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"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 (15^th-21^st 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 (22^nd-28^th 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.

Week 27 (29^th of June - 5^th 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.


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.


Sunday
Present: AR, FF, KDL, RHA and AMK
While waiting to get our own sequences we performed transformation of plasmid BBa_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.

Week 28 (6^th-12^th of July)

Monday
Present: AMK, KDL and RHA
Transformation of BBa_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 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 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 from 2017

	LB	LB+amp
Top10 + water	50 µL	50 µL
Top10 + BBa_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


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 (13^th-19^th 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.


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.

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 (6^th-12^th 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 (13^th-19^th 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 (20^th-26^th 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 (27^th of July - 2^nd 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.


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.



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.


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.


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.

Sunday
Present: none

Week 32 (3^rd-9^th 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.

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.

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.

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 (10^th-16^th 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.

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.

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.

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.

Saturday
Present: none

Sunday
Present: none

Week 34 (17^st-23^rd 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.

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

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

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.

Saturday
Present: none

Sunday
Present: none

Week 35 (24^th-30^th) 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.

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.

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 (31^st of August - 6^th 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.


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.


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.


Thursday
Present: AMK, FT, KDL
Cas12 group:
Part from 2016, plate 3, 5D is used. This contains part BBa_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.

New CCC were made with CaCl₂ protocol.

Sunday
Present: none

Week 37 (7^th-13^th 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.

• 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.


• cas12 without promoter was not shown here, so another gel with Cas12+promoter and Cas12 v1.2 was run.


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:


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.


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 (6A), BBa_B1003 (4O) and BBa_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:



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.

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 (14^th-20^th 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.

• 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.

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.


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.

• 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.


Cas13a group:
The same EcoRI site is attempted to be added by PCR, yielding the same result as for the Cas12a group.


Saturday
Present: none

Sunday
Present: none

Week 39 (21^st-27^th 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.

• 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.

• 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.


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.


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.


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.


• 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


• 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.

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 (28^th of September - 4^th 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.


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.

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 (5^th-11^th 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.



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:


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.

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.

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:
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Week 42 (12^th-18^th 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.
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.


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:
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Week 43 (19^th-25^th)

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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PROTEIN PURIFICATION

Week 40 (28^th of September-4^thd 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” 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 (5^th-11^th 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” We put the sonicated lysates at –20℃.

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

Week 42 (12^th-18^th 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” 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 (19^th-25^th 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 (28^th of September - 4^th 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.

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.


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.


Friday
Present:
See assembling of cas proteins

Saturday
Present:
See assembling of cas proteins

Sunday
Present:
See assembling of cas proteins

Week 41 (5^th-11^th) of October

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

Week 42 (12^th-18^th 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 (19^th-25^th 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

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