Notebook

1/7
The ordered fragments arrived in powder form and were prepared to reach a concentration of 20ng/µL. The E. coli strain containing our entry vector, pYTK095*, was inoculated in LB media with supplemented chloramphenicol in preparation for extraction.

2/7
MoClo assembly was done on the ordered fragments in accordance with protocol . The two constructs we had designed were assembled in parallel, one tube for the CHI92 gene and one for the SP1 gene. The resulting plasmids were transformed into competent E. coli using heat shock, which were then plated on agar plates with LB and chloramphenicol. The entry vector was extracted from the liquid culture using the GeneJET protocol.

3/7
The plates from the previous day were green-white screened and were shown to only contain white colonies. This could suggest that the MoClo and transformation were both entirely successful, but it is more likely that something went wrong. 3 colonies were harvested and placed in liquid culture to confirm.

4/7
Plasmid extraction
Plasmids from the inoculated cells were extracted using the GeneJET miniprep Kit with the plasmid extraction protocol . Only 1.5mL of the liquid was used, which will be changed to all 5mL in the future to obtain higher plasmid concentration.
The plasmid concentration was analyzed with a NanoDrop and one specimen was discarded due to low concentration. The remaining were combined with a primer provided by a supervisor and sent for sequencing.
Four new colonies from the same CHI92 and SP1 plates were inoculated and will be extracted after the weekend.

This week, we adjusted some of our methods

• The restriction enzymes BsmBI and BcuI were added to the MoClo reaction. Both the ordered vector and pYTK095* have the same resistance gene. By letting the enzymes cut into the ordered vector we are reducing the likelihood that cells without our gene of interest survive
• Instead of extracting plasmids outright, we do colony PCR.
• We scaled up the MoClo reactions to 30µL

7/7
A new MoClo assembly was done according tho protocol . Both of the genes were done in parallel once again. The assembled plasmids were transformed into E. coli which were plated and set to incubate overnight.

8/7
Colony PCR was done on the E. coli colonies from yesterday using colony PCR . A 1% agarose gel was casted in preparation for gel electrophoresis. The gel ran clear, indicating an error in our design. After investigation, it appears like there was a BcuI cut site in the finished construct. Another error might be the primer design. This left us with three options, and we decided to move ahead with all three.

• Order everything again, but this time as a complete construct ready to put into the backbone
• Inoculate colonies, extract the plasmids and hope for the best
• Re-do the MoClo without BcuI, but with BsmBI and BsaI

9/7
MoClo assembly and transformation
A new MoClo assembly was done according to protocol . Both of the genes were done in parallel once again. The assembled plasmids were transformed into E. coli which were set to incubate over night.
After the thermocycler finished the plasmids were transformed into E. coli using heat shock. Two plates for each construct were made, one for a lower concentration of cells and one for a higher concentrations. The plates were left to incubate overnight.

Plasmid extraction
The tubes that were inoculated yesterday were collected and their plasmids extracted in accordance with the GeneJET protocol. The resulting concentrations were high enough for five of the tubes, which were diluted to more usable concentrations and placed in the freezer for storage.

10/7
Result of yesterday's MoClo
The colonies were screened with UV light. From the high concentration plate, one colony containing SP1 was white and 7 containing CHI92 were white. Of the colonies on the low concentration plates, none were visible.

Colony PCR Colony PCR was done on the white E. coli colonies using colony PCR protocol and Saphire polymerase. A template-negative control was included.
Gel electrophoresis
The product from the colony PCR was run through a gel which resulted in no bands, indicating an unsuccessful PCR. The PCR will be redone with other primers.

11/7
Colony PCR
A new round of colony PCR was done with colonies both from the day before and from this day. The protocol was modified in the following ways:

• Used a new master mix called "Phire Master Mix"
• The template DNA was boiled with the CTREAT program

Gel electrophoresis
The gel electrophoresis showed bands for two of the tested colonies. One colony with CHI92 showed a band at 1500bp, which suggested a successful transformation. One colony with SP1 also showed a band, but at around 4000bp which was not expected. The remaining cells from both of these colonies were inoculated for further testing.

14/7
Result of last week's inoculation of Chi92
The inoculation of colonies from Friday showed bacterial growth of the Sp1 protein, but not the Chi92. It is likely that the remaining colony was too small to survive. As a last resort, the area of the colony was swabbed and inoculated. The remaining liquid media with cells transformed with CHI92 were given fresh media and inoculated further. If this fails the MoClo will have to be repeated.
Investigation of the SP1 transformation
We did a plasmid extraction and a GeneJet plasmid purification of the colony that had a band at 4000bp yesterday. A restriction enzyme digestion was run according to protocol with NotI which should result in two fragments. The gel electrophoresis protocol resulted in a single band at 4000bp again. This made it evident that the transformation had failed and that the MoClo would have to be repeated for SP1 as well.

15/7
B. subtilis transformation media
A new culture medium for future transformation of plasmids into B. subtilis was prepared using the
Schaeffer's medium protocol. Inoculation result
The result from yesterday's inoculation of CHI92 cells showed zero growth, so the E.coli culture was discarded.

16/7
Another round of Moclo Assemblies were done for both proteins individually using the MoClo protocol.

17/7
B. subtilis cells were inoculated in 5mL LB media and placed in 37°C over night in preparation for making a glycerol stock for future use.

16/7
The inoculated B. subtilis from yesterday were combined with glycerol diluted with MQ and placed in -80°C for long term storage.

25/7
We received the packages, but the CHI92 construct had a very low concentration of ≤ 0.5ng. To combat this, it was diluted with 10nL MQ amplified with PCR and then transformed into E. coli. The PCR was done in accordance with the PCR protocol with a template-negative control and the transformation into E. coli was done using heat shock.

26/7
The E. coli transformed with CHI92 had not grown, so the procedure was redone with electroshock instead of heat shock according to the electroporation protocol.

28/7
PCR of CHI92
The previous PCRs have been done using Sapphire. Since they have been unsuccessful we switched to Phusion polymerase. A PCR with this polymerase was done on the CHI92 construct, trying both GC buffer and HF buffer. The product was run through a gel together with the Sapphire product from last week. The result was positive, indicating a successful amplification of the plasmid.

Transformation of CHI92
The CHI92 plasmid was transformed into E. coli using heat shock.

29/7
PCR of yeast genome
The PCR of CHS3 from the yeast genome was redone today with 1min 45s extension time (calculated from 30s per kilobases) and an annealing temperature of 62°C. The PCR product will be run on a gel tomorrow.

Plasmid extraction
Plasmid extraction on the inoculated E. coli from yesterday was done according to the GeneJET protocol. The protocol was modified in the following ways:

• Incubated at room temperature for 10min after transferring the solution to the GeneJET spin column to increase plasmid yield.
• Used 30μL of MQ instead of 50μL Elution Buffer for eluting the plasmid, also to increase yield.

The plasmid concentrations were measured with a NanoDrop and ranged from 330ng/μL to 380ng/μL.

Plasmid verification
Restriction digestion was used to cut the plasmids using the FastDigest protocol. Two different restriction enzymes were used: BsmBI (Eco31I) which should cut twice, and BcuI which should cut once.
A gel electrophoresis was done according to the gel electrophoresis protocol and the correct bands seemed to appear. However they were pretty slanted and there were some extra fragments. The gel will be rerun tomorrow at 80V for 45 minutes.

30/7
A new gel electrophoresis of the CHI92 plasmids was done at 80V for 45min. The result was much the same as the previous day; BsmBI cut at two sites and BcuI cut at one site, which was expected. This implies that the colonies we picked not only took up the antibiotic gene from the plasmid, but the CHI92 gene as well.

The PCR product from yesterday was collected and run through a gel. Negative (-Template) controls were included. All specimens, including the controls, seemed to have the same extremely short fragments. The genomic CHS3 will have to be amplified again.

31/7
PCR pf CHS3 and verification
The PCR of CHS3 from the yeast was redone with both Saphire, Phusion, and Phire polymerases. Template-negative controls were included for each polymerase. A gel was cast in preparation for electrophoresis. The gel showed a single band for the Saphire mix at about 3500bp which was the expected length. All of the other columns were empty.

SP1 transformation
Plasmids containing SP1 were transformed into E. coli using heat shock which were left to grow overnight in 37°C on selective plates.

1/8
SP1 transformation
The E. coli containing SP1 that were plated yesterday were placed in the fridge.
CHS3 PCR purification
The PCR product from the Saphire tube that resulted in a band yesterday was purified using the GeneJet purification protocol. It was checked with gel electrophoresis again and the same band at 3500bp appeared.

CHS3 plasmid transformation
The plasmid from the purification was inserted into the pYTK001 backbone using MoClo (PART) and the finished plasmid was transformed into E. coli using heat shock. The cells were plated and left to grow in room temperature over the weekend.

4/8
We began the week by doing a colony PCR for the CHS3 gene both with the high and the low concentrations using the colony PCR protocol. It was realized later that the extension time we used was incorrect.
We still got bands in the gel electrophoresis at the length of our gene of interest for the high concentration group. We still wanted to redo the PCR with the correct PROGRAM to enhance results.

Today we also picked colonies transformed with the SP1 plasmid from the plates that were made on Thursday last week. They were inoculated in 5mL LB medium+kanamycin and incubated in a 37°C shaking incubator overnight.

5/8
The failed colony PCR from yesterday will not be redone since the big size of the fragment may cause problems. Instead, the colonies were inoculated in the hopes that they would work. Tomorrow they will be checked with restriction digestion. Plasmid extraction was done on the SP1 colonies using the GeneJET protocol. These will be mutated to include a stop codon since it had been lost when the gene was produced.

6/8
Plasmids carrying CHS3 were isolated from E. coli colonies using the GeneJET protocol and quantified by NanoDrop (264.6-495.1 ng/μL). For construct verification, the plasmids were digested using the FastDigest protocol with Green buffer. The Eco31I (BsaI) enzyme was predicted to yield three fragments and the SacI was expected to only linearize the plasmid.
Samples (with negative controls for each clone) were run on a gel. As anticipated, BsaI produced three bands and SacI a single band. Although slight lane waviness reduced readability, banding patterns were consistent with the in-silico map.

After confirmation, we performed Mutation PCR to template DNA to remove internal restriction sites using primers 003 and 004.

7/8
CHS3 mutation
A gel was casted according to protocol and was used to run the CHS3 plasmids that were mutated yesterday. It resulted in bands at about 5kb as hoped, but the bands were hard to read. We proceeded with DpnI digestion to remove the unmutated template plasmids and ran the product on another gel. This unfortunately resulted in bands at 3kb instead of 5kb. The result of the NanoDrop was strange, and ranged from -14.1ng/μL to 7.7ng/μL.

After some investigating, it was discovered that the wrong primers had been used to do the mutation. They did not overlap and also lacked the desired mutation, leading to the 3kb bands in the gel.

CHS3 update
Despite the delay, we confirmed today that the CHS3 gene that we ordered a few weeks ago would be delivered tomorrow. This means that MoClo can be done tomorrow before transforming it into E. coli and no more mutation PCR has to be done on it.

SP1 and CHI92 update
On this day, it was also discovered that the SP1 and CHI92 constructs we had ordered lacked stop codons. SP1 requires a point mutation, but CHI92 requires the insertion of an entire stop codon which will impact the temperatures for the mutation PCR. We will probably do a gradient PCR for CHI92 to determine the optimal temperature.

8/8
We received our package with the CHS3 gene, so we proceeded by doing a Moclo assembly according to the MoClo protocol. The parts can be viewed in the Engineering page. This was followed by preparing plates for the plasmid transformation into E.coli, one with amplicilin and two with kanamycin resistance (high and low concentration) and heating them to 37°C. To create the high concentration plate we simply span down the cell mixture and got rid of excess liquid. These plates were then inoculated during the weekend in room temperature.

11/8
Nothing had grown on the CHS3 plates from Friday and they were placed in the 37°C incubator for a while. To be safe, the MoClo assembly was redone using the same parts and protocol. as last time. The finished construct was transformed into E. coli using heat shock and plated on LB+Amp plates and placed in the incubator overnight. Both a plate with a high and a low cell concentration were made. The plates from Friday never grew anything.

12/8
CHS3
Colonies had grown on the plates from yesterday, both white and green ones. 4 colonies from each of the two plates were picked and inoculated in liquid LB and ampicillin overnight.

Stop codon mutation PCR
Mutation PCR was started in order to re-introduce the lost stop codons in both CHI92 and SP1, but two forward primers had accidentally been ordered instead of one forward and one reverse primer for the SP1 gene. This was discovered only after the protocol was started, so it was continued regardless. The primers were diluted to 1muM with sterile MQ and placed in the freezer for future use.
Dilutions of the template DNA for CHI92 and SP1 were made to 1ng/muL and also placed in the freezer.
A gel electrophoresis was done to check the PCR product, with a template-negative control for each of the genes. Both the ladder and the bands looked very odd and smudgy. There seemed to be some primer dimers shorter than 250bp on the CHI92 wells and the control, which was expected. There were none in the SP1 wells which is reasonable since the duplicate primers were not expected to dimerize. This will be redone with the correct primers when they arrive.

13/8
The liquid E. coli culture with CHS3 which was inoculated yesterday was used today. Plasmid extraction using the GeneJET protocol was done for each colony sample, 8 in total. To verify them, restriction digestion was done with either PacI or Esp3I for all the samples. The digestion was done with FastDigest enzymes using the FastDigest protocol. PacI should linearize the plasmids while Esp3I should cut twice, resulting in two fragments.
A gel electrophoresis was run with the 16 samples and ladders on either side. The gel showed a lot more cuts than initially expected. It was discovered that this was due to the use of the wrong primers for gene amplification. The amplification was redone with the correct primers to be checked the next day.

14/8
After testing a few different combinations of agarose concentration, voltage and time we landed on 1% agarose run at 90V for 40 minutes for the CHS3 assembly. The gel resulted in the predicted bands for some of the samples, and these were assembled into the backbone PLE_YTK007 using MoClo. The finished product was placed in the freezer.

15/8
CHS3
The MoClo products from the day before were transformed into E. coli and the cells were plated on LB+Kanamycin plates after an inoculation period of 1h in liquid LB. They were placed in the incubator overnight.

Stop codon mutation PCR
A Mutation PCR was done on CHI92 and SP1 to introduce the stop codons using Phusion polymerase and an HF buffer with two template-negative controls, one for each gene. For CHI92 an entire codon needed to be inserted, making the optimal annealing temperature unknown. Due to this, a gradient PCR was done ranging from 53°C to 63°C with one tube of CHI92 for each temperature. Since SP1 only required a single substitution it was run at 59.1°C annealing temperature, which was the closest temperature we could get to the optimal using the gradient PCR. The extension time was calculated as 25s/kb. The success of the PCR will be checked next week.

18/8
Green-White screening was done on the plated E. coli transformed with CHS3 on Friday and nearly all colonies were white. 2 white colonies from each plate were picked and transferred into liquid LB+Kanamycin. They were placed to incubate overnight.

Colony PCR
More white colonies were picked from the same plates (2 from each). A colony PCR was done following the colony PCR protocol. The PCR products were left in the thermocycler overnight.

Mutation PCR
We checked the mutation PCR product from yesterday using the gel electrophoresis protocol. There was very little liquid left in the tubes, likely due to a gap between the lids and the tubes. Because of this, only 1μL of product was used for the gel electrophoresis, diluted with 4μL MQ and mixed with 1μL loading dye. There were no bands apart from a lot of short fragments.

19/8
The colony PCR from yesterday was run through a gel according to the gel electrophoresis protocol which showed only short fragments, meaning that it has to be redone.
A new Mutation PCR of CHI92 and SP1 was done, since last week yielded no bands. The same gradient of temperatures was used and the tube with SP1 was again placed on the row closest to the optimal temperature. The PCR product was run through a gel, which was pretty hard to read and will be redone tomorrow.

20/8
The gel from yesterday was redone, and indicated that the mutation PCR was unsuccessful and will need to be redone. Next time we will increase the annealing temperature of the gradient PCR and increase the annealing temperature for SP1.
It was discovered that the wrong primers were used for the colony PCR yesterday and there weren't enough of the colonies on the plates to inoculate them again. Plasmid extraction was done again on 6 remaining liquid cultures using the GeneJET protocol and their concentrations measured with a NanoDrop. The concentrations ranged between 178 and 235 ng/muL, which was quite low but expected since we used less liquid culture than usual in case we needed to repeat it again.
The plasmids were linearized with the EcoRI restriction enzyme using the FastDigest protocol at 37°C for 30min. A control was done containing the original backbone pYTK_007. After digestion they were run through a 0.5% agarose gel. Each well showed three bands, which was unexpected since they were only supposed to be cut once. The only exception was the original backbone, which was only linearized. A decision on how to proceed will be done tomorrow with a supervisor.

21/8
A gradient PCR was done for the mutation of CHI92 and SP1 with annealing temperatures ranging from 60C to 70C. The extension time was recalculated to 30s/kb as well to hopefully give better results. A 1% agarose gel was casted for the PCR product which was run as usual. The controls were in a different kind of PCR tube, where the lids are attached in strips. There must have been a gap between the lids and the tubes because they were empty when taken from the thermocycler, likely due to evaporation. The gel showed a lot of bands, some of which could have been our mutated plasmid but we decided to redo the mutation PCR to hopefully get a better result.

A larger volume was used along with the temperature that yielded the best result from this run: CHI92 at 68C and SP1 at 60C. These will be run through a gel tomorrow. Despite the strange bands from yesterday it was decided to send the CHS3 plasmid with the prettiest bands for sequencing. The plasmid was combined with a primer and MQ and sent to Eurofins for sequencing.

22/8
A gel was run with the mutation PCR product from yesterday. There were no bands in the gels, so mutation PCR has to be redone again. This time, extra care was taken to follow the protocol as closely as possible as well as keeping the tubes on ice at all times and foregoing the mastermix. Additionally, the tubes were placed on a pre-warmed PCR machine. SP1 was run at 60C and CHI92 at 68C. After the PCR the samples were run through a gel. This time, good bands at the right size appeared along with some that were assumed to come from primer dimerization. The DNA from the bands were extracted using the GeneJET protocol plasmid extraction and the purified samples were stored in preparation for Gibson assembly and later transformation into E. coli.

25/8
The GenArt Gibson assembly protocol was followed to make the plasmids from Friday circular again. After incubation the plasmids were transformed into competent E. coli using using heat shock which were then plated onto LB+Chloramphenicol plates and left to incubate overnight.

26/8
The plates did not grow as expected and the transformation and plating from yesterday was redone, this time with a higher concentration of the plasmids.

27/8
Colonies grew on the plates from yesterday, so three colonies from each CHI92-containing and SP1-containing plate were picked and inoculated in liquid LB+Chloranphenicol. In preparation for the binding affinity test with chitin beads, a buffer was prepared using:

• 220mL MQ
• 7.3g NaCl
• 0.788g Tris-HCL
• 0.09g EDTA
• 250muL Tween 20

28/8
Plasmids from yesterday’s inoculated E. coli were extracted using GeneJET protocol and placed in the freezer. B. subtilis cells were inoculated in liquid transformation media in preparation for transformation.

29/8
The B. subtilis from yesterday were harvested and their OD450 measured. It was fairly low and they were left to grow further with 1mL fresh transformation media added. After measuring again a while later the OD was still very low and they were left to incubate for even longer. After a discussion with some lab colleagues regarding handling of B. subtilis the inoculation procedure was tweaked. The following procedure will be followed in the future: Plate cells on LB using Quadrant streaking with an inoculation loop and grow over night at 37C Pick a big, well isolated colony and inoculate in 10mL transformation medium overnight at 37C. After growth the OD450 should be 0.5-1 Continue with the transformation protocol. With this in mind, Quadrant streaking was done and placed to incubate.

2/9
B. subtilis cells from the glycerol stock were inoculated in LB media in preparation for transformation.

3/9
The B. subtilis culture from yesterday were placed in the fridge to prevent overgrowth.

4/9
The culture had an OD₄₅₀ of 0.1, so we spun it down at 3000g for 5min. We calculated that we should remove media until we reached a final volume of 1.4mL to reach a better OD₄₅₀ of 0.7 using the following:
OD₁×V₁=OD₂×V₂
→0.1×9900 = 0.7×V₂
→V₂ = 9900/0.7 = 1.4mL
We ended up with an OD₄₅₀ of 0.43 but our supervisor deemed it close enough to proceed with the 1-step transformation protocol. We used the CHI92 plasmid with the highest concentration at 334.4ng/μL. No SP1 plasmids were transformed since there weren't enough cell culture for that.
The transformed cells were plated on LB+chloramphenicol plates and left to grow in the 37°C incubator.

5/9
The plates from yesterday were checked several times over the course of the day, but nothing grew unfortunately.

8/9
B. subtilis was inoculated in LB medium, 2 from the glycerol stock (stored at -80°C) and 2 from the previous colonies. Additional B. subtilis cells were also plated on LB plates in case the liquid cultures don’t grow.

9/9
The plates from yesterday had grown colonies and were placed in the fridge.
OD₄₅₀ measurements were taken of the B. subtilis cells from yesterday. The result was as follows:
Cryo.L: 0.54
Cryo.K: 2.04
Colony.L: 0.64
Colony.L: 1.4
They were deemed high enough to proceed with the transformation. We chose one sample from each of the 4 tubes from the OD measurement. We followed the transform-bsubtilis-2step of B.subtillis. We took 500μL of each sample and placed it in 2mL of Schaeffer's medium in inoculation tubes that were placed in the shaking incubator for 4.5h. After the time had passed, we used a Nanodrop for the three colonies with the SP1 plasmids which had not been done previously. Colony two had the highest concentration (339.7ng/μL) in comparison to C1 and C3 (160.4 ng/μL and 60.5 ng/μL respectively). 3μL of the SP1 plasmid from C2 was added to half of the tubes of cells. The other half of the tubes were combined with 3μL of the CHI92 plasmid from last week (C3). The tubes were placed in a shaking incubator at 37°C for 2.5h.
After 2.5h the cells were plated on selective plates and placed in 37C to grow overnight.
We also started the 1-step transformation protocol again. This was done by taking one colony from each plate from yesterday and placing it in 10mL transformation medium to grow overnight.

10/9
We checked plates from the 2-step transformation and there was unfortunately no growth. We moved on to finishing the 1-step transformation that was started yesterday.
We checked the OD for the inoculated B. subtilis cultures from yesterday:
1: 042
2: 0.8
Those values were close enough to the protocol so genes were introduced. 4 tubes were made, 2 for CHI92 in each of the cultures and 2 for SP1 in each of the cultures. They were left to grow for an hour according to the protocol and were tne plated on LB+chloramohenicol plates.

11/9
Nothing had grown on the plates for the 1step transformation from yesterday. At this point there was no more time for another round of transformation. It was decided that we would send the CHI92 and SP1 plasmids that we extracted from E. coli for sequencing to see if they were assembled correctly.

22/9
Today, two tubes of plasmid DNA were sent to Eurofins for sequencing. One for the CHI92 plasmid and one for the SP1 plasmid.

19/8
The sequencing results came in and were checked on Benchling.