Team:Aachen/Notebook
Week 1
05/29
AVP1: For the Integration of AVP1 we are using the easy clone plasmid pCfB391 which contains the homologous regions, the terminator and a Histidin auxotrophy marker. We received E.coli DH5alpha carrying pCfb391 and made overnights in LB+Ampicillin (To be used by AVP1 Group Jotho)
LAB: Isolation of yeast genome (quick and dirty protocol)
ENA1: PCR of left, right homologous arm from the previously isolated yeast genome and kanMX from pUG6 plasmid were performed, but the gel showed failure. The PCRs failed repeatedly, with the gels looking strange; our troubleshooting concluded that the error was in a wrongly prepared agarose solution.
05/30
LAB: For further use we created sterile 50% Glycerol stock.
AVP1: We created Cryos of the overnights of E.coli carrying pCfB391, Storage at -80°C
ENA1: The production PCR for the left and right homologous arm for the ENA1 knockout finally worked.
Gel right arm:
Gel left arm:
05/31
LAB: Overnight cultures of Escherichia coli carrying the plasmids pUG6, Easy clone plasmid and our Gal1 plasmid were made.
ENA1: A gradient PCR was performed for the kanMX cassette, to determine the annealing temperature of our primers.
06/01
LAB:Cryo cultures of Escherichia coli carrying the plasmids pUG6, Easy clone plasmid and our Gal1 plasmid were made to secure the plasmids.
ENA1:The kanMX cassette was amplified, with a pUG6 plasmid as template.
The PCR worked fine.
The ENA1 knockout transformation was performed with the three previously
amplified fragments into BY4742 wildtype.
kanmx product:
AVP1:We prepared the saving of the AVP1-gene in Escherichia coli. We made four overnights, two of E. coli pCfB 391 (containing the easy clone plasmid) and another two of E. coli containing pUC19. They were incubated at 37°C.
06/02
NHX1: The 4 fragments for the overexpression transformation of NHX1 were amplified: the left homologous sequence “X-Up”, the Gal1 promotor from the pUG6 plasmid, the NHX1 gene from BY4742 genome and the right homologous sequence from an easy clone plasmid, including the marker genes for yeast and e.coli, “X-Down”.
AVP1: We performed a plasmid preparation of pUC19.
Week 2
06/06
mCherry Labelling: To check the correct intracellular position (vacuolar membrane) of the foreign genes we are transforming into S.cerevisiae BY4742 we want to label the native vacuolar protein encoded by the gene ATG22 with mCherry (Fusion Protein). On the other hand, we will label the foreign genes with eGFP and colocalize it with ATG22-mCherry to the vacuolar membrane.
In the following weeks we worked on transforming S.cerevisiae BY4742 with mCherry. Steps included:
- I. Isolation of Plasmids carrying the genes for mCherry, eGFP and a Kanaymcin-Cassette (Selection with Geniticin) (Week 2)
- II. Isolation of Homologous Regions from S.cerevisiae BY4742 for integration into BY4742 Genome via homologous recombination (Week 2)
- III. PCRs to amplifiy the 4 Fragments for the mCherry Transformation: Homologous Region Left, KanMX, mCherry, Homologous Region Right (Week 2-5)
- IV. Transformation of S.cerevisiae BY4742 with the 4 amplified fragments (Week 5)
- V. Verification PCR of transformed colonies which grew on selection Media (Week 5-6)
- VI. Microscopy with Fluorescence Microscope (Week 6)
Results of these steps can be seen at 07/07 (Week 6)
AVP1: We successfully performed a single-digest of our prepped pUC19 with EcoR1.
AVP1 Fragment Preparation: AVP1 Fragment Preparation: We inoculated 2 overnight cultures of e.coli to prepare fragments for the integration of AVP1. Overnight 1 contained an e.coli strain carrying the EasyClone Plasmid (pO391). The other strain contained the pGREGG566 Plasmid with the Gal1 Promoter that is part of one fragment for the AVP1 integration.
ENA1: The plates of the transformation of ENA1 in BY4742 showed growth. The wildtype control plate with G418 antibiotic showed growth too, though – which led to the suspicion that the plates weren’t produced correctly, for example with too little G418. Transformed cells, a control transformation with a plasmid with kanMX and wildtype BY4742 were plated again to test this. We also produced new G418 plates.
NHX1: A PCR-cleanup was performed with the four previously amplified fragments for the NHX1 transformation.
06/07
ENA1: It was discovered that our strain was wrongly labelled and already contained a G418 resistance – which explained the problems with growth of the negative control. The plates turned out not to be the problem.
NHX1: The transformation of NHX1 into BY4742 was performed with the previously amplified fragments according to protocol. This transformation had to be repeated later due to the discoveries about our strain. To be able to repeat he transformation, the fragments for it were to be amplified again. After the Gel, PCR cleanup was performed.
NHX1 PCR product:
AVP1 Fragment Preparation: We isolated the 2 plasmids from the overnights (06/06) and determined the concentration (pGREGG566, carrying Gal1 Promoter: 740 ng/µL; pCfB 391 EasyClone: 208,5 ng/µL).
LAB: We mixed 1L of Tris-HCl buffer and 1L of TE buffer.
06/08
AVP1 Fragment Preparation: We used the pGREGG566 plasmid isolated the day before to amplify the Gal1 Promoter linear fragment. Therefore, we carried out a PCR and Gelelectrophoresis afterwards.
All 3 PCR tubes showed bands at 500 bp, which is the expected length
for our Gal1 Promoter Fragment (462 + 40 bases overhang from the reverse
Primer -> 502 bases long. The negative control tube 4 with no template
(plasmid) showed no band at 500 bp.
We cleaned up the product and determined the concentration: 25,5 ng/L.
06/09
LAB:An overnight culture of E.coli containing a plasmid with eGFP was made.
ENA1:: The kanMX fragment for the transformation was amplified again, as our product from the previous PCR was depleted. The transformation was repeated because of the strain mix-up. Overnight culture of the E.coli containing the plasmid with kanMX was made.
AVP1 Fragment Preparation: We carried out 2 PCRs and
2 Gelelectrophoresis to amplify the Upper Homologous Region +
Terminator (TADH1) = Fragment 1, and the Lower Homologous Region
+ loxP + Marker + loxP = Fragment 4 from the EasyClonePlasmid pCfB 391.
Bought Fragments are necessary for the integration of AVP1
into the yeast genome.
Gel Fragment 1:
all 4 PCR tubes showed bands at around 900 bp, which is
what we expected (our Fragment 1 (Up and TADH1 Terminator)
is 896 bp). The PCR worked in the 50 µL (1,2) as well as in
the 25 µL (M,3) tubes, but not in the negative control. The
negative control tube K with no insert (Easy Clone Plasmid
p0391) showed no band at around 900 bp.
The PCR Product was cleaned up and concentration was determined afterwards.
Gel Fragment 4:
All 4 PCR tubes showed bands at around 2000+ bp, which is what we expected
(our Fragment 4 (Down loxP-Marker-loxP) is 2111 bp long). The PCR worked in
the 50 µL (1,2) as well as in the 25 µL (M,3) tubes, but not in the negative
control. The negative control tube K with no insert (Easy Clone Plasmid p0391)
showed no band at around 2000 bp.
The PCR Product was cleaned up and concentration was determined afterwards.
AVP1: We dephosphorylated puC19 and performed a digestion of the AVP1-gene with EcoR1.
Week 3
06/12
LAB: Plasmid Prep was done for the pUG6 and eGFP plasmids from the overnight cultures.
AVP1: After preparing our parts for the AVP1 back-up in E. coli we finally started the transformation. First our insert (AVP1) and our plasmid (pUC19) were ligated with T4 ligase, we prepared two tubes: the ligation and a negative-control.
Ligation-Mix composition:
| Gene: | AV | P1 |
|---|---|---|
| Components | Ligation | Negative Control |
| Insert (AVP1) | 6,56 µL | - |
| Backbone (pUC19) | 4 µL | 4 µL |
| T4 Ligase | 2 µL | 2 µL |
| Buffer | 2 µL | 2 µL |
| ddH2O | 5,44 µL | 12 µL |
| total | 20 µL | 20 µL |
We incubated the two Eppis for 1h at room temperature and
performed a heat shock at 65°C afterwards.
For the transformation, we used E. coli BL21. We used the
transformation-protocol from NEB.
The transformed cells were spread on Amp-Plates. In addition,
we made a negative and a positive control.
- BL21 with pUC19 + AVP1
- BL21 with pUC19 (without insert)
- wildtype
- dilutions from BL21 with insert
06/13
ENA1: The kanMX fragment was amplified from the previously prepared pUG6 plasmid. The gel shows two bands, the lower one being our long primers (long due to overhangs).
kanMX production PCR:
AVP1: There was no growth on
the plates from the day before, so either the
ligation or the transformation didn't work.
We asked one of our advisors and they gave us
the tip to use a higher insert/backbone-ratio (9:1) and extend the
ligation time.
For our second ligation, we used the same parts as before.
We prepared 3 tubes.
Ligation-Mix composition:
| Gene: | AVP1 | ||
|---|---|---|---|
| Components | Ligation (3:1 ratio) | Ligation (9:1 ratio) | Negative Control |
| Insert (AVP1) | 6,56 µL | 9,84 µL | - |
| Backbone (pUC19) | 4 µL | 2 µL | 4 µL |
| T4 ligase | 2 µL | 2 µL | 2 µL |
| Buffer | 2 µL | 2 µL | 2 µL |
| ddH2O | 5,44 µL | 4,16 µL | 12 µL |
After the ligation we performed the transformation and spread the cells on Amp-Plates.
06/14
LAB: A gradient PCR was done for eGFP to determine the annealing temperature. Although an error was made when creating the primers, the PCR worked quite well.
Annealing Test:
AVP1: The transformation seemed successful. There grew c20 colonies on the 9:1 ratio plate and c5 on the 3:1 ratio plate.
06/15
LAB:Now that the annealing temperature for the eGFP fragments was determined, a production PCR could be performed. The product was cleaned.
production PCR result:
To test the E.coli with pUG6 and eGFP, LB plates with Aminopenicillin for pUG6 and Kanamycin for eGFP were produced, and the bacteria plated on one plate each.
06/16
LAB: In order to produce cryo cultures for pUG6, eGFP and BY4742, we first have to produce overnights from only one colony of each. We used the plates we produced on the day before and the one we received from our advisor (Sandra) (BY4742).
Our overnights contained the following:
1. 1 pUG6 colony + 25 ml LB medium + 25 ul Amp (100%)
2. 1 eGFP colony + 25 ml LB medium + 25 ul Kan (100%)
3. 1 BY4742 colony + 25 ml YPD medium
4. 1 BY4742 colony + 25 ml YPD medium
AVP1: We checked the transformation with a colony-PCR. Therefore, we used the protocol 1.3 Check PCR from the script "Praktikum Molekulare Biotechnologie".
We checked 8 colonies from our 9:1 plate.
Check PCR composition:
| Gene: | AVP1 | ||
|---|---|---|---|
| Components | Volume for one Tube | Volume for ten Tubes | Final Concentration |
| ddH2O | 15 µL | 150 µL | - |
| 5x Green GoTag Buffer | 4 µL | 40 µL | 1x |
| Primer f (10µM) | 0,2 µL | 2 µL | 0,1 µM |
| Primer r (10µM) | 0,2 µL | 2 µL | 0,1 µM |
| dNTP-mix (10mM) | 0,4 µL | 4 µL | 200 µM |
| GoTag Polymerase | 0,2 µL | 2 µL | 2,5 U |
Due to the different melting temperatures, we performed a gradient PCR (T: 57,5-62,0°C).
| Gene: | AV | P1 |
|---|---|---|
| PCR Step | Time | Temperature |
| ID Initial denaturation | 10 min | 94°C |
| D Denaturation | 30 sec | 94°C |
| A Annealing | 30 sec | gradient (57,5-62,0°C) |
| E Extention | 60 sec | 72°C |
| FE Final Elongation | 5 min | 72°C |
| S Storage | unlimited | 8°C |
After the PCR we made a gel-electrophoresis.
Unfortunately, the results didn't meet our expectations, there are bands between 250bp and 500bp, all at a different height. If the transformation had been successful there would be a 2300bp band.
Week 4
06/19
LAB: Overnight cultures from 16.06.17 where used to create cryo cultures. Cryo tubes of pUG6 and eGFP (both in E.coli) were filled with 500µl culture + 500µl 50% glycerin and the cryo of BY4742 with 500µl culture + 500µl 30% glycerin.
Competent BY4742 cryos were created according to the McClean protocol.
AVP1: To check if AVP1 has any other unexpected EcoR1-restriction sites,
we performed a digestion and a gel-electrophoresis afterwards. The
result was one single band with the expected size of c2300bp.
Because of our ambiguous results of the colony PCR we
decided to check our cloning via restriction analysis and
ordered the restriction enzyme Nde1 (cuts in AVP1 & pUC19).
06/20
NHX1: Production PCR for NHX1 was made following the program from the 08.06.17. The gel shows that the PCR worked out well. The bands fit the length of the fragment, which was 1899bp.
Production PCR NHX1:
NHX1 with eGFP: PCR Products for NHX1 with eGFP overhangs and terminator+marker+homologous sequence with eGFP overhangs were produced and PCR cleanup was done.
ENA1: Transformation for the ENA1 knockout was performed. Transformed cells and control were plated on YPD with G418.
AVP1: The restriction analysis showed, that the transformation failed. We talked to our instructor and she recommended us to use the "CloneJET PCR Cloning-Kit" (blunt-end ligation) because we had the fear, that the end digestion of AVP1 didn't work. We performed a new transformation of AVP1 in E. coli DH5a. Ligation mixture:
| Gene: | AVP1 |
|---|---|
| Component | Amount |
| 2x Reaction Buffer | 10 µL |
| Insert | 7 µL |
| pJET 1.2 | 1µL |
| T4 DNA Ligase | 1 µL |
| ddH2O | 1 µL |
| 20 µL |
06/21
LAB: The G418 plates were tested by plating BY4742 wildtype on our YPD+G418 plates.
To test our strains, two overnight cultures were made with BY4742
wildtype, one with G418 and one without.
NHX1: The transformation for the overexpression of NHX1 was performed. The competent cells were tested using a plasmid with kanMX G418 resistance.
ENA1: With a rest of PCR product, PCR cleanup was performed.
AVP1: We transformed the ligation-product and a control-ligation in DH5a, using the protocol from the NEB-Kit "NEB 5-alpha competent E. coli".
06/22
LAB: We made 3 liters of 1,2% Agarose Gel for use in Gel Electrophoresis
NHX1: Product PCR of NHX1, X4-Up homologous sequence,
Gal1 promotor and x4-Down homologous seuqnce with selection marker was
repeated to replenish our stock of the fragments.
The gel showed that the PCR was successful:
PCR-cleanup was performed with the products.
AVP1: The transformation didn't succeed.
06/23
NHX1: The transformation of NHX1 into BY4742 wildtype seemed to work, as colonies started to show on the plates and the control transformation showed no growth. The cells looked very wet and yellow-ish, wish led us to the conclusion that they weren’t yeast cells. Microscoping the cells and comparing them to reference yeast cells confirmed our suspicion that our transformation was contaminated.
AVP1: To perform another transformation, we amplified the AVP1 gene via PCR.
Composition of 100µL master mix:
PCR-programm:
| Gene: | AVP1 |
|---|---|
| Component | Amount |
| B2 PfuS Buffer | 10 µL |
| Primer f | 8 µL |
| Primer r | 8 µL |
| dNTP-mix | 2 µL |
| Polymerase (PfuS) | 1 µL |
| Template (AVP1) | 2,2 µL |
| ddH2O | 71 µL |
| Gene: | AV | P1 |
|---|---|---|
| PCR Step | Time | Temperature |
| ID Initial denaturation | 2 min 20 sec | 98°C |
| D Denaturation | 20 sec | 98°C |
| A Annealing | 30 sec | 61°C |
| E Extention | 2 min 30 sec | 72°C |
| FE Final Elongation | 3 min | 72°C |
| S Storage | unlimited | 8°C |
The PCR was successful. [DNA]= 18ng/µL
Week 5
06/26
NHX1: To have some backup material, we decided to make some cryo cultures for our transformed NHX1 overexpression cells. We made 5 cryo cultures in 30% glycerin solutions and stored them in a -80°C container.
We suspected that our transformation was contaminated because of a rather
unusual smell for a yeast culture. Using a microscope, we found out that our
cultures were in fact e. colis. Several colonies were tested, each with the
same misfortune.
Our cryo cultures were discarded.
ENA1: After several days, no growth was observed on our transformation plate. We decided to use a method called “Abschwämmen”. A small amount of YPD+G418 medium is added to the plate. Using a sterile toothpick, the colonies are carefully picked and dissolved in the medium. This medium was then pipetted into a new YPD+G418 plate.
We tested our SC-Histidine free plates and concluded they were functional.
AVP1 Fragment Preparation:To refill our stock of
Gal1 promoter we repeated the PCR carried out on 06/07:
We used the pGREGG566 plasmid isolated the day before to
amplify the Gal1 Promoter linear fragment. Therefore, we carried out a
PCR, which failed.
AVP1: Because all teams had problems with the PCRs
we performed a PCR-test series to check the polymerases. Our suspicions
were confirmed and we had to throw some of the tubes away.
Simultaneously to the PCRs we performed a second transformation with AVP1
ligated in pJET. this time we used a AVP1 with a higher
concentration (18 ng/µL instead of 9 ng/µL).
06/27
NHX1: Due to the contaminated plates we had to discard, we made a new transformation. In order to test our plates, we did a test transformation using a KanMX plasmid.
LAB: New SC plates were produced, histidine free.
AVP1: This time 5 colonies grew on the plate. We performed a colony PCR to check the transformation success.
Result:
We prepared overnight cultures for a restriction analysis.
06/28
AtHENA: We produced three different overnights; two different knockout strains and one wild type.
AVP1: We performed a second colony PCR and changed
the annealing temperature to 60°C.
In addition, we performed a restriction analysis with Nde1.
Both methods showed, that the transformation failed. Because
the saving of AVP1 in E. coli didn't seemed very promising in the
future we decided to skip this part and begin with the transformation in yeast.
06/29
AVP1: We performed another PCR to amplify all 4 fragments for the integration of AVP1 into S.cerevisiae BY4742 which failed.
LAB: Selection media without histidine and without uracil were produced.
ATHENA: We resuspended our new primers for KanMX extraction in TE – Buffer. Overnights from our plate #2 were made. The antibiotic G418 was added as selective marker.
06/30
Week 6
07/03
AVP1 Fragment Preparation: To refill our stock of Gal1
promoter we repeated the PCR carried out on 06/07 :
We used the pGREGG566 plasmid isolated the day before (
to amplify the Gal1 Promoter linear fragment. Therefore, we
carried out a PCR, which failed.
NHA1: We dissolved our new primers in DMSO with a 25 mMolar
solution. We produced new YPD Medium because of a contamination. These new
primers were used in a gradient PCR to produce the right and left arms beside
the NHA1 gene. A verification PCR for ENA1 was also introduced in our gradient
PCR.
This where our results:
The NHA1 PCRs did not work.
ENA1: We decided to prepare some overnight cultures. Overnight #1 contained G418 (antibiotic) and the ENA1 transformation, overnight #2 contained G418 (antibiotic) and wild type and overnight #3 with contained YPD and the ENA1 transformation.
07/04
AVP1 Fragment Preparation: We repeated the PCR frp, 07/03. This time the gel showed 1 intense band at around 500 bp, which fits the Gal1 fragment length (502 bp). The product was therefore cleaned up and the concentration was determined.
07/05
AVP1 Fragment Preparation: We carried out PCR to amplify
the codon optimized gene from IDT. The test gel showed 1 intense band at
around 2350 kb, which fits the length of the AVP1 Gene (2375 bp in length).
The product was cleaned up using PCR clean-up and concentration was determined
via NanoVue afterwards (123 ng/µL).
Furthermore, we repeated the PCRs from 06/09 to refill our stock of
Homologous Region Fragments for the genome integration.
LAB:We wanted to use a FACS Machine to do a cell analysis. To do this, we needed to produce PBS medium for this analysis and as a medium for fluorescent microscopy.The pH was set to 7.4 using NaCl. After this the overall volume was increased to 1000 ml using cold ddH2O and autoclaved.
NHA1:We found the results from yesterday´s gradient PCR somewhat strange. We decided to rapidly test the template DNA with an electrophoresis. This were our results:
No template was found in our NHA1 left and right arms. That wild type BY4742 genome was disposed of and new genome was isolated from a new overnight culture. Another reason why our gradient PCR wasn´t working was the solution in which the primers were dissolved in, DMSO. A primer solution, whichever the concentration is, should never include more than 3.5 % DMSO, otherwise the polymerase will react with said solute and denaturate.
07/07
mCherry Integration Results: The last weeks we worked on transforming mCherry into S.cerevisiae BY4742 to create the fusion protein ATG22-mCherry. Amplification of the 4 fragments (Homologous Region Left, KanMX, mCherry, Homologous Region Right) was successful, the Transformation as well. Verification PCRs of the integrated fragments showed, that mCherry was integrated correctly, however microscopy did not show fluorescence at all (Used Laser Wavelength was: 561). Control of Codon Usage showed, that mCherry is Codon Optimized for E.coli and therefore not expressed correctly. We will withdraw mCherry and use a S.cerevisiae codon optimized mRFP Gene from now on.
Week 7
07/10
ENA1:Since we had many different mutant colonies where our transformation did seem to work, we decided to store them in case something went wrong along the way. We produced 10 different overnights for ENA1 K.O transformations and wild type BY4742.
Interlab Study:We transformed samples of competent E. coli cells. We transformed with three different controls; one negative, one positive and another positive with a RFP marker. We produced 30 LB plates containing chloramphenicol from which 18 plates were used.
07/11
AVP1: We created his-drop out medium (plates) for our transformation of AVP1 in yeast.
NHA1:We produced some YPD medium flasks for later use.
We proceeded to new gradient PCRs (10 samples for each) containing XII-Up
(left arm), XII-Down (right arm) and the URA3 + loxP marker.
The following gel electrophoresis shows our results:
The XII-Up PCR did not work. The temperatures that barely seem to work for X-Down were 49.3 °C, 51.2 °C, 53.3 °C and 57.2 °C.
Interlab Study:On all our plates, some E. coli colony growth was observed. However, we could only count the colonies on 4 of these plates. Because of this we decided to replate on LB + Chloramphenicol most of the E. colis on new plates to obtain single colonies.
07/12
AVP1: For the transformation, we first performed a PCR of fragment 3 (Gal1-Promotor). After that we transformed our fragments into Saccharomyces cerevisiae. 3 tubes were made:
- yeast + fragments 1-4
- yeast + control plasmid
- yeast from the other group for testing + control plasmid
| Gene: | AVP1 | ||
|---|---|---|---|
| Component | Tube 1 | Tube 2 | Tube 3 |
| Fragment 1 (TADH1) | 11,8 µL | - | - |
| Fragment 2 (AVP1) | 12,5 µL | -td> | - |
| Fragment 3 (Gal1) | 13,9 µL | - | - |
| Fragment 4 (his-aux-marker) | 7,3 µL | - | - |
| plasmid p0391 | - | 0,96 µL | 0,96 µL |
| ddH2O | - | 33,04 µL | 33,04 µL |
The transformation-mixture contained 720µL 50% PEG 350; 108 µL 1M LiOAc and 150µL carrier DNA (salmon sperm). For the transformation, we used the protocol: "Heatshock transformation S. cerevisiae (modified after Joska al. 2014)" from Sandra.
NHA1:A gradient PCR was made for the XII-Up, XII-Down and URA3+ loxP exactly as yesterday. The results of the gel electrophoresis show that no product was present in our mixtures.
Interlab Study:We wanted to be sure our LB plates weren’t working well so we tested them. We plated E. coli dH5d and E. coli with PuG6 on our plates.
07/13
mRFP Labelling: Since the expression of mCherry failed due to wrong Codon usage
we are repeating the labeling with a codon optimized mRFP.
We performed PCRs of the for the Transformation needed fragments 1 (Left Homologous Region),
3 (mRFP) and 4 (Right Homologous Region). Fragment 2 did not change.
PCR Mix:
| Gene: | Left Homologous Fragment | mRFP | Right Homologous Fragment |
|---|---|---|---|
| Component | µl | µl | µl |
| Template | 1,314 | 0,348 | 1,104 |
| Buffer | 15 | 15 | 15 |
| Primer f | 12 | 12 | 12 |
| Primer r | 12 | 12 | 12 |
| dNTP | 3 | 3 | 3 |
| Polymerase (PfuS) | 1,5 | 1,5 | 1,5 |
| ddH2O | 105,2 | 106,15 | 105,4 |
PCR Programm:
| Gene: | Left Homolo | gous Fragment | mR | FP | Right Homolo | gous Fragment |
|---|---|---|---|---|---|---|
| PCR Step | Time | Temperature | Time | Temperature | Time | Temperature |
| ID Initial denaturation | 180 sec | 98°C | 180 sec | 98°C | 180 sec | 98°C |
| D Denaturation | 20 sec | 98°C | 20 sec | 98°C | 20 sec | 98°C |
| A Annealing | 40 sec | 58°C | 40 sec | 63°C | 40 sec | 53°C |
| E Extention | 60 sec | 72°C | 60 sec | 72°C | 60 sec | 72°C |
| FE Final Elongation | 5 min | 72°C | 5 min | 72°C | 5 min | 72°C |
| S Storage | unlimited | 8°C | unlimited | 8°C | unlimited | 8°C |
Afterwards we analyzed the PCRs via a Gel Electrophoresis, performed a PCR Clean-Up and determined the DNA-concentration via NanoVue.
Gel:
ENA1: We did a verification PCR using the isolated genome of our ENA1 mutated colonies. We then followed this with a gelelectrophoresis.
NHA1: Due to the crazy results from the past days, we decided to do 2 separate PCRs. One production PCR for the XII-Down and URA3 + loxP fragments and one gradient PCR for the XII-Up fragment. We also used the new primers (now diluted in TE buffer to a 25-mM concentration) for every PCR. The following gel shows our results:
URA3 + loxP and XII-Down seem to work fine. Nonetheless XII-Up gradient PCR showed now promising bands. Both successful products were cleaned up and stored.
AVP1: We created competent S. cerevisiae BY4742, using the protocol "McClean: Making and Using Frozen Yeast Competent Cells".
07/14
mRRP Labelling: We transformed S.cerevisiae BY4742 with the 4 mRFP Fragments to integrate mRFP into the genome. YPD-Plates containing Genticin were used as a selection marker.
DNA-Mix:
| Gene: | mR | FP |
|---|---|---|
| Fragment | m needed [ng] | V needed [µl] |
| Homologous Region Left | 1000 | 23,81 |
| KanMX | 500 | 10 |
| mRFP | 13,42 | 1000 |
| Homologous Region Right | 1000 | 23,81 |
| H2O | 0 | 0 |
Trafo-Mix:
| Trafo: | mRFP into Wildtype (WT) |
|---|---|
| Component | Amount [µl] |
| 50% PEG 3350 | 240 |
| 1M LiAC | 36 |
| Carrier DNA | 50 |
AVP1: Transformation results (07/12): Because there was growth
on the plates with the transformed yeast, the transformation seemed to be
successful.
We isolated the genome of 5 colonies from plate 1 for further tests.
NHA1: The only fragment missing to continue was XII-Up. We followed a production PCR for XII-Up with a PCR clean up while we waited for our gel electrophoresis.
Our products did contain the wished fragment size.
Week 8
07/17
mRFP Labelling: Check of Transformation done at 07/14. 4 colonies grew. No fluorescence under UV-Lamp. Verficiation PCR needed.
AVP1: Our transformed yeast cells were contaminated with E. coli, so we performed a new transformation. This time we used the competent yeast cells we made the week before. The amounts of the four DNA-fragments were the same as last time (tube 1). Again, we used the protocol "Heat shock transformation S. cerevisiae".
NHA1: We produced 2 liters x1 PBS-Buffer (100 ml x10 PBS per 900 ml ddH2O).
07/18
mRFP Labelling: Check of Transformation done at 07/14. 4 colonies grew. No fluorescence under UV-Lamp. Verficiation PCR needed.
NHA1: We finally started with our transformation tests. Our NHA1 transformed cells and wild type BY4742 were then plated on a URA- plates. We also produced some PEG 50% 3350 solution for further yeast transformations.
ENA1: We did a verification PCR for our ENA1 K.O followed by a gel electrophoresis. No bands were observed.
NHX1: To check if our NHX1 overexpression transformation we did a verification PCR with our isolated genome. None of our products worked.
AVP1: We successfully amplified our 4 fragments for further transformations.
07/19
mRFP Labelling: Gel Electrophoresis of the PCR Products was unsuccessful. We did another PCR with the right and left parts of the integrated fragments, which failed as well. Therefore, mRFP was not integrated into BY4742 during Transformation.
AVP1: We made competent Ena1-kock-out BY4742 mutants and performed a transformation with these cells to integrate AVP1. We used the same amounts of fragment as before.
NHX1: Since our verification PCR did not work yesterday we decided to isolate a new genome and use it for a new verification PCR. This again did not work.
07/20
mRFP Labelling: We performed another Transformation of BY4742 with the 4 mRFP Fragments (Left Homologous Fragment, KanMX, mRFP, Right Homologous Fragment) as done at 07/14.
DNA-Mix:
| Gene: | mR | FP |
|---|---|---|
| Fragment | m needed [ng] | V needed [µl] |
| Left Homologous Region | 500 | 7,1 |
| KanMX | 250 | 5 |
| mRFP | 500 | 6,71 |
| Homologous Region Right | 500 | 11,9 |
| H2O | 4 |
Trafo-Mix:
| Trafo: | mRFP into WT |
|---|---|
| Component | Amount [µl] |
| 50% PEG 3350 | 240 |
| 1M LiAC | 36 |
| Carrier DNA | 50 |
AVP1: To check the success of our transformation (07/17)
we performed a genome-isolation and a check-PCR with 5 of the c50 colonies.
For the genome-isolation we used the protocol "Quick and Dirty
(Mans et. al 2015)" from our advisor Sandra.
(The check-PCR failed.)
NHA1: We needed some XII-Up fragments again. Instead of doing a gradient PCR, we decided to set our temperature to 47.7 °C and make larger mixtures. Our gel electrophoresis showed the result of said PCR.
Our products were cleaned up and used for a new NHA1 transformation (transformation #2). The transformed yeast cells were again plated (along with a wild type) on URA+ plates.
07/21
NHA1:After not seeing any culture growth in the past days, we decided to do a third transformation just to be sure. We plated our transformation and wild type on URA- plates.
Our XII-Down and XII-Up fragments were accidentally thrown away, therefore we needed to produce more. Since we already knew the ideal annealing temperatures for our fragments, we decided to do a production PCR. We analyzed our products with a gel electrophoresis. The results showed that none of the PCR products worked.
Week 9
07/24
mRFP Labelling: 40 colonies grew on the Transformation from 07/20. We isolated the Genomes and performed Verification PCRs of 5 colonies. Primers included both left and right part of the integrated fragments and additional 100bp left and right of the integration sites.
PCR Mix:
| Strain: | BY4742+mRFP |
|---|---|
| Component | µl |
| Template | 1,5 per Tube |
| Buffer | 5 |
| Primer f | 2 |
| Primer r | 2 |
| dNTP | 1 |
| Polymerase (PfuS) | 0,5 |
| ddH2O | 37,2 |
PCR Programm:
| Strain: | BY474 | 2+mRFP |
|---|---|---|
| PCR Step | Time | Temperature |
| ID Initial denaturation | 180 sec | 98°C |
| D Denaturation | 15 sec | 98°C |
| A Annealing | 30 sec | 57°C |
| E Extention | 50 sec | 72°C |
| FE Final Elongation | 5 min | 72°C |
| S Storage | unlimited | 8°C |
AtNHXS1: We want to integrate the Arabidopsis Thaliana vacuolar Sodium/Proton Antiporter AtNHXS1 into S.cerevisiae. Therefore, we use the Easy Clone Plasmid pCfb257, carrying the TCYC1 Terminator, Integrationsides for Chromsome X-3 and a Leucin Marker. We received E.coli DH5alpha carrying pCfb257 on a plate containing Ampicillin and incubated it. Additionally we performed PCRs to amplify the Fragments 2 (Gal1 Promoter) and 3 (AtNHXS1 synthesized by IDT) for Transformation, which failed.
AVP1: We repeated the check-PCR. It failed again.
We isolated another five genomes from different colonies (BY4742+AVP1 & Ena1-kock-out + AVP1).
07/25
mRFP Labelling: Gel Electrophoresis of the Verification PCRs from 07/24. Colonies 2 showed correct bands. We microscoped colonies 2 and 4 other colonies (6, 7, 8 and 9) which had not been PCR verified to check fluorescence (Laser wavelength: 561nm). Whereas colony 2, 7, 8 and 9 showed no to small fluorescence, colony 6 showed high fluorescence. 20 other microscoped colonies showed fluorescence as well. We will go on working with colony 6.
AtNHXS1: We prepared an overnight culture of E.coli DH5alpha carrying pCfB257 to amplify the plasmid. Additionally, we performed another PCR to amplify Fragments 2 and 3, which failed.
AVP1: We performed another cPCR. It failed again.
Because all our PCRs failed, we received a different polymerase (GoTag)
from our advisor and repeated the cPCR. We had 12 tubes.
Gelelectrophoresis:
- 1, 8: ladder
- 2, 3, 4: cPCR AVP1
- 5, 6, 7: cPCR Ena1
- 9-14: control
07/26
AtNHXS1: We performed a plasmid prep of the E.coli+pCfB257 Overnight to isolate pCfB257. We performed PCRs of all 4 Fragments (Integrationside Up, Gal1, AtNHXS1, Terminator-Leucin Marker-Integrationside Down)
PCR Mix:
| Gene: | Fragment 1: Up | Fragment 2: Gal1 | Fragment 3: AtNHXS1 | Fragment 4: T-Down |
|---|---|---|---|---|
| Component | µl | µl | µl | µl |
| Template | 0,52 | 0,34 | 1,5 | 0,52 |
| Buffer | 25 | 25 | 25 | 25 |
| Primer f | 20 | 20 | 20 | 20 |
| Primer r | 20 | 20 | 20 | 20 |
| dNTP | 5 | 5 | 5 | 5 |
| Polymerase (PfuS) | 2,5 | 2,5 | 2,5 | 2,5 |
| ddH2O | 177 | 177,2 | 176 | 177 |
PCR Programm:
| Gene: | Fragme | nt1:Up | Fragment | 2:Gal1 | Fragment3 | :AtNHXS1 | Fragment | 4:T-Down |
|---|---|---|---|---|---|---|---|---|
| PCR Step | Time | Temperature | Time | Temperature | Time | Temperature | Time | Temperature |
| ID Initial denaturation | 30 sec | 98°C | 30 | 98°C | 30 sec | 98°C | 30 sec | 98°C |
| D Denaturation | 8 sec | 98°C | 8 sec | 98°C | 8 sec | 98°C | 8 sec | 98°C |
| A Annealing | 20 sec | 59°C | 20 sec | 61°C | 30 sec | 63°C | 30 sec | 58°C |
| E Extention | 20 sec | 72°C | 20 sec | 72°C | 60 sec | 72°C | 30 sec | 72°C |
| FE Final Elongation | 5 min | 72°C | 5 min | 72°C | 5 min | 72°C | 5 min | 72°C |
| S Storage | unlimited | 8°C | unlimited | 8°C | unlimited | 8°C | unlimited | 8°C |
Afterwards we performed a Gel Electrophoresis, a PCR-Clean Up and determined the DNA-concentrations of the fragments.
Gel:
AVP1: We checked the other homologies recombination locus of the tested Ena1-mutants and both loci of the AVP1-mutant.
Gel-electrophoresis
- 1, 16: ladder
- 2, 3, 4, 5: posterior homologues region
- 6, 7, 8 ,9: AVP1+Gal
- 10-13: control
- 14, 15: anterior homologues region
Both strains integrated the homologues DNA-sequences.
We performed another PCR to prove the right order of the integrated fragments.
Gel-electrophoresis
07/27
mRFP: We performed Verification PCRs to verify the integration of mRFP into colony 6 which was fluorescing on 07/25. The PCRs failed. We made an overnight of colony 6 in YPD containing G418.
AtNHXS1: We transformed the 4 amplified AtNHXS1 Fragments into BY4742 WT and BY4742 containing Ena1 Knock-Out. We used SC-Plates without Leucin as selection media.
DNA Mix:
| Gene: | AtNH | XS1 |
|---|---|---|
| Fragment | m needed [ng] | V needed [µl] |
| Up | 500 | 4,46 |
| Gal1 | 500 | 4,33 |
| AtNHXS1 | 500 | 3,38 |
| T-loxP-Leu-loxP-Down | 350 | 4,76 |
| H2O | 17,07 |
Trafo Mix:
| Trafo: | AtNHXS1 into WT & Ena-Mutant |
|---|---|
| Component | Amount [µl] |
| 50% PEG 3350 | 480 |
| 1M LiAC | 72 |
| Carrier DNA | 100 |
AVP1: The integration of AVP1 in the Ena1-knockout-mutant was successful, the integration of AVP1 in the wildtype failed.
We isolated the genome of 8 other colonies and performed a cPCR.
Gelelectrophoresis:
07/28
mRFP: We performed new PCRs to verify the integration of mRFP into BY4742. We checked the left and right part of the fragments and additionally 100bp on each side of the fragment of the genome.
PCR Mix:
| Gene: | Left Part | Right Part |
|---|---|---|
| Component | µl | µl |
| Template | 1 | 1 |
| Primer f | 0,2 | 0,2 |
| Primer r | 0,2 | 0,2 |
| One Taq 2x Mastermix | 5 | 5 |
| ddH2O | 3,4 | 3,4 |
PCR Programm:
| Gene: | Left | Part | Right | Part |
|---|---|---|---|---|
| PCR Step | Time | Temperature | Time | Temperature |
| ID Initial denaturation | 30 sec | 94°C | 30 sec | 94°C |
| D Denaturation | 15 sec | 94°C | 15 sec | 94°C |
| A Annealing | 25 sec | 57°C | 25 sec | 59°C |
| E Extention | 75 sec | 68°C | 120 sec | 68°C |
| FE Final Elongation | 5 min | 68°C | 5 min | 68°C |
| S Storage | unlimited | 8°C | unlimited | 8°C |
Afterwards we performed a Gel Electrophoresis.
Gel:
We made competent cells of BY4742 + mRFP from the overnights from 07/27.
AVP1: We checked the 5 colonies that contained the upper homologues sequence. They didn't integrated the other fragments.
Week 10
07/31
AtNHXS1: Colonies grew on AtNHXS1 Transformations from 07/27. We performed a Genome Isolation and verification PCR (Left and Part of Fragments, 100bp of Genome on left side) of 5 colonies.
PCR Mix:
| Gene: | AtNHXS1 |
|---|---|
| Component | µl |
| Template | 1 per Tube |
| Primer f | 2 |
| Primer r | 2 |
| OneTap 2x Mastermix | 50 |
| ddH2O | 36 |
PCR Programm:
| Gene: | AtNH | XS1 |
|---|---|---|
| PCR Step | Time | Temperature |
| ID Initial denaturation | 30 sec | 94°C |
| D Denaturation | 15 sec | 94°C |
| A Annealing | 25 sec | 56°C |
| E Extention | 110 sec | 68°C |
| FE Final Elongation | 5 min | 68°C |
| S Storage | unlimited | 8°C |
We performed a Gel Electrophoresis (WT left, Ena middle, Positive Control right)
We prepared Overinghts of WT+AtNHXS1 (Colony 1) and Ena+AtNHXS1 (Colony 3) to make cryos and competent cells.
AVP1: None of the colonies we checked the day before integrated all of the four fragments.
Therefore, we isolated 12 others from the same plate and performed a check
-PCR.
08/01
AtNHXS1: We performed vericifaction PCRs of the left and right side of WT+AtNHXS1 (Colony 1) and Ena+AtNHXS1 (Colony 3). Primers included whole integrationsite and 100bp Genome on each side.
PCR Mix:
| Gene: | AVP1 |
|---|---|
| Component | µl |
| Template | 1 per Tube |
| Primer f | 0,4 |
| Primer r | 0,4 |
| One Taq 2x Mastermix | 10 |
| ddH2O | 7,2 |
PCR Programm:
| Gene: | Up-At | NHXS1 | T-D | own |
|---|---|---|---|---|
| PCR Step | Time | Temperature | Time | Temperature |
| ID Initial denaturation | 30 sec | 94°C | 30 sec | 94°C |
| D Denaturation | 15 sec | 94°C | 15 sec | 94°C |
| A Annealing | 25 sec | 56°C | 25 sec | 57°C |
| E Extention | 110 sec | 68°C | 200 sec | 68°C |
| FE Final Elongation | 5 min | 68°C | 5 min | 68°C |
| S Storage | unlimited | 8°C | unlimited | 8°C |
Gel Electrophoresis was successful for both strains (Left WT, middle Ena, right positive control)
Gel:
We made 25 competent cryos per strain (WT+AtNHXS1 and Ena+AtNHXS1) from the overnights made 07/31.
AtNHXS1-eGFP: We are planning to label AtNHXS1 with eGFP to colocalize it with ATG22-mRFP to the vacuolar membrane. We performed a PCR to amplify Fragment 3 (AtNHXS1 with Overhang for eGFP) and Fragment 5 (Failed).
PCR Mix:
| Gene: | AtNHXS1-eGFP |
|---|---|
| Component | µl |
| Template | 0,6 |
| Buffer | 10 |
| Primer f | 4 |
| Primer r | 4 |
| dNTP | 2 |
| Polymerase (PfuS) | 1 |
| ddH2O | 70,8 |
PCR Programm:
| Gene: | AtNH | XS1 |
|---|---|---|
| PCR Step | Time | Temperature |
| ID Initial denaturation | 30 sec | 98°C |
| D Denaturation | 8 sec | 98°C |
| A Annealing | 30 sec | 63°C |
| E Extention | 60 sec | 72°C |
| FE Final Elongation | 5 min | 72°C |
| S Storage | unlimited | 8°C |
We performed a Gel Electroophoresis (Fragment 3 left, 5 right), Clean-Up and determined the DNA-concentration.
AVP1: Because the last cPCR didn’t work, we performed a ramp PCR (AVP1 in Ena1 mutant). We prepared two mastermixes, split it in two tubes and added 1uL template DNA each.
| Gene: | AVP1 | |
|---|---|---|
| Component | Mix 1 | Mix 2 |
| Template | 1 µL | 1 µL |
| OneTag Mix | 10 µL | 10 µL |
| Primer f | 0,4 µL | 0,4 µL |
| Primer r | 0,4 µL | 0,4 µL |
| ddH2O | 9,2 µL | 9,2 µL |
PCR-program:
| Gene: | AV | P1 |
|---|---|---|
| PCR Step | Time | Temperature |
| ID Initial denaturation | 30 sec | 94°C |
| D Denaturation | 20 sec | 94°C |
| A Annealing | 30 sec | ramp 45-55°C |
| E Extention | 2 min 50 sec | 68°C |
| FE Final Elongation | 5 min | 68°C |
| S Storage | unlimited | 8°C |
Gelelectrophoresis:
The gelelectrophoresis showed that the transformation was sucessful.
In addition, we performed a cPCR with the AVP1 in wildtype genome.
It showed that this transformation failed.
08/02
AtNHXS1: We made cryos of WT+AtNHXS1 and Ena+AtNHXS1 from the Overnights made 07/31.
AtNHXS1-eGFP: We performed another PCR to amplify Fragment 5 (T-Down with eGFP Overhang).
PCR Mix:
| Gene: | T-Down |
|---|---|
| Component | µl |
| Template | 0,1 |
| Buffer | 10 |
| Primer f | 5 |
| Primer r | 5 |
| dNTP | 2 |
| Q5 High-Fidelity DNA Polymerase | 1 |
| ddH2O | 69,9 |
PCR Programm:
| Gene: | T-D | own |
|---|---|---|
| PCR Step | Time | Temperature |
| ID Initial denaturation | 30 sec | 98°C |
| D Denaturation | 8 sec | 98°C |
| A Annealing | 25 sec | 66°C |
| E Extention | 90 sec | 72°C |
| FE Final Elongation | 2 min | 72°C |
| S Storage | unlimited | 8°C |
We performed a Gel Electrophoresis, Clean-Up and determined the DNA-concentration.
Gel:
AVP1: We performed a new transformation to integrate AVP1 in wildtype using the protocol “Heat shock transformation S. cerevisiae” from Sandra.
08/03
AtNHXS1-eGFP: We performed a Transformation of AtNHXS-eGFP (5 Fragments) into BY4742 mRFP. No colonies grew, the Transformation failed.
LAB: We prepared the following Buffers for isolation of vacuoles: YPD-Media, Spheroplasting Buffer, DEAE-dextran solution, PS-Buffer with 4%, 8% and 15& Ficoll.
AVP1: We performed three PCRs to amplify our fragments for the transformation.
08/04
LAB: We performed a PCR to amplify the homologous region Down of the BY4742 genome for the Knock-Out of NHA1. The PCR failed.
AVP1: Transformation results (08/02): 21 colonies had grown on our plate. We isolated the genome and performed a check-PCR
Week 11
08/07
AtNHXS1-eGFP: We performed another Transformation of AtNHXS1-eGFP into BY4742 mRFP.
DNA Mix:
| Gene: | AtNHXS | 1-eGFP |
|---|---|---|
| Fragment | m needed [ng] | V needed [µl] |
| Up | 500 | 4,46 |
| Gal1 | 500 | 4,33 |
| AtNHXS1 | 500 | 7,87 |
| eGFP | 500 | 7,75 |
| T-Down | 350 | 5,98 |
| H2O | 3,61 |
Transformation Mix:
| Trafo: | AtNHXS1-eGFP into BY4742 mRFP |
|---|---|
| Component | Amount [µl] |
| 50% PEG 3350 | 240 |
| 1M LiAC | 36 |
| Carrier DNA | 50 |
AtNHXS1: We performed a Transformation of AtNHXS1 into AVENA (WT+Ena+AVP1)
DNA Mix:
| Gene: | AtNH | XS1 |
|---|---|---|
| Fragment | m needed [ng] | V needed [µl] |
| Up | 500 | 4,46 |
| Gal1 | 500 | 4,33 |
| AtNHXS1 | 500 | 3,38 |
| T-Down | 350 | 4,76 |
| H2O | 17,07 |
Transformation Mix:
| Trafo: | AtNHXS1 into BY4742 AVENA |
|---|---|
| Component | Amount [µl] |
| 50% PEG 3350 | 240 |
| 1M LiAC | 36 |
| Carrier DNA | 50 |
08/10
Biobrick Assembly AtNHXS1: We will integrate AtNHXS1 as a part into the Biobrick Plasmid pSB1C3. We performed a Restriction Digest of AtNHXS1 and pSB1C3 with EcoRI and PStI.
Digestion Mix:
| Gene: | pSB1C3 | AtNHXS1 |
|---|---|---|
| Component | µl | µl |
| Template | 4 | 2 |
| Buffer Cutsmart (19x) | 1 | 1 |
| EcoRI-HF | 0,5 | 0,5 |
| PstI-HF | 0,5 | 0,5 |
| ddH2O | 4 | 6 |
We performed a PCR Clean-Up, determined the DNA-concentrations performed a Ligation of Backbone and Insert.
Ligation Mix:
| Gene: | AtNHXS1 into pSB1C3 |
|---|---|
| Component | µl |
| Backbone (25ng) | 5 |
| Insert (90ng) | 18 |
| Buffer | 1 |
| T4 DNA Ligase | 1 |
We transformed the Ligation Mixture into E.coli DH5alpha and transformed the backbone as a negative control. Selection on LB-Plares with Cloramphenicol.
08/11
AtNHXS1-eGFP: We microscoped the transformed colonies of AtNHXS1-eGFP into BY4742 mRFP (Transformation: 08/07). Only around 1% of the cells fluoresced red. Every cell that fluoresced red, fluoresced green as well. Resolution was too low to colocalize transporters.
Week 12
08/14
AtNHXS1: We performed verification PCRs of the left part (+100bp from Genome) of 6 transformed colonies BY4742 AVENA+AtNHXS1 (ATAVENA).
PCR Mix:
PCR Program:
| Gene: | ATAVENA |
|---|---|
| Component | µl |
| Template | 1 per Tube |
| Primer f | 1,2 |
| Primer r | 1,2 |
| One Taq 2x Mastermix | 30 |
| ddH2O | 21,6 |
| Gene: | ATAV | ENA |
|---|---|---|
| PCR Step | Time | Temperature |
| ID Initial denaturation | 30 sec | 94°C |
| D Denaturation | 15 sec | 94°C |
| A Annealing | 25 sec | 56°C |
| E Extention | 110 sec | 68°C |
| FE Final Elongation | 5 min | 68°C |
| S Storage | unlimited | 8°C |
Gel Electrophoresis was successful for colonies 1 and 2.
AtNHXS1-eGFP: AtNHXS1-eGFP: Verification PCRs of 7 growing colonies failed.
AVP1: We prepared an overnight-culture to create competent cells, using the protocol McClean: Making Yeast competent cells.
08/15
AtNHXS1: We performed Verifiecation PCRs of both the left and right part of the Fragments (+100bp of the Genome on each side) of colonies 1 and 2.
PCR Mix:
PCR Program:
| Gene: | Left | Right |
|---|---|---|
| Component | µl | µl |
| Template | 1 per Tube | 1 per Tube |
| Primer f | 0,4 | 0,4 |
| Primer r | 0,4 | 0,4 |
| One Taq 2x Mastermix | 10 | 10 |
| ddH2O | 7,2 | 7,2 |
| Gene: | Le | ft | Rig | ht |
|---|---|---|---|---|
| PCR Step | Time | Temperature | Time | Temperature |
| ID Initial denaturation | 30 sec | 94°C | 30 sec | 94°C |
| D Denaturation | 15 sec | 94°C | 15 sec | 94°C |
| A Annealing | 25 sec | 56°C | 25 sec | 58°C |
| E Extention | 110 sec | 68°C | 200 sec | 68°C |
| FE Final Elongation | 5 min | 68°C | 5 min | 68°C |
| S Storage | unlimited | 8°C | unlimited | 8°C |
Afterwards we performed a Gel Electrophoresis, both colonies show correct results.
AtNHXS1-eGFP: We performed another verification PCR of the 7 growing colonies, which failed, the Transformation was unsuccessful.
BioBrick AtNHXS1-eGFP: We made Overnights of the 8 grown colonies from the Transformation from 08/11. Purpose: Plasmid Prep.
AVP1: We completed the making of the competent cells.
08/16
AtNHXS1-eGFP: We performed another Transformation of AtNHXS1+eGFP into BY4742 mRFP with a new eGFP Fragment.
DNA Mix:
Trafo Mix:
| Gene: | AtNHXS | 1-eGFP |
|---|---|---|
| Fragment | m needed [ng] | V needed [µl] |
| Up | 500 | 4,46 |
| Gal1 | 500 | 4,33 |
| AtNHXS1 | 500 | 7,87 |
| eGFP | 500 | 5,56 |
| T-Down | 350 | 5,98 |
| H2O | 1,42 |
| Trafo: | AtNHXS1-eGFP into BY4742 mRFP |
|---|---|
| Component | Amount [µl] |
| 50% PEG 3350 | 240 |
| 1M LiAC | 36 |
| Carrier DNA | 50 |
Biobrick AtNHXS1: We made a Plasmid Prep and Cryos of 8 colonies with the Overnights from 08/15. Afterwards we performed a Single Digest of the Plasmids with EcoRI.
Digestion Mix:
| Gene: | AtNHXS1-pSB1C3 |
|---|---|
| Component | µl |
| Cut Smart Buffer | 1 |
| Template DNA | 5 |
| EcoRI | 1 |
| ddH2O | 3 |
We performed a PCR Clean-Up. Gel Electrophoresis was successful for colonies 1-5 and 8.
LAB:We made Overnights of several mutants (WT,Ena,Avp,Avena,AtNHXS1,AtEna,AtAvena) for Cryos, Galactose Growth Check, Genome Isolation and making competent cells.
08/17
AtNHXS1: We performed a Transformation of AtNHXS1 into AVP (AVPA).
DNA Mix:
Trafo Mix:
| Gene: | AtNH | XS1 |
|---|---|---|
| Fragment | m needed [ng] | V needed [µl] |
| Up | 500 | 4,46 |
| Gal1 | 500 | 4,33 |
| AtNHXS1 | 500 | 3,38 |
| T-Down | 350 | 4,76 |
| H2O | 17,07 |
| Trafo: | AtNHXS1 into BY4742 AVP |
|---|---|
| Component | Amount [µl] |
| 50% PEG 3350 | 240 |
| 1M LiAC | 36 |
| Carrier DNA | 50 |
We made 10 eppis competent cells of ATAVENA from the Overnight of 08/16.
AVP1: We performed a transformation to integrate AVP1 together with eGFP in our mRFP mutant. We used the same protocol as always.
After the plate was incubated at 28°C for 4 days, 6 colonies grew.
08/18
Galactose Growth Check: We plated 6 different strains (WT, AVP, AVENA, AtNHXS1, AtENA, AtAVENA) on YPD-Agar containing 60%, 80% and 100% Galactose as carbon source.
LAB: We made Cryos of 7 BY4742 strains (WT, ENA, AVP, AVENA, AtNHXS1, AtENA, AtAVENA). We isolated the genome of 6 BY strains (AVP, AVENA, AtNHXS1, AtENA, AtAVENA, mRFP) and dterermined the DNA-concentrations.
AtNHXS1: We performed PCRs to amplify template DNA for Sequencing of AtNHXS1 of the strains AtNHXS1, AtENA, AtAVENA (Promoter – 40bp of Terminator) and additionally BY mRFP. We want to sequence 500bp of ATG22 and the whole mRFP gene. The PCRs failed.
Week 13
08/21
AtNHXS1: We performed new PCRs to amplify the templates for sequencing of the AtNHXS1 part of AtNHXS1, AtENA, AtAVENA and additionally BY mRFP. The PCRs failed.
AVP1: (We checked our transformation of the mRFP mutant. It failed.)
08/22
AtNHXS1: We performed new PCRs to amplify the templates for sequencing of the AtNHXS1 part of AtNHXS1, AtENA, AtAVENA and additionally BY mRFP. The PCRs failed.
AVP1: We send the genome fragments of our transformed wildtype
and the Ena1-knock-out mutant to eurofins to get it sequenced.
We repeated the transformation with AVP1 + eGFP with a higher AVP1
concentration. The used protocol we used was the same as always.
08/24
AVP1: We got our sequencing results: the integration of AVP1 in wildtype was successful, the AVP1 gene in the Ena1-kock-out had a point-mutation. (The transformation of the mRFP mutant with AVP1 + eGFP failed.)
Week 14
08/28
AVP1: We performed a PCR to amplify our fragments.
08/29
AVP1: We integrated the AVP1 fragment into four different mutants form the other teams:
- ATENA (AtNHXS1 + Ena1-knockout)
- Ena1 (Ena1 knockout)
- HATENA (ATENA + NHA1)
- NHNA (NHA1 + Ena1-kockout
The protocol we used was the same as always.
08/31
AtNHXS1: We performed Verifiecation PCRs of both the left and right part of the Fragments (+100bp of the Genome on each side) of colonies 1 and 2.
PCR Mix:
PCR Program:
| Gene: | Left | Right |
|---|---|---|
| Component | µl | µl |
| Template | 1 per Tube | 1 per Tube |
| Primer f | 0,4 | 0,4 |
| Primer r | 0,4 | 0,4 |
| One Taq 2x Mastermix | 10 | 10 |
| ddH2O | 7,2 | 7,2 |
| Gene: | Le | ft | Rig | ht |
|---|---|---|---|---|
| PCR Step | Time | Temperature | Time | Temperature |
| ID Initial denaturation | 30 sec | 94°C | 30 sec | 94°C |
| D Denaturation | 15 sec | 94°C | 15 sec | 94°C |
| A Annealing | 25 sec | 56°C | 25 sec | 58°C |
| E Extention | 110 sec | 68°C | 200 sec | 68°C |
| FE Final Elongation | 5 min | 68°C | 5 min | 68°C |
| S Storage | unlimited | 8°C | unlimited | 8°C |
Afterwards we performed a Gel Electrophoresis, both colonies show correct results.
AtNHXS1-eGFP: We performed another verification PCR of the 7 growing colonies, which failed, the Transformation was unsuccessful.
BioBrick AtNHXS1-eGFP: We made Overnights of the 8 grown colonies from the Transformation from 08/11. Purpose: Plasmid Prep.
09/01
AVP1: There was growth on all plates. We performed a check-PCR with 5 colonies of each mutant.
- ATENA (AtNHXS1 + Ena1-knockout): colony 5
- Ena1 (Ena1 knockout): colony 3
- HATENA (ATENA + NHA1): colony 3
- NHNA (NHA1 + Ena1-kockout: colony 1
We send the fragments containing our integrated genes to eurofins for sequencing.
Week 15
09/05
AVP1: We amplified the fragments 1, 2, 3 & 4 via PCR and repeated the transformations of Ena1 and ATENA, using the same protocol as always.
09/06
mRFP: We performed a new Transformation of mRFP into BY4742 WT. Selection with Geniticin.
DNA Mix:
| Gene: | mR | FP |
|---|---|---|
| Fragment | m needed [ng] | V needed [µl] |
| Homologous Region Left | 500 | 1,48 |
| KanMX | 250 | 5 |
| mRFP | 500 | 1,03 |
| Homologous Region Right | 500 | 4 |
| H2O | 22,49 |
Trafo Mix:
| Trafo: | mRFP into BY WT |
|---|---|
| Component | Amount [µl] |
| 50% PEG 3350 | 240 |
| 1M LiAC | 36 |
| Carrier DNA | 50 |
AtSultr1;2: We performed a Transformation of AtSultr1;2 (4 Fragments) into BY4742 WT. Selection with SC-Plates without Lysin. The Transformation failed because no colonies grew.
AVP1: Due to low concentrations, we amplified the fragments 1, 2 & 3 again.
09/07
AVP1:We integrated the fragments in the genome of the mutants Ena1, ATENA, HATENA and NHNA, using the same protocol as always.
Results after two days: growth on all plates.
Week 16
09/11
AVP1: We performed a cPCR of the colonies transformed 09/08. Five colonies per mutant were checked.
Gel-electrophoresis:
The bands were at the expected height.
We send the desired gene-fragments to eurofins to get it sequenced.
09/12
AVP1: Sequencing results: two of the mutants (AVENA & HATAVENA) contained point mutations, the other two (ATAVENA & PHA) couldn't be sequenced due to too low DNA-concentrations.
(We performed a new PCR to amplify the desired fragment for the sequencing. It failed.)
09/13
AVP1: To check the expression of our protein AVP1 we decided to perform a SDS-page. See protocol.
09/14
AVP1: We performed the SDS-page. See protocol.
09/15
AVP1: We performed the SDS-page. See protocol.
Week 17
09/18
AVP1: We integrated the AVP1 fragment into four different mutants form the other teams:
- ATENA (AtNHXS1 + Ena1-knockout)
- Ena1 (Ena1 knockout)
- HATENA (ATENA + NHA1)
- NHNA (NHA1 + Ena1-kockout
The protocol we used was the same as always.
09/20
AtNHXS1-eGFP: We microscoped the transformed colonies of AtNHXS1-eGFP into BY4742 mRFP (Transformation: 08/07). Only around 1% of the cells fluoresced red. Every cell that fluoresced red, fluoresced green as well. Resolution was too low to colocalize transporters.
09/22
AtNHXS1: We performed Verifiecation PCRs of both the left and right part of the Fragments (+100bp of the Genome on each side) of colonies 1 and 2.
PCR Mix:
PCR Program:
| Gene: | Left | Right |
|---|---|---|
| Component | µl | µl |
| Template | 1 per Tube | 1 per Tube |
| Primer f | 0,4 | 0,4 |
| Primer r | 0,4 | 0,4 |
| One Taq 2x Mastermix | 10 | 10 |
| ddH2O | 7,2 | 7,2 |
| Gene: | Le | ft | Rig | ht |
|---|---|---|---|---|
| PCR Step | Time | Temperature | Time | Temperature |
| ID Initial denaturation | 30 sec | 94°C | 30 sec | 94°C |
| D Denaturation | 15 sec | 94°C | 15 sec | 94°C |
| A Annealing | 25 sec | 56°C | 25 sec | 58°C |
| E Extention | 110 sec | 68°C | 200 sec | 68°C |
| FE Final Elongation | 5 min | 68°C | 5 min | 68°C |
| S Storage | unlimited | 8°C | unlimited | 8°C |
Afterwards we performed a Gel Electrophoresis, both colonies show correct results.
AtNHXS1-eGFP: We performed another verification PCR of the 7 growing colonies, which failed, the Transformation was unsuccessful.
BioBrick AtNHXS1-eGFP: We made Overnights of the 8 grown colonies from the Transformation from 08/11. Purpose: Plasmid Prep.
Week 18
09/25
Since the labelling of the vacuole with mRFP and mCherry did not showed the expected results (gene was integrated correct, but there was no fluorescence) we decided to use the fluorescenceprotein mOrange Part BBa_E2050 and create a fusionprotein with the VNX-channel, that only appears in the vacuoles. Therefore we designed and ordered the respective primers.
09/26
We performed Cryos of ATAVENA to back up the transformated cells, after we recieved the sequencing results that showed, that the gene is correct.
We sequenced new colonies of HATAVENA (col 5, 16 and 18) from the transformation that we performed on 08.09.2017. Therefore we performed PCRs with the genomes to isolate the AVP1-cassette for the sequencing.
Furthermore we prepared a RNA-isolation and q-PCR that we want to perform with our AVP1-mutante and the wildtype to check the transcription rate. Therfore we prepared Overnights: a) 2 Overnights of AVP1-mutante in YPD+Galactose b) 2 Overnights of AVP1-mutante in YPD+Glucose and c) 2 Overnights of BY4742 wildtype. We also prepared the buffers and solution we will need for the RNA-Isolation (see protocol!).
09/27
We isolated the RNA of AVP1-mutante and the BY4742 wildtype and performed the reverse transcription by following the protocol “qPCR”. The isolation was successful, the samples were freezed at -80°C.
The sequencing results of HATAVENA showed, that colony 5 and 18 integrated the AVP1-cassette correctly.
09/28
We performed PCRs to amplify the fragments for the integration of AtSultr1;2.
(Biotech-Day)
09/29
(Biotech-Day)
We performed a transformation of AtSult1;2 in BY AVP1 and a transformation of AVP1 and AKT1 in BY XENA (-Cre).
We performed PCRs to amplify the 5 fragments for the integration of the fusion protein VNX+mOrange.
Week 19
10/02
We performed the qPCR using our cDNA which we generated from our isolated RNA (see 27.09.2017) to verify the transcription of the integrated AVP1-gene. The results showed, that the wildtype transcribate no AVP1, the RNA-amount of the induced AVP1-mutante was about 3 time higher than the amount of uninduced AVP1-mutante. (Check our result-page!)
We performed a transformation of mOrange and VNX (fusionprotein) in BY 4742 wildtype.
10/04
We isolated the genomes of the colonies containing mOrange-VNX (transformation see 02.10.2017) for verification.
We performed verification PCRs of AVP1 and AKT1 with the genomes of the transformated colonies (see transformation 20.09.2017). The results showed that only AVP1 was integrated successfully. We performed PCRs for sequencing.
10/05
The sequencing results of AVP1 in BY XENA showed that the gene was integrated correctly. The new mutants are named BY XAVENA.
We performed further verification PCRs of the genomes containing VNX+mOrange, because the PCRs of 04.10.17 showed unexpected results.
We isolated genomes of new colonies containing VNX+mOrange of the transformation see 02.10.2017 and performed verification PCRs.
Week 20
10/11
We performed several transformations: we transformed AKT1-gene into BY NHNA ( a)with the transformation kit and also b) by following the transformation protocol), we transformed AtSult1;2 into BY 4742 wildtype and we transformed mOrange+VNX also in BY2742 wildtype.
10/12
ENA1:Since we had many different mutant colonies where our transformation did seem to work, we decided to store them in case something went wrong along the way. We produced 10 different overnights for ENA1 K.O transformations and wild type BY4742.
Interlab Study:We transformed samples of competent E. coli cells. We transformed with three different controls; one negative, one positive and another positive with a RFP marker. We produced 30 LB plates containing chloramphenicol from which 18 plates were used.
10/13
AVP1: We integrated the AVP1 fragment into four different mutants form the other teams:
- ATENA (AtNHXS1 + Ena1-knockout)
- Ena1 (Ena1 knockout)
- HATENA (ATENA + NHA1)
- NHNA (NHA1 + Ena1-kockout
The protocol we used was the same as always.
Week 21
10/16
AtNHXS1: We performed Verifiecation PCRs of both the left and right part of the Fragments (+100bp of the Genome on each side) of colonies 1 and 2.
PCR Mix:
PCR Program:
| Gene: | Left | Right |
|---|---|---|
| Component | µl | µl |
| Template | 1 per Tube | 1 per Tube |
| Primer f | 0,4 | 0,4 |
| Primer r | 0,4 | 0,4 |
| One Taq 2x Mastermix | 10 | 10 |
| ddH2O | 7,2 | 7,2 |
| Gene: | Le | ft | Rig | ht |
|---|---|---|---|---|
| PCR Step | Time | Temperature | Time | Temperature |
| ID Initial denaturation | 30 sec | 94°C | 30 sec | 94°C |
| D Denaturation | 15 sec | 94°C | 15 sec | 94°C |
| A Annealing | 25 sec | 56°C | 25 sec | 58°C |
| E Extention | 110 sec | 68°C | 200 sec | 68°C |
| FE Final Elongation | 5 min | 68°C | 5 min | 68°C |
| S Storage | unlimited | 8°C | unlimited | 8°C |
Afterwards we performed a Gel Electrophoresis, both colonies show correct results.
AtNHXS1-eGFP: We performed another verification PCR of the 7 growing colonies, which failed, the Transformation was unsuccessful.
BioBrick AtNHXS1-eGFP: We made Overnights of the 8 grown colonies from the Transformation from 08/11. Purpose: Plasmid Prep.
10/17
AtNHXS1: We performed verification PCRs of the left part (+100bp from Genome) of 6 transformed colonies BY4742 AVENA+AtNHXS1 (ATAVENA).
PCR Mix:
PCR Program:
| Gene: | ATAVENA |
|---|---|
| Component | µl |
| Template | 1 per Tube |
| Primer f | 1,2 |
| Primer r | 1,2 |
| One Taq 2x Mastermix | 30 |
| ddH2O | 21,6 |
| Gene: | ATAV | ENA |
|---|---|---|
| PCR Step | Time | Temperature |
| ID Initial denaturation | 30 sec | 94°C |
| D Denaturation | 15 sec | 94°C |
| A Annealing | 25 sec | 56°C |
| E Extention | 110 sec | 68°C |
| FE Final Elongation | 5 min | 68°C |
| S Storage | unlimited | 8°C |
Gel Electrophoresis was successful for colonies 1 and 2.
AtNHXS1-eGFP: AtNHXS1-eGFP: Verification PCRs of 7 growing colonies failed.
10/20
ENA1:Since we had many different mutant colonies where our transformation did seem to work, we decided to store them in case something went wrong along the way. We produced 10 different overnights for ENA1 K.O transformations and wild type BY4742.
Interlab Study:We transformed samples of competent E. coli cells. We transformed with three different controls; one negative, one positive and another positive with a RFP marker. We produced 30 LB plates containing chloramphenicol from which 18 plates were used.
Week 22
10/23
We performed two double digests of a) the AKT1-gene b) the AtSultr1;2 and c) the pSB1C3-backbone with EcoRI and PstI. Afterwards we ligated a) AKT1 with pSB1C3-backbone and b) the AtSultr1;2 with pSB1C3-backbone and finally performed the transformation of the ligated plasmids into competent E.coli DH5α.
10/25
We performed Overnights of the grown colonies of the transformation with the pSB1C3-AtSultr1;2-plasmid.
10/27
ENA1:Since we had many different mutant colonies where our transformation did seem to work, we decided to store them in case something went wrong along the way. We produced 10 different overnights for ENA1 K.O transformations and wild type BY4742.
Interlab Study:We transformed samples of competent E. coli cells. We transformed with three different controls; one negative, one positive and another positive with a RFP marker. We produced 30 LB plates containing chloramphenicol from which 18 plates were used.
