification](https://2020.igem.org/Team%3ASDU-Denmark/Human_Practices/Gamification)
[Early Detection Game](https://2020.igem.org/Team%3ASDU-Denmark/Human_Practices/Card_game)
[Proposed Implementation](https://2020.igem.org/Team%3ASDU-Denmark/Implementation)

Hardware

[Overview](https://2020.igem.org/Team%3ASDU-Denmark/Hardware)
[Integrated Design](https://2020.igem.org/Team%3ASDU-Denmark/Design)
[Modeling](https://2020.igem.org/Team%3ASDU-Denmark/Model)
[Entrepreneurship](https://2020.igem.org/Team%3ASDU-Denmark/Entrepreneurship)

Videos

[Project Promotion](https://2020.igem.org/Team%3ASDU-Denmark/Promotion)
[Music Video](https://2020.igem.org/Team%3ASDU-Denmark/Excellence)
[Final Presentation](https://2020.igem.org/Team%3ASDU-Denmark/Presentaion)

# Results

Results and future perspectives

## Results

### Assembly of Cas plasmids

![agarose gel showing bands representing correct assembly of both cas12a and cas13a](https://static.igem.org/mediawiki/2020/6/63/T--SDU-Denmark--Cas-assembly.png)

**Figure 1. Confirmation of cas12a+terminator and cas13a+terminator plasmids, by way of agarose gel electroforesis.**  Bands at 2000 bp indicate linear backbone + terminator, bands at 4000 bp indicate linear cas13a or cas12a genes and bands at 6000 indicate linear part backbone + terminator with cas13a or cas12a inserted. Lane 1) Undigested backbone + terminator. Lane 2) Backbone + terminator digested with FastDigest restriction enzyme EcoRI. Lane 3) Backbone + terminator digested with FastDigest restriction enzymes, EcoRI, and SpeI. Lane 4) Undigested backbone + terminator with Cas13a gene inserted. Lane 5) Backbone + terminator with Cas13a gene inserted, digested with FastDigest restriction enzyme EcoRI. Lane 6) Backbone + terminator with Cas13a gene inserted, digested with FastDigest restriction enzyme EcoRI and SpeI. Lane 7) Undigested backbone + terminator with Cas12a gene inserted. Lane 8) Backbone + terminator with Cas12a gene inserted, digested with FastDigest restriction enzyme EcoRI. Lane 9) Backbone + terminator with Cas13a gene inserted, digested with FastDigest restriction enzyme EcoRI and SpeI.

Genes for the Cas12a and Cas13a proteins were assembled through PCR and ligated into a backbone. Cas13a and Cas12a are proteins that require sgRNAs to enable their functions as RNase and DNase, respectively. Successful insertion of *cas13a* and *cas12a* into biobrick [BBa\_B1006](http://parts.igem.org/Part%3ABBa_B1006) (Backbone) was observed on a 1% agarose gel (Figure 1). Backbone was shown, both undigested and digested, using two of the restriction enzymes used in the ligation process. The plasmid digested only with EcoRI was used to show the size of the linear plasmid, while digestion on both EcoRI+SpeI was used together to show the expected length of the plasmid backbone when *cas13a* or *cas12a* is cut out. As seen from the restriction of the [BBa\_B1006](http://parts.igem.org/Part%3ABBa_B1006) backbone with EcoRI and SpeI no part was cleaved out as expected.

Furthermore, *cas13a* or *cas12a* inserted into the backbone is also seen in Figure 1. It can be observed that the band for undigested plasmid is noticeably longer in size than the backbone without an inserted gene. Figure 1 lanes 6 and 9 show the cas-plasmids digested with EcoRI. This shows a single band at 6000 bp, indicating *cas13a* or *cas12a* was inserted into the plasmid. For both *cas13a* and *cas12a*, there are two bands each at 2000 bp and 4000 bp for the plasmids digested with both EcoRI+SpeI. The band at ~2000 bp corresponds to the band of the backbone that was digested with EcoRI+SpeI, showing *cas13a* or *cas12a* being cut out. Likewise, the band at 4000 bp shows the expected length of the gene being approx. 4000 bp.

In conclusion, the *cas13a* and *cas12a* genes have both been inserted into their respective backbones, part [BBa\_B1006](http://parts.igem.org/Part%3ABBa_B1006), as seen in Figure 1.

### Cas protein purification and tag removal

After successful transformations of both Cas12a and Cas13a into *E. coli*  ER2566. The SOP for [protein purification with His-tag](https://2020.igem.org/Team%3ASDU-Denmark/Experiments%20) was followed.
Both transformants were grown to OD600≈0.7 in LB-media with chloramphenicol (50 µg/mL). At OD600≈0.7 both cultures were induced with IPTG 1mM for ~3.5 hours, to then reach anOD600≈1.4. Both cultures were harvested, sonicated, and resuspended in TMN and anion-buffer corresponding to the concentration of ions in wash-buffer.

![SDS-gel protein purification of cas12a](https://static.igem.org/mediawiki/2020/b/bb/T--SDU-Denmark--protein_purification_cas12.png)

**Figure 2. Successful protein purification of Cas12a.** The first lane displays a sample of lysed *E. coli*  ‘ER2536 + Cas12a plasmid’ flow-through before induction of IPTG. The second lane displays a sample of lysed *E. coli*  ‘ER2536 + Cas12a plasmid’ flow-through after induction of IPTG. The third lane displays flow-through from wash-buffer. The fourth lane displays pre-pre-elution-buffer flow-through. The fifth lane displays pre-elution buffer flow-through. The sixth lane displays 0.5 mL elution-buffer flow-through. Lanes 7-12 display 1 mL elution-buffer fractions. In the lines with elution fractions, there is a clear band above 140 kDa correlating well with the molecular weight of Cas12a+SUMO-Histag = 156,31 kDa. Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”; Gel: Bis-Tris 4-12%; Running buffer: MOPS.

![SDS-gel protein purification of Cas13a](https://static.igem.org/mediawiki/2020/1/18/T--SDU-Denmark--protein_purification_cas13.png)

**Figure 3. Successful protein purification of Cas13a.** The first lane displays a sample of lysed *E. coli*  ‘ER2536+ Cas13a plasmid’ flow-through before induction with IPTG. The second lane displays a sample of lysed *E. coli*  ‘ER2536 + Cas13a plasmid’ flow-through after induction of IPTG. The third lane displays flow-through from wash-buffer. The four lane displays pre-pre-elution-buffer flow-through. The fifth lane displays pre-elution buffer flow-through. The sixth lane displays 0.5 mL elution-buffer flow-through. Lane 7-12 displays 1 mL elution-buffer fractions.In the lines with elution fractions, there is a clear band above 140 kDa correlating well with the molecular weight of Cas13a+SUMO-Histag = 153,8 kDa. Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”; Gel: Bis-Tris 4-12%; Running buffer: MOPS.

The following applies both for Cas12a and Cas13a illustrated in Figures 2 and 3 respectively. 1 mL Ni-NTA Agarose (Qiagen) per culture was prepared. Ni-NTA Agarose was incubated with the lysed cells over-night (O/N, approx. 10 hrs). After the incubation, the mixtures were washed (lane 4), pre-pre eluted (lane 5), pre-eluted (lane 6), and eluted (lane 7-12) from the chromatography column.
The elution was made in fractions of ≈ 1 mL. The protein concentration was estimated with [Bradford assay](https://2020.igem.org/Team%3ASDU-Denmark/Experiments%20) . Concentrations for Cas12a (Figure 2) at a range 55-90 ng/mL for fraction 8-11 and Cas13a (Figure 3) at a range 50-80 ng/mL for fraction 8-11 were achieved. Lane 2 is lysate before IPTG-induction and lane 3 is lysate after IPTG-induction.

![SDS-gel protein purification of cas13a](https://static.igem.org/mediawiki/2020/4/49/T--SDU-Denmark--SumoProteaseCas13.png)

**Figure 4. Successful cleaving of SUMO-6x His-tag from Cas13a.**
The first lane displays Cas13a above 140 kDa as expected. The second lane displays cas13a after incubation with SUMO protease following the SUMO-protease assay from Invitrogen. The SUMO-6Xhis-tag hasa molecular weight of 5 kDa. In the second lane, a band is seen at around 140 kDa, fitting relatively well with the cleaving of SUMO-6Xhis-tag, isolation a Cas13a with the molecular weight of 148,8 kDa. Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”Gel: Bis-Tris 4-12% Running buffer:MOPS.

The 6xHis-SUMO-tag was cleaved off using the  [SUMO protease assay from Invitrogen.](https://www.thermofisher.com/order/catalog/product/12588018#/12588018)  Successful cleaving was achieved only for Cas13a (Figure 4).

### *In-vitro* transcription

RNA targets and sgRNAs were produced through *in-vitro* transcription. Products of this were run on a 13% polyacrylamide gel and were shown to all have length of around 64 bases, as seen by UV shadowing (results not shown).

### Cas13a function

We tested the ability of Cas13a to become activated through complementary binding between sgRNA and the RNA biomarker. This was analyzed with the use of ribosomal RNA (rRNA) and agarose gel electrophoresis, where the rRNA is used as a reporter since active Cas13a collaterally cleaves ssRNA leaving it as small fragments.

Purified Cas13a protein ([BBa\_K3602020](http://parts.igem.org/Part%3ABBa_K3602020)) and the *in-vitro* transcribed rs6983267-G SNP gRNA ([BBa\_K3602004](http://parts.igem.org/Part%3ABBa_K3602004)) were mixed for co-incubation in 10 minutes at 37°C, to ensure the formation of Cas13a-sgRNA complex. Different concentrations of sgRNA were used (1.7 ng/ µL, 0.43 ng/µL, 0.043 ng/µL, and 0.0043 ng/µL) to examine the dependency of sgRNA on Cas13a activity. After co-incubation, the matching target RNA ([BBa\_K3602007](http://parts.igem.org/Part%3ABBa_K3602007)) and rRNA were added to a concentration of 0.43 ng/µL and 20 ng/µL respectively. Then these were incubated at 37°C for the collateral cleavage to proceed. Samples were taken out after 15 min, 40 min and 2 hours and put on ice until being examined with agarose gel electrophoresis. The different time steps were included to get an idea of the RNase activity time.

![](https://static.igem.org/mediawiki/2020/f/f0/T--SDU-Denmark--cas13_sgRNA_conc.png)

Figure 5. Proof of Cas13a collateral cleavage being dependent on sgRNA concentration. All samples contain 20 ng/µL rRNA as a marker. Lane 1 and 2 are the positive and negative controls receptively. The positive control contains 1 µg/µL RNase A and the negative only contains rRNA and nuclease-free water. Lane 3-6 were incubated in 15 mins at 37°C, lane 7-19 were incubated for 40 mins and lane 11-14 were incubated for 3 hours. For each time samples with four different sgRNA concentrations were used. Thus, the samples contained 35 ng/µL Cas13a, 0.43 ng/µL target and 1.7 ng/µL. The varying concentration of sgRNA are lane 3, 7, and 11, = 0.43 ng/µL for lane 4, 8, and 12 = 0.043 ng/µL for lane 5, 9, and 13, = for lane 6, 10, and 14 = 0.0043 ng/µL.

When comparing to the negative control (lane 2) in Figure 5, it is seen that all rRNA is degraded to some extent in all other lanes. The rRNA in the positive control (lane 1) is totally degraded thereby leaving no signal. For the samples with Cas13a, sgRNA and target it is observed that the more time the samples were incubated the more rRNA is degraded. This indicates that the prolonged time gives Cas13a more time to degrade rRNA or that the rRNA is degraded because of the heat. Within the different time intervals, it is generally observed that the rRNA in the samples with 0.043 ng/µL sgRNA are most degraded ones. However, it is seen that the 0.0043 ng/µL samples are the less degraded ones besides from the negative control. This indicates, that for Cas13a to be fully activated an amount greater than 0.0043 ng/µL is needed. Thus, Cas13a is dependent on sgRNA binding upon binding to target and then activation.

To be able to conclude if Cas13a is also dependent on the target sequence and thus if Cas13a is only active in the presence of a matching target sequence, a new experiment was set up. In this experiment, the rs6983267-G target RNA and the matching sgRNA was also used. The experiment included two samples with Cas13a, target sequences, and 0.043 ng/µL or 0.0043 ng/µL sgRNA. In this experiment, five controls were included to properly ensure that the rRNA degradation was caused by collateral cleavage from Cas13a. The controls include a positive control with RNase A and four negative controls only with Cas13a (lane 2), sgRNA+target (lane 3), Cas13a+sgRNA (lane 4), and nuclease-free water (lane 7).  Then the samples were incubated at 37°C and aliquots were put on ice after 20 mins and 1 hour before running on the gel electrophoresis.

![](https://static.igem.org/mediawiki/2020/f/fc/T--SDU-Denmark--Cas13_POC_results_page.png)

Figure 6. Proof that Cas13a is activated by the sgRNA and target sequence for rs6983267-G. All samples contain 1 µg/µL RNase A, 40 ng/µL Cas13a or water, 0.043 ng/µL sgRNA, 0.0043 ng/µL target and 20 ng/µL rRNA as a reporter unless other is specified. Lane 1 is a positive control containing 2µL RNase A and 2 µL Tris-HCl. Lane 2 only contains Cas13a and rRNA. Lane 3 contains sgRNA, target and water instead of protein. Lane 4 contains Cas13a and sgRNA. Lane 5 contains Cas13a, 0.043 ng/µL sgRNA and target. Lane 6 contains Cas13a, 0.0043 ng/µL sgRNA and target. Lane 7 is a negative control only containing water and the rRNA. It is seen that Cas13a is only fully activated and cleaves the rRNA when target and sgRNA are also present (lane 5-6). It is also seen that some rRNA is cleaved in the negative controls but not as much as in lane 5 and 6.

The experiment in Figure 6 showed that upon addition of target RNA to the sgRNA-Cas complex solution, collateral cleavage was induced. This can be observed as a smear in lane 5 and 6 thereby indicating degradation of rRNA. It is possible to see some degraded rRNA in all samples. This is assumed to be because the samples were incubated at 37°C for 40 mins as RNA is easily degraded when heated. Lane 4 is without target and was therefore expected to be negative as the Cas protein should not be activated without the matching target. As more RNA is degraded in lane 4 than lane 2 it is expected that Cas13a has intrinsic collateral RNase activity when bound to the sgRNA. The sgRNA might induce conformational change activating the protein. In our PMT this would lead to false positives and it should therefore be optimized. Optimization includes shortening the heating time and optimizing the sgRNA. Heating time can easily be shortened as the results in Figure 5 show collateral cleavage of Cas13a even after 15 mins. By optimizing the sgRNA, the conformational changes might be avoided thus the sgRNA-Cas13a complex is only activated in presence of a target. However, as it is possible to see that lane 5 and 6 are more degraded than the rest, Cas13a depends on sgRNA and target for full activation.

### Cas12a function

To test for the activation and enzymatic activity of Cas12a, we used our Cas13a plasmid as a reporter, both in its plasmid form, but also linearized form through digestion with EcoRI. Cas12a only cleaves ssDNA nonspecifically, so we boiled the dsDNA from the plasmid for 10 minutes at 95°C and cooled the sample down directly on ice water to maintain the integrity of the now ssDNA. After producing the ssDNA, we used a 2% agarose gel to check for enzymatic activity under different conditions.

![](https://static.igem.org/mediawiki/2020/f/fa/T--SDU-Denmark--Cas12_POC_3.png)

**Figure 7. Cas12a activity on 2% agarose gel.** All samples were tested on the rs16901979 C;A genotype and received the same amounts of target and reporter DNA. Lanes 1-4 contain the controls used, where double stranded Cas13a was either treated with DNAse or not and was digested on an EcoRI restriction site and thereafter boiled at 95°C to open the strand. Lanes 5-8 show the samples of either ds- or ssDNA that received 1 µM sgRNA+target and lanes 9-13 contain samples that received 2µM sgRNA+target. Lastly, lanes 13-16 show bands for samples that did not receive any target sequence yet received 2 µM sgRNA. The results of the gel show that Cas12a is actively degrading the reporter DNA in samples that used ssDNA and recevied 2 µM sgRNA+target. This is confirmed by the band exhibited in lane 13, where Cas12a was not active due to the absence of a target sequence.

Here we tested for Cas12a activity using the target sequence ([BBa\_K36002012](http://parts.igem.org/Part%3ABBa_K36002012)) and sgRNAs that we designed for the rs16901979 C;A genotype and measured this through the degree of degraded DNA in the gel. As shown in Figure 7, controls were made consisting of dsDNA+DNase (lane 1), dsDNA without DNase (lane 2), DNA digested on an EcoRI restriction site (lane 3), and ssDNA (lane 4). Two different incubation times were used to illustrate how fast the endonuclease activity is and different concentrations of gRNA were also used, namely 1µM and 2µM sgRNA (lanes 5-16).

It can be observed in lanes 9-12 that Cas12a was only activated when ssDNA was used as a reporter together with 2µM sgRNA and target DNA. No activity can be seen with only 1µM concentration of gRNA. A no-target control was made with 2µM sgRNA found on lanes 13 and 14, which showed no change in ssDNA levels compared to the control with only ssDNA.

Through the ssDNA, we were also able to observe collateral activity of Cas12a, which can cut only if there is a match between the gRNA and dsDNA target sequence. Based on the controls, it can be concluded that it was the target sequence that induced collateral ssDNA cleavage, and not due to foreign DNases.

### RNase inactivation and reintroduction

We tested whether it was possible to inhibit naturally occurring RNases in urine and saliva with proteinase K. This inhibition is especially important for PMT, as RNases would cleave the RNA reporter, giving false positive readouts. Firstly, inhibition of RNases was tested with the RNase Alert kit from ThermoFisher [1]. The kit contains a fluorophore quenched by a quencher attached by an RNA strand. If the strand is cut by an RNase, a signal will be emitted. A saliva and urine sample were each incubated for three hours with proteinase K at a concentration of 400 µg/mL. Afterwards, 5 µL of RNase Alert was added to a 45 µL sample. After 30 min incubation, fluorescence was scanned using the Typhoon FLA 9500 with excitation at 490 nm and emission at 520 nm.

![RNase inhibition results](https://static.igem.org/mediawiki/2020/d/d1/T--SDU-Denmark--RNases_3h_4.png)

**Figure 8. Saliva and urine samples after 3-hour or overnight incubation with proteinase K.**  Controls were not treated with proteinase K and the black spot reflects the light emitted and thus the RNases present.

As the tests show in Figure 8, a 3-hour treatment was effective in removing RNase activity from the samples, as seen from the clear decrease in intensity of the spots. However, some level of RNase activity was still present in the saliva, so a sample of saliva was incubated O/N at 55°C with proteinase K. As seen in Figure 8, the sample did not emit any light after O/N incubation and extensive vortexing, meaning that all RNase activity was halted.

The treated urine and saliva had 1 mM phenylmethylsulfonyl fluoride (PMSF) added to inhibit proteinase K. To ensure that proteinase K activity had been properly neutralized, untreated urine or saliva was reintroduced to the samples. The solution was then incubated for three additional hours and analyzed with RNase Alert. As seen in Figure 9, the inhibition of proteinase K was successful, as the RNase Alert shows a clear presence of active RNases.

![RNAse reintroduction](https://static.igem.org/mediawiki/2020/3/31/T--SDU-Denmark--RNases_reintro_2.png)

**Figure 9. Reintroduction of untreated urine and saliva to previously proteinase K treated urine and saliva with added PMSF to a 1 mM concentration.** The solution was incubated at 55°C for 3 hours after the reintroduction. After the 3 hours, the solution was again mixed with RNase Alert to show if the reintroduced RNases from the urine and saliva were still active.

**Flow strip**
After testing the inhibition of RNases with RNase Alert, we also tested it with the flow strip read-out as this read-out was preferred for the PMT. Samples of urine and saliva were treated with proteinase K and PMSF prior to testing, and were then reintroduced with fresh urine or saliva. For each step, an aliquot was taken, and all were tested on flow strips in the end. The result can be seen in Figure 8.

![Flow strips results with saliva](https://static.igem.org/mediawiki/2020/a/a0/T--SDU-Denmark--flow_strips_with_saliva.png)

**Figure 10. Flow strips after treatment with proteinase K and PMSF.**  All samples contain 20 µL sample (nuclease free water, urine or saliva), 1 pmol RNA-reporter and 100 µL HybridDetect Assay Buffer. 1) positive control containing RNase A and Tris HCL. 2) negative control only containing water, RNA reporter and buffer. 3) untreated urine. 4) urine treated with proteinase K. 5) Urine treated with proteinase K and PMSF. 6) Urine treated with proteinase K, PMSF, and reintroduced urine to ensure inhibition of proteinase K. The same treatments are done for the four saliva strips (7-10). As seen, the RNases in urine are efficiently inhibited by proteinase K, which is again inhibited by PMSF allowing for reintroduced RNases to be active. This is not seen for the saliva as 3 hours with proteinase K did not inhibit the RNases.

As seen in Figure 10, the strips with only urine or saliva are positive, which shows the problem that RNases are already present in the samples. For the urine samples, it is seen that when treated with proteinase K the strip is negative denoting that RNases in urine was effectively inhibited. Treatment with PMSF still results in a negative test, and when urine is reintroduced the test is positive again. Thus, showing that the proteinase K was efficiently inhibited as the reintroduced RNases was still active. The reintroduction of urine reflects the introduction of Cas13a after inhibition of already present RNases.

For the strips with saliva it is seen that all strips are positive. The intensity of the test line is falling with treatment of proteinase K and strengthened when saliva is reintroduced. This indicates that 3-hours was not enough time to inhibit the RNases in saliva fully but that they are generally more inhibited when proteinase K is added.

 **RNAse conclusion**
These results suggest that proteinase K is responsible for inhibition of RNase in urine after 3 hours at 55°C and that the activity of proteinase K can be stopped again after the treatment before addition of Cas13a. Three hours for a home test is a long time and optimization of this step should be done in the future. Optimization includes changing of temperature and time and possibly look more into RNase inhibitors to investigate the possibilities of a better solution.

Inhibition of RNases in saliva is seen when proteinase K treatment is prolonged overnight. This time span is not ideal for a home test and we therefore introduced the use of Cas12a instead of Cas13a when testing saliva. Cas12a cleaves single-stranded DNA rather than RNA and the read-out will not be affected by RNases. Cas12a will be affected by DNases instead and the inactivation of these prior to introduction of Cas12a also has to be investigated.

As seen on the flow strip results, it is possible to detect whether the RNases are active on the Universal Lateral Flow strips [2]. However, the negative tests are seen to show signal at both lines, while the positive tests show signal at one line. In this set-up, it is not a problem since it is still possible to see the difference between the two, as all or most of the RNA reporters are cleaved by the RNases leaving only a signal in the test line of the positive strips. In other experiments, this might not be the case as the RNases might not always cleave all RNA reporters. Therefore, the flow strip read-out needs further optimization. A visible T-line in the negative test is a common problem when combining flow strips with CRISPR/Cas detection. Therefore, many different strategies for eliminating the T-line are possible, as discussed lated in the "Future Perspectives" section [3].

## Future perspectives

Finished products mostly require a great amount of work, and even though our team has been working hard from the beginning of our journey, there is still much work left before PROSTATUS is on the market.

Here you can find a list of things that still needs to be done to realize PROSTATUS . Some of this could very well be solved by a new iGEM team:

Make the flowstrips give a clear result

As of now, our flowstrips give 2 lines on negative sample because of extra gold particles that bind non-specifically to the FAM reporter. The goal is to optimize the flowstrip, so that only one line will appear. This could be done by changing the amount and quality of RNA reporter, increasing the viscosity of the buffer, and/or changing the ratio and dilution of Cas and gRNA.

Generate new biomarkers (for urine and saliva samples)

The biomarkers our team has focused on are biomarkers that are mostly present in Caucasian people. Design of biomarkers, that are more present in other ethnicities and developing the test to other areas of the world, would be optimal.

 Test on urine

When the flowstrips have been optimized the test should be performed on urine from healthy men and women, women with breast cancer, men with benign cancer, men with malignant prostate cancer. All of these should be performed on samples from people with multiple ethnicities.

An application for access to a urine biobank was sent into Palle Osther at “Sygehus Lillebælt”. However, time constraints kept us from gaining access to patient urine samples. The full application can be found  [here](https://static.igem.org/mediawiki/2020/0/0e/T--SDU-Denmark--ApplicationBiobank.pdf).

 Freezedry and compress components into pills

To commercialize our product the different biological components will need to be compressed in an inactive pill form, that will become activated when it comes into contact with the sample fluid.

 Apply the biological aspects in the designed PROSTATUS hardware

When the whole biological process has been optimized in the lab, the test should be performed in PROSTATUS hardware to ensure that the materials do not interfere with any of the biological processes.

 End-user testing of hardware/final product

The test step and hardware components will have to be tested by the end-user. This could be done by observing the end-user implementation with the finalized product, to see whether the end-user can interact with the product the same way we expect them to. A round of interviews would also be an option to hear about the end-users opinions and possible suggestions.
This can also be done by focus group or pre-launch approach.

If the design is intuitive, the component has to go through a design team for manufacturing and assembly process, where the components will have to be standardized and simplified to ensure low production cost.

 Market launch/market implementation

Before the final product can be launched, it will have to be certified and approved by medical institutions. Moreover, the intellectual property rights (IPR) will have to be protected by either patenting, trade secrets, etc. to minimize new entrance threats. Furthermore, the estimated supply chain in the business model canvas will have to be confirmed by forming production and distribution partnerships. Lastly, a marketing strategy should be created and an entry mode should be considered.

## References

[1] ThermoFisher. RNaseAlertTMLab test Kit. From https://www.thermofisher.com/order/catalog/product/4479768#/4479768
[2] Milenia biotec. Hybridetect - Universal Lateral Flow Assay Kit. From https://www.milenia-biotec.com/en/product/hybridetect/
[3] Breitbach, A (2020). Lateral Flow Readout for CRISPR/Cas-based detection strategies. From https://www.milenia-biotec.com/en/tips-lateral-flow-readouts-crispr-cas-strategies/

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