iGEM 2019 — Team MSP-Maastricht

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# Team:MSP-Maastricht/Notebook

ROCKIT

[![](https://static.igem.org/mediawiki/2019/4/47/T--MSP-Maastricht--RocKitLogo.jpg)](https://2019.igem.org/Team%3AMSP-Maastricht)
* Project
  + [Inspiration & Description](https://2019.igem.org/Team%3AMSP-Maastricht/Description)
  + [Background & Design](https://2019.igem.org/Team%3AMSP-Maastricht/Design)
  + [RocKit](https://2019.igem.org/Team%3AMSP-Maastricht/Demonstrate)
  + [Experiments & Results](https://2019.igem.org/Team%3AMSP-Maastricht/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AMSP-Maastricht/Notebook)
* [The Team](https://2019.igem.org/Team%3AMSP-Maastricht/Team)
* Acknowledgements
  + [Collaborations](https://2019.igem.org/Team%3AMSP-Maastricht/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3AMSP-Maastricht/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3AMSP-Maastricht/Sponsors)
* Parts
  + [Overview](https://2019.igem.org/Team%3AMSP-Maastricht/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3AMSP-Maastricht/Basic_Part)
  + [Composite Parts](https://2019.igem.org/Team%3AMSP-Maastricht/Composite_Part)
* [Software](https://2019.igem.org/Team%3AMSP-Maastricht/Software)
* Human Practices
  + [Integrated Human Practices](https://2019.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Education & Engagement](https://2019.igem.org/Team%3AMSP-Maastricht/Public_Engagement)
  + [Outreach](https://2019.igem.org/Team%3AMSP-Maastricht/Outreach)
* Business
  + [Business plan](https://2019.igem.org/Team%3AMSP-Maastricht/BusinessPlan)
  + [Intellectual Property](https://2019.igem.org/Team%3AMSP-Maastricht/IP)

## The Notebook

* [Week 1](#week1)
* [Week 2](#week2)
* [Week 3](#week3)
* [Week 4](#week4)
* [Week 5](#week5)
* [Week 6](#week6)
* [Week 7](#week9)
* [Week 8](#week10)
* [Week 9](#week11)
* [Week 10](#week12)
* [Week 11](#week13)
* [Week 12](#week14)
* [Week 13](#week15)
* [Week 14](#week16)
* [Lab Pictures](#labpictures)

The experiments in the notebook are color coded. The green entries correspond to the artificial introns experiment. The red entries correspond to the enzymatic mutation experiment. The black entries correspond to both experiments.

### Week 1 (July 1 - July 7)

---

* ![]()

  #### Monday

  1 vial of competent DH5-alpha E. coli cells were transformed with the empty pESC-LEU vector backbone for amplification, since we only had a limited supply.
  The transformation was carried out using the standard protocol from New England Biolabs. The transformed cells were plated on agar plates with and without ampicillin and left at 37C overnight.
* ![]()

  #### Wednesday

  2 overnight cultures were made from the transformed DH5-alpha with pESC-LEU. These were made with 5mL of LB broth with ampicillin inoculated with a single colony from the ampicillin agar plate and shaken at 30C overnight. It should be noted that the shaker incubator was broken, otherwise they would have been incubated at 37C.
* ![]()

  #### Thursday

  A miniprep DNA extraction was performed on the 2 overnight cultures. For this, the QIAprep Spin Miniprep Kit and accompanying protocol from QIAgen was used to obtain 2 minipreps of 500uL volume in MiliQ water. In the final elution stage, only 50uL of sterile water should have been added to the column, but due to a pipette mix up, 500uL was added instead, making the minipreps much more diluted than they should be.

### Week 2 (July 8 - July 14)

---

* ![]()

  #### Monday

  Gel electrophoresis with 1% agarose and GelRed in 1x TAE buffer was run to confirm the presence of the plasmid in the minipreps. The plasmid appeared to be present in low concentration in both. Several buffers were also prepared in advance of future experiments, such as SDS-page running buffer, staining and de-staining solutions, Tris-HCl 0.5M and 1M, PBS, 10% SDS and a stock of 50x TAE.

### Week 3 (July 15 - July 21)

---

* ![]()

  #### Tuesday

  The DNA concentration of the 2 pESC-LEU minipreps was measured with a plate reader and the concentration was found to be too low to measure. At this time, the previous mistake of adding 500uL instead of 50uL was discovered. The mistake was rectified by repeating the QIAprep protocol from the step were buffer N3 (neutralisation buffer) is added to the cell lysate (in this case, our diluted miniprep). Instead of centrifuging to obtain a pellet and transferring the supernatant to the column, the solution was transferred directly and the rest of the protocol was followed normally to obtain a 50uL elution. The concentration of DNA was measured again and was found to be 31ng/mL and 48ng/mL in samples 1 and 2 respectfully.
* ![]()

  #### Wednesday

  The Gal4 gene with and without introns was assembled into pESC-LEU from miniprep 2 and transformed into DH5-alpha E. coli for amplification.
  The New England Biolabs Gibson assembly kit and transformation protocol was for this procedure. The vector was first linearized with BamHI. It was calculated that 20.8uL of miniprep 2 were needed in the reaction volume of 50uL to obtain 1ug of plasmid.
  Since both the intron and the wild type Gal4 were ordered as gBlocks of 4 fragments, it was decided that 0.025pmol of each fragment would be used. The volumes needed to obtain this amount for each fragment were calculated and 2 Gibson assemblies were performed, 1 with introns (INT) and 1 without (WT).
  2 vials of competent DH5-alpha E. coli cells were transformed with 2uL from each of the assembled products and plated with and without ampicillin to incubated overnight.
* ![]()

  #### Thursday

  The transformations from both assemblies were successful, giving small, but multiple colonies on the ampicillin plates. 2 overnight cultures of 5mL of LB broth with ampicillin were inoculated with 10 randomly selected colonies each. This was due to an error in the communication of instructions: instead of 10x 5mL cultures each inoculated with a single colony, 2 cultures were inoculated with 10 from only the Gal4 INT plate.
* ![]()

  #### Friday

  2 miniprep extractions were performed on the overnight cultures. After elution, 5uL of the minipreps were removed and linearized with 1uL BamHI and 1uL XbaI to a reaction volume of 20uL to cut out the Gal4 INT fragment. A 1% agarose gel with 1x TAE was run with both the cut and the uncut miniprep DNA to test for the presence of the correct fragment. Although a fragment of the right size was detectable, there were multiple other unexpected fragments, and the previous mistake of taking too many colonies was realised. It was agreed that the miniprep needed to be repeated to properly confirm the results.
  10x 10mL LB-Amp cultures were inoculated with single colonies from the Gal4 INT plate, and 10 were made with colonies from the GGal4 WT plate to give a total of 20 overnight cultures. These were left at room temperature since it was a Friday and they needed to grow over the weekend.

### Week 4 (July 22 - July 28)

---

* ![]()

  #### Monday

  Miniprep extractions of all 20 overnight cultures were performed (10 INT and 10 WT). Again, 5uL of each were linearized with BamHI and XbaI to cut out the fragment. The 1% agarose gel was run with 1x TAE buffer with the cut and uncut version of each sample against a 1kb ladder and the samples that contained the correct fragment were identified.

  Gibson Assembly of dCas9-FokI into pESC-LEU vector:
  The dCas9-FokI gene was split into 5 fragments (F1, F2, F3, F4, F5) which were ordered as gBlocks from IDT. Fragments were resuspended to a concentration of 10ng/µL according to IDT protocol. pESC-LEU vector was linearized with BamHI-HF restriction enzyme. The linearized plasmid and the fragments were added together in the Gibson Assembly reaction mixture so that the total amount of DNA was 0.2 pmol with each fragment present in the same amount. Assembly was carried out according to New England Biolabs (NEB) protocol. Competent DH5α E. coli cells were transformed with the assembled pESC-LEU\_dCas9-FokI according to the NEB protocol and plated on LB agar Amp+ plates (2 plates: G1 and G2).
* ![]()

  #### Wednesday

  All the plates, broths and chemical buffers needed for the yeast transformation were prepared. This included YPD liquid media, SD media without leucine, SD media without uracil, YPD agar, SD agar without leucine and SD agar without uracil. Since the autoclave was discovered broken that day, the materials were left until it could be fixed.

  Made 10x overnight (ON) cultures from G1 (5x) and G2 (5x).
* ![]()

  #### Friday

  All materials for yeast transfection were autoclaved and the agar plates were poured. A streak plate of MaV203 yeast cells was made on YPD agar, and a YPD liquid culture of 15mL was inoculated with yeast from the MaV203 stock. Although it would have been more ideal to grow colonies on the streak plate and make the liquid culture from a single source, the time constraint was such that an extra day would have been lost.

  Plasmid DNA was extracted from the ON cultures (G1, G2) using Qiagen MiniPrep Kit. The extracted DNA was digested with BamHI-HF and XbaI. Digested and undigested DNA samples were subjected to electrophoretic analysis on a 1% agarose gel. All samples seemed to show that the gene was correctly assembled in the plasmid (further analysis will reveal this to not be true). Sample 4 (S4) from G1 showed to have the highest concentration of DNA (brighter luminescence on the gel).

### Week 5 (July 29 - August 4)

---

* ![]()

  #### Monday

  The OD600 reading for the liquid yeast culture left to grow over the weekend was 1.3. This was diluted 1mL into 14mL fresh YPD to give an OD600 of 0.15. This was left to grow throughout the day for approximately 6 hours with OD600 readings taken every 30 minutes. At the end of the day, the OD600 was only 0.41, which was not high enough to perform the transfection, therefore the culture was left to grow at room temperature overnight.
  Solutions of 50% PEG, 1M and 0.1M lithium acetate were also made and sterilised during this time.

  Competent DH5α E. coli cells were transformed with S4 DNA sample (pESC-LEU\_dCas9-FokI) according to the NEB protocol and plated on a LB agar Amp+ plate.
* ![]()

  #### Tuesday

  The OD600 of the culture in the morning was 1.56. This was diluted only by 50% (7.5mL of culture into 7.5mL fresh YPD) to give an OD600 of 1.12, then again 10mL into 5mL YPD to give 0.66. This was left to grow for 2 generations (about 2 hours), then pelleted in a centrifuge for 10 minutes at 2000rpm. The transformation mixture was prepared with 260uL 50% PEG, 36uL 1M LiOAc and 10uL of 2ug/mL salmon DNA. The cell pellet was washed with 7.5mL sterile water, re-pelleted, and washed again with 7.5mL of 0.1M LiOAc. It was then pelleted again, re-suspended in 300uL of 0.1M LiOAc and transferred to a microcentrifuge tube for pelleting at 8000rpm. The pellet was then suspended in 33uL of DNA from the miniprep containing the Gal4 INT plasmid, then mixed with 306uL of transformation mix. This was incubated at 30C for 30 minutes then heat shocked at 42C for 17 minutes. The mixture was then centrifuged at 8000rpm for 2 minutes and the supernatant was removed. The cells were re-suspended in 200uL of sterile water and plated on 1 YPD plate (control) and 1 SD -Leu (selection) plate. These were grown at 30 degrees overnight.

  Made 2x 5mL ON cultures from pESC-LEU\_dCas9-FokI plate.
* ![]()

  #### Wednesday

  The yeast transformation appeared unsuccessful as there was no visible growth on either the control or selection plates. Another vial of competent DH5-alpha was transformed with the remaining 2uL of miniprep DNA with Gal4 INT and 2x 5mL YPD cultures were inoculated with single yeast colonies from the streak plate. The pH of the TE buffer used to dissolve the salmon DNA for the yeast transformation was tested, and it was discovered to be at pH 6 instead of the desired pH 8. Drops of NaCl were added to raise the pH to 8 for subsequent transformations.

  Plasmid DNA was extracted from the ON cultures (E1, E2) using Qiagen MiniPrep Kit. The extracts were quantified on a Synergy HTX Multi-Mode Reader using UV-VIS spectrometry A260/280 (E1=33.6 ng/uL; E2=42.6ng/uL). The extracted DNA was digested with BamHI-HF and XbaI. Digested and undigested DNA samples were subjected to electrophoretic analysis on a 1% agarose gel. Again, on first analysis the plasmid seemed to contain the gene; further analysis will prove that to be wrong [19.09].
* ![]()

  #### Thursday

  Another yeast transformation was attempted with the Gal4 INT DNA. The overnight cultures were back diluted to an OD600 of approximately 0.5 and grown for 2 hours to an OD600 of about 0.75. The same procedure detail on Tuesday 30thwas followed, but this time only 5uL of miniprep DNA was used. 2 transformations were performed, 1 using salmon DNA in pH 8 TE for the transformation mix and 1 using pH 6, to see if there would be any difference. 2 more 5mL YPD cultures were made, with the intention of using them for the Gal4 INT if this transformation was unsuccessful or repeating the same protocol with Gal4 WT if it was.
  2 5mL overnight cultures of LB-amp were made from 2 colonies of DH5-alpha transformed with Gal4 INT from the miniprep.
* ![]()

  #### Friday

  The yeast plates from the previous day did not have distinct colonies, but the control plates showed some smearing. It was decided to leave them in the incubator for a few days longer since yeast have a slower generation time that E. coli. Due to time constraints, the overnight YPD cultures were only back diluted to an OD600 of 0.7 and grown for 90 minutes. The yeast transformation protocol from the previous day was repeated.
  2 miniprep extractions were performed on the transformed DH5-alpha with Gal4 INT, but this time 1mL of each LB culture was saved to make a glycerol stock. After extraction, the samples were digested with BamHI and XbaI and tested for the presence of the fragment on a 1% agarose gel.

### Week 6 (August 5 - August 11)

---

* ![]()

  #### Monday

  It was found after the weekend that all yeast plates with Gal4 INT had produced colonies. The plate with the Gal4 WT had not, but since this transformation was done after all the others it was left in the incubator to have more time to grow. Another transformation of E. coli was performed with the remaining Gal4 WT miniprep to amplify it and make a glycerol stock of the cells containing it.
  For the testing of the Gal4 INT introns, several drop out cultures were made. Three types of liquid cultures were inoculated: YPD for the positive control, SD without Leu for selection of the plasmid and SD without Ura for testing if the introns were working. Each of these types of cultures were inoculated either with colonies from the Gal4 INT plate or with wild type yeast as a control. Gal4 INT and wild type yeast were also streaked onto SD -Ura plates as a secondary growth test.
* ![]()

  #### Tuesday

  All of the Gal4 INT cultures in YPD and SD -Leu had grown overnight. The SD -Ura cultures did seem to have grown, but significantly less than the positive control, though it was expected that they would grow slower due to the introns needing to be spliced. The wild type yeast grew in YPD but not in SD -Leu or -Ura, which was expected. All cultures were left in the incubator for another night. At this time, the Gal4 WT selection plate had small colonies, and these were used to make drop out cultures of the GAL4 WT too.
  2x overnight cultures in 5mL LB-amp were made from the transformed DH5-alpha with Gal4 WT.
* ![]()

  #### Wednesday

  Miniprep extractions of the Gal4 WT were performed, saving 1mL of overnight culture for a glycerol stock. This was also restricted with BamHI and XbaI and tested on a gel.
  The -ura yeast cultures seemed to be growing but were left for a few more days to be sure
* ![]()

  #### Friday

  The OD600 readings of all the liquid cultures were measured, and the results were collected in the table below.

  | Sample | SD-Leu | SD-Ura | YPD (Control) |
  | --- | --- | --- | --- |
  | Gal4 INT no1 | 1.913 | 0.364 | 2.665 |
  | Gal4 INT no2 | 1.889 | 0.299 | 2.349 |
  | Gal4 INT no3 | 1.849 | 0.196 | 2.309 |
  | Gal4 WT | 1.814 | 0.139 | 2.155 |
  | Wild Type (no Gal4) | 0.1 | - | 2.240 |

  From this is was confirmed that the yeast cells transformed with Gal4 INT were still able to grow in -Ura, if not as efficiently as they can in YPD. The lower value with the Gal4 WT in -Ura could be accounted for by the extra day that the Gal4 INT cultures had to grow.

### Week 7 (August 26 - September 1)

---

* ![]()

  #### Thursday

  Overnight cultures of plated DH5-alpha transformed with pESC-Leu were made.
* ![]()

  #### Friday

  Gibson assemblies

  **dCas9-FokI\_CSY4**

  The dCas9-FokI gene required an additional fragment necessary for its functioning (F0). F0 was ordered as a gBlock from IDT and resuspended according to their protocol. pESC-LEU\_dCas9-FokI plasmid was linearized (LP) with BamHI-HF. A Gibson Assembly reaction mixture was prepared containing LP and F0 in equal amounts and the assembly was carried out according to NEB protocol.

  **dCas9-AID**

  The fragment containing AID was ordered as a gBlock from IDT and resuspended according to their protocol (A1). pESC-LEU plasmid was linearized (LP) using BamHI-HF. A reaction mixture containing LP, A1, F2, F3, F4, F5 in equal amounts was prepared. Gibson Assembly was performed as detailed by NEB protocol.

  **dCas9-eAID**

  The fragment containing eAID was ordered as a gBlock from IDT and resuspended according to their protocol (E1). pESC-LEU plasmid was linearized (LP) using BamHI-HF. A reaction mixture containing LP, E1, F2, F3, F4, F5 in equal amounts was prepared. Gibson Assembly was performed as detailed by NEB protocol. The assembled plasmid (E) was stored in the -20°C freezer.
  The assembled plasmids containing pESC-LEU\_dCas9-FokI\_CSY4 (F), pESC-LEU\_dCas9-AID (A) and pESC-LEU\_dCas9-eAID (E) were stored in the -20°C freezer.

  * ![]()

    Miniprep extractions were performed on the overnight cultures to obtain more plasmid. Various materials such as more LB broth, plates and SOC media were prepared.

### Week 8 (September 2 - September 8)

---

* ![]()

  #### Monday

  Competent DH5α E. coli cells were transformed with plasmids F, A and E according to the NEB protocol and plated on a LB agar Amp+ plates and on standard LB agar plates for control.
* ![]()

  #### Wednesday

  Plates were checked for colonies. Only the plate with dCas9-FokI (F) showed a very small growth. The others were completely empty. 2x ON cultures (F1, F2) from plate F were made.
* ![]()

  #### Thursday

  Plasmid DNA was extracted from cultures F1 and F2 using Qiagen MiniPrep Kit. The extracts were quantified on a Synergy HTX Multi-Mode Reader using UV-VIS spectrometry A260/280 (F1=1.29 ng/uL; F2=1.497 ng/uL).
* ![]()

  #### Friday

  Extracts F1 and F2 were digested with BamHI-HF and XbaI according to NEB protocol and analysed on a 1% agarose gel. No results were visible on the gel.

### Week 9(September 9 - September 15)

---

* ![]()

  #### Thursday

  Plasmids pESC-LEU (PL) and pESC-LEU\_dCas9-FokI (FL) were linearized with BamHI-HF according to NEB protocol and stored for subsequent use in Gibson assembly.

  3x ON cultures of colonies containing pESC-LEU\_dCas9-FokI plasmid (F1,F2,F3) were prepared as well as 3x ON cultures of pESC-LEU plasmid (P1, P2, P3).
* ![]()

  We decided to test the introns again after changing the sequence slightly. We took this opportunity to also test if they would still work with a higher number of intron pairs. New Gal4 gBlocks for each fragment were ordered, each with 2 introns inserted, so that different combinations of intron-containing and wild type fragments could be combined to form Gal4 assemblies with 2, 4, 6 or 8 introns.
  Gibson assemblies were carried out for each of these combinations using the standard protocol.
* ![]()

  #### Friday

  Plasmid DNA was extracted from cultures F1, F2, F3, P1, P2, P3 using Qiagen MiniPrep Kit. The extracts were quantified on a Synergy HTX Multi-Mode Reader using UV-VIS spectrometry A260/280 (F1=38 ng/uL; F2=74 ng/uL; F3=71 ng/uL; P1=93 ng/uL; P2=94 ng/uL; P3=39 ng/uL).

  Gibson assemblies of dCas9-FokI, dCas9-AID and dCas9-eAID were performed, as previously described [30.08].

  The assembled plasmids containing pESC-LEU\_dCas9-FokI\_CSY4 (F), pESC-LEU\_dCas9-AID (A) and pESC-LEU\_dCas9-eAID (E) were transformed into competent DH5α E. coli cells following NEB protocol and subsequently plated on LB Agar Amp+ plates for overnight incubation at 37°C.
* ![]()

  All 4 assemblies prepared the previous day were transformed into competent DH5-alpha cells and plated on LB agar.

### Week 10 (September 16 - September 22)

---

* ![]()

  #### Monday

  Plates were checked for growth, but no colonies were observed.
* ![]()

  #### Thursday

  No colonies were growing on any of the transformation plates after the weekend, indicating the transformation was unsuccessful. The cells were plated again with 200 instead of 100uL.
* ![]()

  #### Tuesday

  Gibson assemblies of dCas9-FokI, dCas9-AID and dCas9-eAID were performed, as previously described [30.08].

  The assembled plasmids containing pESC-LEU\_dCas9-FokI\_CSY4 (F), pESC-LEU\_dCas9-AID (A) and pESC-LEU\_dCas9-eAID (E) were transformed into competent DH5α E.coli cells following NEB protocol and subsequently plated on LB Agar Amp+ plates for overnight incubation at 37°C.

  SOC media was prepared as well as 24 LB Agar Amp+ plates.
* ![]()

  The new plates still showed no growth, so it was decided that the most likely reason was that the Gibson assembly had failed. The assemblies were performed again for each of the Gal4 intron variations after re-calculation of molar ratios and transformed into competent DH5-alpha cells.
* ![]()

  #### Wednesday

  Plasmid DNA containing pESC-LEU\_dCas9-AID was extracted using Qiagen MiniPrep Kit. The extracts were quantified on a Synergy HTX Multi-Mode Reader using UV-VIS spectrometry A260/280 (A=96.87 ng/uL).
* ![]()

  There was still no growth on the transformed Gal4 plates, except for 1 very large colony on the Gal4 6int plate from the first transformation on 13.09. An overnight culture was made from this single colony. More competent cells were prepared from a plate of un-transformed DH5-alpha for future transformations, using a standard CaCl2 protocol. Overnight cultures for glycerol stocks were also made in LB with ampicillin.
* ![]()

  #### Thursday

  The competent cell protocol was continued for day 2. A miniprep extraction was performed on the overnight culture from Gal4 Int6, but when the DNA concentration was measured it was near 0 and indicated no plasmid. It is therefore likely that the single colony was due to contamination after the plate was left in the incubator for a few days.
* ![]()

  #### Friday

  Digestion with BamHI-HF and XbaI followed by electrophoretic analysis was performed on the following samples:
  GAL4-8introns (extracts 1, 2 and 3); pESC-LEU plasmid (extracts 1, 2 and 3); pESC-LEU\_dCas9-FokI plasmids (extracts 1, 2 and 3); pESC-LEU\_dCas9-AID.
  All GAL4 and AID DNA was degraded. pESC-LEU and dCas9-FokI plasmids showed very similar bands, suggesting that the latter does not actually contain the dCas9-FokI gene.

### Week 11 (September 23 - September 29)

---

* ![]()

  #### Wednesday

  In order to check if pESC-LEU and pESC-LEU\_dCas9-FokI were actually the same, they were linearized with BamHI-HF and NcoI. Electrophoretic analysis showed that in fact they were the same and that the dCas9-FokI gene was never actually assembled in the pESC-LEU plasmid.
* ![]()

  #### Friday

  24 LB Agar Amp+ plates were prepared

### Week 12 (September 30 - October 6 )

---

* ![]()

  #### Monday

  Gibson assemblies of dCas9-FokI, dCas9-AID and dCas9-eAID were performed, as previously described [22.07 for dCas9-FokI, 30.08 for AID and eAID].
* ![]()

  In a last attempt at transformation, the assemblies were re-transformed into the freshly prepared competent cells to see if it was the cell line that was the problem.
* ![]()

  #### Friday

  The pESC-Leu vector was linearized with either BamHI-HF or SpeI-HF. The assemblies of dCas9-AID dCas9-eAID were re-done
* ![]()

  The assemblies of Gal4 with 2, 4, 6 and 8 introns were re-done.

### Week 13 (October 7 - October 13)

---

* ![]()

  #### Monday

  The assemblies from 07.10 were transformed into competent DH5-alpha cells.
* ![]()

  #### Wednesday

  Nothing had grown on the transformation plates after 2 days at 37C. It was suspected by this point that the plasmid backbone may be the problem, as it was the only common feature between all the assembly products.

### Week 14 (October 14 - October 20)

---

* ![]()

  #### Wednesday

  A transformation of Gal4 8int was performed with a different type of E. coli, BL21, in a last attempt to get any colonies.
* ![]()

  #### Thursday

  Nothing had grown on the plate. A new Gibson assembly of AID and eAID was performed with double the fragment/plasmid ratio to send to the Wageningen iGEM team for our scientific collaboration, to see if they had any success with different transformation protocols.

### Laboratory Pictures

---

![](https://static.igem.org/mediawiki/2019/4/4c/T--MSP-Maastricht--Lab_Image_1.jpeg)

![](https://static.igem.org/mediawiki/2019/0/08/T--MSP-Maastricht--Lab_Image_2.jpeg)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:MSP-Maastricht/Experiments

ROCKIT

[![](https://static.igem.org/mediawiki/2019/4/47/T--MSP-Maastricht--RocKitLogo.jpg)](https://2019.igem.org/Team%3AMSP-Maastricht)
* Project
  + [Inspiration & Description](https://2019.igem.org/Team%3AMSP-Maastricht/Description)
  + [Background & Design](https://2019.igem.org/Team%3AMSP-Maastricht/Design)
  + [RocKit](https://2019.igem.org/Team%3AMSP-Maastricht/Demonstrate)
  + [Experiments & Results](https://2019.igem.org/Team%3AMSP-Maastricht/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AMSP-Maastricht/Notebook)
* [The Team](https://2019.igem.org/Team%3AMSP-Maastricht/Team)
* Acknowledgements
  + [Collaborations](https://2019.igem.org/Team%3AMSP-Maastricht/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3AMSP-Maastricht/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3AMSP-Maastricht/Sponsors)
* Parts
  + [Overview](https://2019.igem.org/Team%3AMSP-Maastricht/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3AMSP-Maastricht/Basic_Part)
  + [Composite Parts](https://2019.igem.org/Team%3AMSP-Maastricht/Composite_Part)
* [Software](https://2019.igem.org/Team%3AMSP-Maastricht/Software)
* Human Practices
  + [Integrated Human Practices](https://2019.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Education & Engagement](https://2019.igem.org/Team%3AMSP-Maastricht/Public_Engagement)
  + [Outreach](https://2019.igem.org/Team%3AMSP-Maastricht/Outreach)
* Business
  + [Business plan](https://2019.igem.org/Team%3AMSP-Maastricht/BusinessPlan)
  + [Intellectual Property](https://2019.igem.org/Team%3AMSP-Maastricht/IP)

## Experiments

### & Results

* [Experiment 1: Introns](#Experiment1)
* [Experiment 2: Mutations](#Experiment2)
* [Experiment 3: Receptor](#Experiment3)
* [Protocols](#Protocols)

### Experiments

#### Introns

---

Before continuing with other experiments, we first needed to ensure that the artificial introns containing the binding sites for the dCas9 complexes worked. In other words, we needed to make sure they were recognised by the yeast cells as being introns and spliced out of the mRNA transcript to give the correct sequence for the receptor.
We first constructed a basic sequence for our artificial introns. The skeleton sequence was taken from Gatermann et al (1989):

5’ – GTAGGT(19N)CTAAT(4N)AG – 3’

Since the introns needed to contain sgRNA binding sites, these were inserted into the 19N section and the 4N section was removed. The sgRNA sequences including the PAM sites inserted into a pair of introns were:

5' TCTAACCCCCACCTCCTGTTCGG 3' and 5' GGCAAGCGCCATGTCTAGTGAGG 3'

To prove the introns worked, we designed a proof of concept experiment using the Gal4 transcription factor protein. A pair of artificial introns were inserted into the gene encoding the Gal4 with Gibson assembly. The Gal4 protein was ordered as 4 gBlocks, 1 of which was ordered as 2 different versions, one with and one without the intron pair. This was to allow us to assemble a wild type Gal4 as a control, as well as the one containing the intron pair. The strain of yeast that we used, MaV203, is a Gal4 knockout, so it does not contain its own copy of the gene. The Ura3 gene, which encodes an enzyme involved in pyrimidine ribonucleotide synthesis, is under the control of a Gal4 promotor and can therefore only become active when a functioning Gal4 is present. This strain of yeast, when un-transformed, can only grow if there is uracil added to the media. The vector we used to insert the Gal4 was the pESC-LEU plasmid, and therefore after transforming the yeast we were able to select for the plasmid by growing the cells on SD agar plate deficient in Leucine. Once we knew the transformation was successful, we inoculated liquid SD -Ura cultures with single yeast colonies. These cultures were deficient in uracil, and therefore the cells should only have been able to grow if there was a functioning Gal4. Yeast growth in this media therefore indicated that the Gal4 we had inserted was functioning, implying that the introns were being properly spliced out of the transcript.

##### Results The yeast containing wild type and intron Gal4 were grown in 3 types of liquid media, YPD (non-selective), SD -Leu (selective for the plasmid) and SD -Ura (selective for the Gal4), as well as on YPD and SD -Ura plates. After 4 days, the YPD plates and liquid cultures showed a lot of growth, as expected, as did the SD -Leu cultures. The SD -Ura cultures started to show visible cloudiness after 4 days, and colonies appeared on the plates after 5. The OD600 readings for the liquid were taken after 5 days and are shown in the Table below. | Sample | SD-Leu | SD-Ura | YPD (Control) | | --- | --- | --- | --- | | Gal4 INT rep1 | 1.913 | 0.364 | 2.665 | | Gal4 INT rep2 | 1.889 | 0.299 | 2.349 | | Gal4 INT rep3 | 1.849 | 0.196 | 2.309 | | Gal4 WT | 1.814 | 0.139 | 2.155 | | Wild Type (no Gal4) | 0.1 | - | 2.240 |

#### Mutations

---

The aim of this experiment is to characterize the type and amount of mutations generated by the three different synthetic mutagenic enzymes across different time-frames and against varying lengths of target sequence, to determine the best enzyme and the best overall mutagenic conditions.

Three mutagenic enzymes are being tested, as described in the Components section: dCas9-FokI, dCas9-AID and dCas9-eAID (hereafter FokI, AID and eAID respectively). Their genes and the gRNA expression cassettes are assembled within the pESC-LEU plasmid vector as described in the Lab Notebook Section. The genes are placed under the control of the inducible GAL1,10 promoters, whose expression is regulated by the presence of galactose.

The target gene is a synthetic version of GAL4, containing synthetic introns within its sequence which in turn contain the gRNA complementary sequence that allows for FokI, AID and eAID to precisely target their mutating activity. A total of four pESC-LEU plasmids are prepared with four different versions of the GAL4 gene -as described in the Lab Notebook Section- each of which contains either 2, 4, 6, or 8 synthetic introns inserted at varying locations throughout the gene.

S. cerevisiae (MaV203 strain) cells are transformed with a combination of 2 plasmids, containing either one of the three enzymes and either one of the four target genes, so that each enzyme is tested against all four versions of GAL4:

* FokI + GAL4-8int
* FokI + GAL4-8int
* FokI + GAL4-4int
* FokI + GAL4-2int

* AID + GAL4-8int
* AID + GAL4-6int
* AID + GAL4-4int
* AID + GAL4-2int

* eAID + GAL4-8int
* eAID + GAL4-6int
* eAID + GAL4-4int
* eAID + GAL4-2int

The transformed cells are plated on SD-LEU plates for selection of correctly transformed colonies and on YPD plates as controls.

The selected colonies are then transferred to LEU deficient liquid medium supplemented with 2% raffinose instead of glucose. Right before the cells reach the log growth phase (OD600 = 0.3), galactose is added to the medium to induce the expression of the mutagenic enzyme. Different amounts of galactose are used to test the activity of the enzymes across different time intervals. The rationale behind this is that, since the expression of mutating enzymes is dependent on the presence of galactose in the media, higher amounts of the latter will be consumed to completion later than lower amounts, thus allowing mutations to occur for a longer time. Galactose concentrations are 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 3%, 4%. Cells are harvested during late log phase (OD600 = 0.9-2) and plasmid DNA is extracted.

The extracted DNA from all conditions (96 in total) is sent for sequencing with primers flanking the synthetic GAL4 gene. Each condition will contain multiple reads that differ from each other depending on how they were mutated. By aligning those reads the distribution of the mutations along the targeted gene can be mapped.

#### Receptor

---

The aim of this experiment is to build a calibration curve for bioluminescent signal generated by the dimerization of the synthetic vascular endothelial growth factor receptor 1 (VEGFR1) coupled to the NanoLuc® system occurring when the ligand (Human hVEGF-A) is correctly recognized by the receptor.

The VEGFR1 gene is a homodimer protein which dimerizes only on correct binding of the ligand. To monitor its activation, each of the two components of the NanoLuc® system (SmBit and LgBit) had to be coupled to either one of the receptor monomers. Therefore, it is necessary to code the two receptor dimers into two separate genes: the same sequence is used for the extracellular and transmembrane domains, while the intracellular domain either has the SmBit (VEGFR1-S gene) or the LgBit (VEGFR1-L). Both genes are placed under the control of TEF1 constitutive promoter and assembled into the two multiple cloning sites of the plasmid vector pESC-LEU.

The plasmid is transformed into Saccharomyces Cerevisiae cells, the cells are grown at 30°C to mid log phase (OD600 = 0.6) and then transferred to a 12-well plate. Each well is supplemented with a different concentration of hVEGF-A purified recombinant protein (0.01nM, 0.02nM, 0.05nM, 0.1nM, 0.2nM, 0.5nM, 0.8nM, 1.0nM, 1.2nM, 1.5nM, 1.8nM, 2.0nM).

Cells are incubated at 30°C inside the Synergy™ HTX Multi-Mode Microplate Reader, which is programmed to measure bioluminescence every 15 minutes for 6 hours.
Once the calibration curve is built for the intensity of the bioluminescence signal caused by receptor dimerization, it can be used to determine the binding affinity of the mutated receptor to any other ligand: simply compare the bioluminescence levels generated by the target ligand binding to the mutated receptor with the calibration curve and determine the amount of ligand bound.

#### Protocols

---

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### References

1. Gatermann, K. B., Hoffmann, A., Rosenberg, G. H., & Käufer, N. F. (1989). Introduction of functional
artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe. Molecular and cellular biology, 9(4), 1526-1535.

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# Team:MSP-Maastricht/Results

ROCKIT

[![](https://static.igem.org/mediawiki/2019/4/47/T--MSP-Maastricht--RocKitLogo.jpg)](https://2019.igem.org/Team%3AMSP-Maastricht)
* Project
  + [Inspiration & Description](https://2019.igem.org/Team%3AMSP-Maastricht/Description)
  + [Background & Design](https://2019.igem.org/Team%3AMSP-Maastricht/Design)
  + [RocKit](https://2019.igem.org/Team%3AMSP-Maastricht/Demonstrate)
  + [Experiments & Results](https://2019.igem.org/Team%3AMSP-Maastricht/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AMSP-Maastricht/Notebook)
* [The Team](https://2019.igem.org/Team%3AMSP-Maastricht/Team)
* Acknowledgements
  + [Collaborations](https://2019.igem.org/Team%3AMSP-Maastricht/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3AMSP-Maastricht/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3AMSP-Maastricht/Sponsors)
* Parts
  + [Overview](https://2019.igem.org/Team%3AMSP-Maastricht/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3AMSP-Maastricht/Basic_Part)
  + [Composite Parts](https://2019.igem.org/Team%3AMSP-Maastricht/Composite_Part)
* [Software](https://2019.igem.org/Team%3AMSP-Maastricht/Software)
* Human Practices
  + [Integrated Human Practices](https://2019.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Education & Engagement](https://2019.igem.org/Team%3AMSP-Maastricht/Public_Engagement)
  + [Outreach](https://2019.igem.org/Team%3AMSP-Maastricht/Outreach)
* Business
  + [Business plan](https://2019.igem.org/Team%3AMSP-Maastricht/BusinessPlan)
  + [Intellectual Property](https://2019.igem.org/Team%3AMSP-Maastricht/IP)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the part's Main Page on the Registry.** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)