The team received mandatory safety training and general lab routine information.

A really cool video about Chalmers' chemical safety routine (recorded at our very own lab!) can be seen in http://www.chalmers.se/insidan/EN/about-chalmers/environmental-work/policies/chemical-safety-routine

As a part of the mandatory training required by the SysBio group, the team received instructions on how to operate the centrifuges and autoclaves of the labs.

We also worked on our risk declaration; a mandatory document containing our experiments descriptions and information about the chemicals we are going to use, their handling, storage and associated risk, as well as waste handling.

Our Risk Declaration was approved by the lab manager and our supervisors. We also sign a safety certificate stating that we are aware of the safety instructions and with that, completed the mandatory "check-in list for new incomers" of the lab.

On the first day of laboration, we prepared LB, YPD and SC-Ura/His/Trp-Leu and YPD media.

Succesfully PCR amplified five out of the seven parts from yeast genome via a Phusion High-Fidelity DNA Polymerase.

The PCR program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. Gradient from 52 to 61ºC for 30s
4. 1 min at 72ºC
5. Go to step 2 (repeat 39 times)
6. 10 min at 72ºC.

A diagnostic gel was performed and the purification of the succesfully amplified parts was done using a PCR Purification Kit from GenJet following the instructions of the manufacture's manual but with no addition of isopropanol.

Plasmid Purification

Performed plasmid purification on E. coli cells containing plasmids p413, p414, p415, p416, p2055 and a plasmid containig our Cas9 sequence (pTPG1-dCas9-UPGP)via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Amplification and purification of parts

Performed a PCR amplification of the non-synthetic parts that failed on the day before and on the dCas9 sequence and the three fluorescent proteins -Yellow, Cyano and Red (YFP, CFP and RFP, respectively).

The PCR was done using the Phusion High-Fidelity DNA Polymerase (see protocol here) and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. Gradient from 50 to 55ºC for 30s
4. 30s at 60ºC
5. 2min 15s at 72ºC
6. Go to step 2 (repeat 39 times)
7. 10 min at 72ºC.

A diagnostic gel was performed and the purification of the succesfully amplified parts was done using a PCR Purification Kit from GenJet following the instructions of the manufacture's manual but with no addition of isopropanol.

Gel extraction

Extracted the fluorecent proteins amplified on the day before with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

PCR amplification and merging

Performed a PCR to fuse the pDSE4, pHO and dCas9 parts together.

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. Gradient from 50 to 55ºC for 30s
4. 30s at 60ºC
5. 2min 15s at 72ºC
6. Go to step 2 (repeat 39 times)
7. 10 min at 72ºC.

A diagnostic gel was performed. The bands did not seem correct, so this PCR was repeated on the next day.

PCR amplification and merging

Repeated the PCR to fuse the pDSE4, pHO and dCas9 parts together.

The PCR was done using the Phusion High-Fidelity DNA Polymerase (see protocol here) and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. 50ºC for 30s
4. 58ºC for 30s
5. 2min 15s at 72ºC
6. Go to step 2 (repeat 39 times)
7. 10 min at 72ºC.

A diagnostic gel was performed and this did not seemed to work either.

Plasmid restriction

Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI.

Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water.

This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.

PCR amplification and merging

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. 50ºC for 30s
4. 58ºC for 30s
5. 2min 30s at 72ºC
6. Go to step 2 (repeat 40 times)
7. 10 min at 72ºC.

A diagnostic gel was performed, however the sizes of the resulting fragments did not match the expected.

Plasmid restriction

Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI.

Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water.

This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.

Samples were loaded into a gelGreen, that showed p413, p415 and p416 approximately in the 5000bp size band, while p414 presented two bands, one around 4000bp and another at 2000bp.

this indicates that the 5000bp band corresponds to the uncut plasmid, while the 4000bp and 2000bp are probably contaminations.

Plasmid restriction

Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI.

Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water.

This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.

PCR amplification

Amplified the parts pDSE4, cas9 and pHO. The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. 50ºC for 30s
4. 58ºC for 30s
5. 2min 15s at 72ºC
6. Go to step 2 (repeat 40 times)
7. 10 min at 72ºC.

A diagnostic gel was performed, however the sizes of the resulting fragments did not match the expected.

PCR amplification

Repeated the amplification of the parts pDSE4, cas9 and pHO. The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. 50ºC for 30s
4. 58ºC for 30s
5. 2min 15s at 72ºC
6. Go to step 2 (repeat 40 times)
7. 10 min at 72ºC.

A diagnostic gel was performed and the sizes of the fragments matched for the pDSE4 and pHO, but not for the Cas9.


Gel extraction

Extracted the DNA fragments previously amplified with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Concentration determination

The concentration of all the fragments obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 8.5 ng of DNA per UL to 42ng/UL.

Plasmid purification

Performed plasmid purification on E. coli cells containing plasmids p413, p414, p415, p416 and p2055 via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step. Each plasmid was purified in duplicates.

Plasmid restriction

Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI.

Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.

This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.

A diagnostic gelGreen was performed and showed that the cleavage of plasmids p413, p415 and p416 was successful.

Gel extraction

Extracted the cleaved plasmids with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Concentration determination

The concentration of the circular and linear plasmids obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 6.55 ng of DNA per UL to 543.4ng/UL.

Plasmid restriction

Cleaved the plasmids p414TEF with the enzymes SacI and XbaI.

Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.

This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.

A diagnostic gelGreen was performed and showed multiple size bands for the samples treated with XbaI. Consultation to the literature showed that this enzyme has not an unique restriction site for p414.

Therefore the restriction was repeated with the enzyme SpeI, that has an unique cleavage site in p414 located 6bp away from the XbaI's desirable cleavage site. The same procedure described above was used, substituting XbaI for SpeI.

The disgnostic gel performed on this second cleavage showed correct band sizes, but with too low lines. This indicates low concentration of the plasmid, therefore it was not extracted.

Plasmid restriction

Cleaved the plasmids p414TEF with the enzymes SacI and SpeI.

Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of SpeI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.

This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.

A diagnostic gelGreen was performed and showed again too light bands or no bands at all.

Plasmid restriction

Repeated the cleavage the plasmids p414TEF with the enzymes SacI and SpeI.

Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of SpeI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.

This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.

A diagnostic gelGreen was performed and SacI seemed to be leaving an excessive amount of uncut plasmid while SpeI apparently degrades the sample.

Plasmid restriction

Repeated the cleavage the plasmids p414TEF with the enzymes SacI with SpeI or BamHI.

Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of SpeI or BamHI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each and without phosphatase were also prepared.

This reaction mixture was incubated at in a water bath at 37ºC for 20 minutes and then enzymatic inactivation was performed for 5min in a heating block at 75ºC for samples containing BamHI and at 65ºC for samples containing SpeI.

A diagnostic gelGreen was performed and SacI again seemed to be leaving an excessive amount of uncut plasmid. However, samples treated with SacI and SpeI with and without phosphatase and one of the samples treated with SacI, BamHI and phosphatase seemed to work and were extracted.

Gel extraction

Extracted the succesfully cleaved plasmids with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

PCR amplification and merging

Performed a PCR to fuse the following fragments:

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. 50ºC for 30s
4. 30s at 60ºC
5. 2min 15s at 72ºC
6. Go to step 2 (repeat 39 times)
7. 10 min at 72ºC.

A diagnostic gelRed was performed and revealed the expected band sizes for the merging of D-tag with CFP and pHO with Sic1.

Gel extraction

Extracted the successfully merged fragments with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

PCR amplification and merging

Performed a PCR to fuse the following fragments:

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. Gradient from 47 to 55ºC for 30s
4. 30s at 60ºC
5. 2min 15s at 72ºC
6. Go to step 2 (repeat 39 times)
7. 10 min at 72ºC.

A diagnostic gelRed was performed and revealed the expected band sizes for the merging of D-tag with YFP.

Gel extraction

Extracted the successfully merged fragments with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Dissolution of synthetic parts

The received synthetic parts (g-blocks) were centrifuged at 3000g for 3min and then dissolved with 10UL of sterile water. The solution was transferred to 10UL of TE buffer, resulting in a final concentration of 10ng/UL.

Amplification and purification of synthetic parts

Performed a PCR amplification of the synthetic parts previously diluted. Namely

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. 54ºC for 30s
4. 30s at 60ºC
5. 20s at 72ºC
6. Go to step 2 (repeat 39 times)
7. 10 min at 72ºC.

A diagnostic gel was performed and showed the expected band sizes for all the fragments except pCYC1.

Gel extraction

Extracted the successfully merged parts with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Preparation of competent cells

Following the instructions of the protocol "Frozen-EZ Yeast Transformation II" by ZymoResearch, Saccaromyces cerevisiae competent cells were prepared and stored at -80ºC.

Amplification and purification of synthetic parts

Repeated the PCR amplification of the synthetic part pCYC1 m1.

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. 54ºC for 30s
4. 30s at 60ºC
5. 20s at 72ºC
6. Go to step 2 (repeat 39 times)
7. 10 min at 72ºC.

A diagnostic gel was performed and showed the expected band sizes.

Gel extraction

Extracted the successfully merged part with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Yeast Transformation

Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".

Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:

Plasmid restriction

Cleaved the plasmids p413TEF and p416TEF with the enzymes SacI and XbaI.

Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.

This reaction mixture was incubated at in a water bath at 37ºC for 30 minutes and then enzymatic inactivation was performed for 5min in a heating block at 65ºC.

A diagnostic gelGreen was performed and showed fragments with the correct sizes.

Gel extraction

Extracted the successfully restricted plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Dissolution of synthetic parts

The received synthetic part pCYC1 m2 was centrifuged at 3000g for 3min and then dissolved with 10UL of sterile water. The solution was transferred to 10UL of TE buffer, resulting in a final concentration of 10ng/UL.

Amplification of synthetic parts

Performed PCR amplification of the synthetic part pCYC1 m2.

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. 54ºC for 30s
4. 30s at 60ºC
5. 20s at 72ºC
6. Go to step 2 (repeat 39 times)
7. 10 min at 72ºC.

A diagnostic gel was performed and showed the expected band sizes.

PCR purification

The purification of the succesfully amplified part was done using a PCR Purification Kit from GenJet following the instructions of the manufacture's manual but with no addition of isopropanol and elution was done with 30UL of sterile water.

Concentration determination

The concentration of the pCYC1 m2 samples obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 15.1 ng of DNA per UL to 34.2ng/UL.

Yeast Transformation

Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".

Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:

Observation of the previously transformed cells

One of the plates with cells transformed on the 28th showed growth, but with contamination by filamentous fungi, while the other presented no growth at all.

A single yeast colony from the contaminated plate was replated on a YPD-agar plate.

Plasmid restriction

Cleaved the plasmids p413TEF, p414TEF and p416TEF with the enzymes SacI and XbaI.

Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.

This reaction mixture was incubated at in a water bath at 37ºC for 30 minutes and then enzymatic inactivation was performed for 5min in a heating block at 65ºC.

A diagnostic gelGreen was performed and showed fragments with the correct sizes.

Gel extraction

Extracted the successfully restricted plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Preparation of Competent cells

Competent E.coli cells were prepared according to the protocol described by Hanahan(1983) and stored at -80ºC.

Inoculation of cells

The single colony replated on the day before was inoculated in 4mL YPD medium as an overnight culture for transformation.

Yeast Transformation

Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".

Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:

Inoculation of cells

Cells transformed on the 28th that showed growth after 3 days were plated on new adequate selective media.

PCR amplification and merging

Performed PCR to fuse the following fragments:

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 30s at 98ºC
3. Gradient from 43 to 53ºC for 30s
4. 1min 30s at 72ºC
5. Go to step 2 (repeat 40 times)
6. 10 min at 72ºC.

A diagnostic gelGreen was performed and the sizes of all the resulting fragments match the expected.

Gel extraction

Extracted the successfully merged parts with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Plasmid restriction

While reviewing primer design documents, the team realized we were using the wrong enzyme to cleave the plasmids: it should be SacI with XhoI instead of XbaI. Therefore, our plasmids did not have the correct overlap with the constructs.

Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XhoI.

Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XhoI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water.

This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.

A diagnostic gelGreen was performed and the cleavage showed to be unsuccessful.

Plasmid restriction

Repeated the cleavage of the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XhoI.

Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XhoI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water.

This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.

A diagnostic gelGreen was performed and the expected band sizes were observed.

Gel extraction

Extracted the successfully cleaved plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Concentration determination

Used a NanoDrop spectrometer to determinate the concentration of the previously extracted plasmids. The concentrations ranged from 5.95 to 7.75ng/UL.

PCR amplification and merging

Performed PCR to fuse the following fragments:

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 30s at 98ºC
3. Gradient from 43 to 60ºC for 30s
4. 2min 30s at 72ºC
5. Go to step 2 (repeat 40 times)
6. 10 min at 72ºC.

A diagnostic gelGreen was performed and the sizes of all the resulting fragments did not match the expected.

PCR amplification and merging

Repeated the PCR to fuse the following fragments:

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. Gradient from 45 to 62ºC for 30s
4. 2min 30s at 72ºC
5. Go to step 2 (repeat 40 times)
6. 10 min at 72ºC.

A diagnostic gelGreen was performed and the sizes of all the resulting fragments were smaller than expected and the bands were difuse.

Gibson Assembly

Attempted to transform the Daughter Resetter construct (pDSE4,YFP,D-tag and Counter 1) into E. coli following the Gibson Assembly protocol from New England Biolab.

The cells were inoculated in LB-Amp medium and let to grow overnight.

Plasmid Purification

Performed plasmid purification on E. coli cells containing plasmids p413, p414, p415, p416, p2055 and a plasmid containig our Cas9 sequence (pTPG1-dCas9-UPGP)via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Plasmid restriction

Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XhoI.

Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XhoI, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water.

This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.

A diagnostic gelGreen was performed and the expected band sizes were observed.

Gel extraction

Extracted the successfully cleaved plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Gibson Assembly

Continued with the Gibson Assembly protocol (New England Biolab) and used p414 in the cells with the assembly poducts. A positive control with cells and the circular plasmid was also prepared.

All the inoculated cultures were placed at 37ºC.

Observed results from the Gibson Assembly

The positive control showed several monoclonal colonies.

However, the plate with the actual construct showed only one colony which is unlikely to contain the construct.

Yeast Transformation

Performed transformation on the competent cells previously prepared according to the protocol "Frozen-EZ Yeast Transformation II".

Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:

PCR amplification and merging

Performed PCR to fuse the following fragments:

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 30s at 98ºC
3. 53ºC for 30s
4. 2min 30s at 72ºC
5. Go to step 2 (repeat 40 times)
6. 10 min at 72ºC.

The samples were stored under refrigeration and analysed on the next day.

E. coli transformation

Added 10UL of the registry Cas9 plasmid (after a 1:10 dilution) to competent E.coli cells. A control with no DNA addition was also prepared.

The cells were kept on ice for 30min and then heat shocked at 42ºC for 60s and then kept on ice again for 3min.

250mL of LB medium were then added and cells were incubated at 37ºC for 90min, under 200rpm shaking.

After incubation, the cells were centrifuged at 9000rpm for 1min and the supernatant was discarded. The pellet was plated in LB+chlorophenicol plates and let to grow overnight at 37ºC.

Diagnostic gel and gel extraction

A gelRed was run to evaluate the PCR products from the day before. The fragments of dCas9, YFP-Counter 1, D-tag-pCYC1 and D-tag-CFP presented the expected band sizes.

The successfully merged and amplified were extracted fragments with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

E. coli transformation

The succesfully transformed colonies from the day before were replated on new LB medium.

The transormation procedure was repeated for dCas9 and p413TEF.

Added 10UL of the registry Cas9 and p413TEF plasmids (after a 1:10 dilution) to competent E.coli cells. A control with no DNA addition was also prepared.

The cells were kept on ice for 30min and then heat shocked at 42ºC for 60s and then kept on ice again for 3min.

250mL of LB medium were then added and cells were incubated at 37ºC for 90min, under 200rpm shaking.

After incubation, the cells were centrifuged at 9000rpm for 1min and the supernatant was discarded. The pellet was plated in LB+chlorophenicol plates and let to grow overnight at 37ºC.

Concentration determination

The concentration of the fragments obtained on the day before was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 15.3 ng of DNA per UL to 101.6ng/UL.

Yeast Electroporation

Prepared an overnight culture according to the "Yeast electroporation" protocol.

Yeast Electroporation

Following the "Yeast Electroporation" protocol, Saccaromyces cerevisiae were transformed with the Daughter Resetter construct (D-tag, YFP, pDSE4 and Counter 1 synthetic part fragments into p414 plasmid), the Cas9 construct (pHO, Sic1 and Cas9 fragments into the plasmid p413) and the Counter 2 construct (D-tag, CFP, pCYC1 and Counter 2 synthetic part fragments into p416 plasmid).

Cells were plated in the adequate restrictive media.

Plasmid Purification

Performed plasmid purification on the overnight culture of E. coli cells containing the registry Cas9 via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

PCR amplification and merging

Performed PCR to fuse and amplify the following fragments:

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. 58ºC for 30s
4. 2min 30s at 72ºC
5. Go to step 2 (repeat 40 times)
6. 10 min at 72ºC.

The samples were stored under refrigeration and analysed on August 18th.

Diagnostic gel

A gelGreen was run to evaluate the PCR products from the 15th. The fragments showed the expected band sizes.

PCR purification

The purification of the successfully amplified part was done using a PCR Purification Kit from GenJet following the instructions of the manufacture's manual but with no addition of isopropanol and elution was done with 30UL of sterile water.

Plasmid restriction

Cleaved the plasmids p413TEF, p414TEF, p416TEF and the registry cas9 plasmid by using the enzymes SpeI and XbaI (for the resgistry plasmid) and SacI and XhoI (for the others).

Approximately 500ng of each plasmid was mixed with o.5UL of each enzyme, 2UL of FastDigest buffer, 1UL of FastAP (alkaline phosphatase) and up to 20 UL of deionized sterile water.

This reaction mixture was incubated at in a water bath at 37ºC for 30 minutes and then enzymatic inactivation was performed for 5min in a heating block at 65ºC.

A diagnostic gelGreen was performed and the sizes of all the resulting fragments match the expected except p415TEF.

Gel extraction

Extracted the successfully restricted plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Gibson Assembly

Attempted to transform the following constructs into E. coli following the Gibson Assembly protocol from New England Biolab:

The cells were inoculated in LB-Amp medium and let to grow overnight.

Yeast Transformation

Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".

Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:

The cells were inoculated in LB-Amp medium and let to grow overnight at 30ºC.

E. coli transformation

Added 10UL of the registry Cas9 plasmid (after a 1:10 dilution) to competent E.coli cells. A control with no DNA addition was also prepared.

The cells were kept on ice for 30min and then heat shocked at 42ºC for 60s and then kept on ice again for 3min.

250mL of LB medium were then added and cells were incubated at 37ºC for 90min, under 200rpm shaking.

After incubation, the cells were centrifuged at 9000rpm for 1min and the supernatant was discarded. The pellet was plated in LB+chlorophenicol plates and let to grow overnight at 37ºC.

Colony PCR

Ran colony PCR on the colonies obtained so far (from different transformation attempts and both in E.coli and yeast). A single colony from each plate was suspended in 100mL LiAc, 1% SD solution and incubated at 70ºC for 5min. Then 300mL of 96% ethanol were added and the solution was centrifuged at 15000g for 3min.

The pellet obtained was washed with 70% ethanol and dissolved in 100mL of water and then centrifuged at 15000g for 15s. 1mL of the obtained supernatant was used for the PCR.
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The PCR program used was:

1. 10 min at 98ºC
2. 10s at 98ºC
3. Gradient from 50 to 59ºC for 30s
4. 2 min at 72ºC
5. Go to step 2 (repeat 39 times)
6. 10 min at 72ºC.

A diagnostic gel was performed and correct band sizes were observed for counter 1 and counter 2 transformations done in 18/8.

Plasmid Purification

Performed plasmid purification on E. coli cells containing the plasmid containing the Cas9 part improvement (from 19/8) via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
instructions were followed, except the elution buffer was substituted by water on the last step.

Concentration determination

The concentration of the obtained plasmids was measured via a NanoDrop. The samples were measured in four replicates. The results ranged from 35.4 ng of DNA per UL to 289ng/UL.

Fluorescence microscopy

Checked cells transformed with the Daughter Resetter construct under a fluorescent microscope and fluorescent yellow cells were observed, as well as dead cells under the green and yellow filter.

PCR amplification

Performed a PCR to amplify Csy4 cconstruct, dCas9, pHO, Sic1 and pCLN1.

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. Gradient from 50 to 55ºC for 30s
4. 30s at 60ºC
5. 2min 15s at 72ºC
6. Go to step 2 (repeat 39 times)
7. 10 min at 72ºC.

A diagnostic gel was performed. The bands match the expected size for all samples except one of the replicates of dCas9.

Gel extraction

Extracted the successfully amplified constructs with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

PCR amplification

Performed a PCR to amplify the D-tag of the intended part submission.

The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:

1. 3 min at 98ºC
2. 10s at 98ºC
3. Gradient from 50 to 60ºC for 10s
4. 7s at 72ºC
5. Go to step 2 (repeat 39 times)
6. 5 min at 72ºC.

A diagnostic gel was performed and the purification of the succesfully amplified parts was done using a PCR Purification Kit from GenJet following the instructions of the manufacture's manual but with no addition of isopropanol.

Yeast plasmid extraction

Extracted plasmids from yeast cells transformed with the Daughter Resetter and Counter 2 constructs via a Zymoprep Yeast plasmid Miniprep II, following the instructions on the kit.

Concentration determination

The concentration of the D-tag and plasmid samples was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 17.1 ng of DNA per UL to 54.6ng/UL.

Restriction digestion

Using a FastDigest kit with enzymes XbaI and SpeI, attempted to cleave p413, p415, D-tag and D-tag-CFP and linearized psB1C3 backbone.

A diagnostic gelGreen was performed and the obtained fragments did not match the expected sizes.

Restriction digestion

Repeated the cleavage of p413, p415, D-tag and D-tag-CFP and linearized psB1C3 backbone, using a FastDigest kit with enzymes EcoRI and PstI.

A diagnostic gelGreen was performed and the obtained fragments did not match the expected sizes.

A diagnostic gelGreen was performed and the obtained fragments match the expected sizes.

Gel extraction

Extracted the successfully restricted constructs with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Concentration determination

The concentration of the restricted constructs was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 1.9 ng of DNA per UL to 39.15ng/UL.

Yeast Transformation

Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".

Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:

E. coli transformation

Added 3UL of the linearize biobrick backbone and 0.5UL of D-tag to competent E.coli cells. A control with only the linearized backbone was also prepared.

The cells were kept on ice for 30min and then heat shocked at 42ºC for 60s and then kept on ice again for 3min.

250mL of LB medium were then added and cells were incubated at 37ºC for 90min, under 200rpm shaking.

After incubation, the cells were centrifuged at 9000rpm for 1min and the supernatant was discarded. The pellet was plated in LB+chlorophenicol plates and let to grow overnight at 37ºC.

Inoculation of plates

Replated transformed yeast cells that showed growth (constructs Csy4 and dCas9 from August 8th and 14th, respectively), as well as E.coli cells transformed with the D-tag part submission.

Colony PCR

Ran colony PCR on the replated plates from the day before. A single colony from each plate was suspended in 100mL LiAc, 1% SD solution and incubated at 70ºC for 5min. Then 300mL of 96% ethanol were added and the solution was centrifuged at 15000g for 3min.

The pellet obtained was washed with 70% ethanol and dissolved in 100mL of water and then centrifuged at 15000g for 15s. 1mL of the obtained supernatant was used for the PCR.
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The PCR program used was:

1. 10 min at 98ºC
2. 10s at 98ºC
3. Gradient from 50 to 59ºC for 30s
4. 2 min at 72ºC
5. Go to step 2 (repeat 39 times)
6. 10 min at 72ºC.

A diagnostic gel was performed and correct band sizes were not observed for Csy4 and dCas9. Degradadion and difused bands were observed for the D-tag.

Restriction digestion

Restriction of possible fully constructed Csy4 construct from the 22nd of August using a FastDigest kit with enzymes XhoI and SpeI.

A diagnostic gelGreen was performed and the obtained fragments did not match the expected sizes.

A diagnostic gelGreen was performed and the obtained fragments did not match the expected sizes.