Template:Groningen/Notebook
Notebook
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Week 25
- Monday June 18th
Who:
Aim
- Tuesday June 19th
Who:
Aim
- Wednesday June 20th
Who:
Aim
- Thursday June 21st
Who: Owen
Aim Preparation of 200 ml YPD and 200 ml YPD with agar. Preparation of 200 ml LB medium and 200 ml LB medium with 2% agar
For preparation of 200 ml YPD add to demi water:
- 2 g yeast extract
- 4 g peptone
- 4 g glucoseFor preparation of 200 ml YPD 2% agar add to demi water:
- 2 g yeast extract
- 4 g peptone
- 4 g glucose
- 4 g agarFor preparation of 200 ml LB add to demi water:
- 4 g LBFor preparation of 200 ml LB 2% agar add to demi water:
- 4 g LB
- 4 g agar - Friday June 22nd
Who: Owen
Aim Prepare 4 ml 1000x ampicillin stock (100mg/ml in MQ)
- Add 400 mg ampicillin to 4 ml MQ and filter sterilize.
- Aliquot and store at - 20 degrees C.Who: Owen
Aim Pour YPD plates and LB+Amp plates
Autoclave media at 121 degrees C for 15 minutes and cool to hand warm.
- Add 200 ul 1000x ampicillin to 200 ml LB medium with agar.
- Pour LB + Amp plates.
- Pour YPD platesWho: Owen
Aim Grow strain BY4741 for cloning
Strain provided by Molecular Microbiology group RUG.
- Plate BY4741 on YPD and grow over the weekend at 30 degrees.
25-6-18 Colonies visible for BY4741 on YPD plate. - Saturday June 23rd
Who:
Aim
- Sunday June 24th
Who:
Aim
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Week 26
- Monday June 25th
Who:
Aim
- Tuesday June 26th
Who:
Aim
- Wednesday June 27th
Who:
Aim
- Thursday June 28th
Who:
Aim
- Friday June 29ththing we did
- Saturday June 30th
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- Sunday July 1st
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Week 27
- Monday July 2nd
Who:
Aim
- Tuesday July 3rd
Who:
Aim
- Wednesday July 4th
Who:
Aim
- Thursday July 5th
Who:
Aim
- Friday July 6th
Who: Rianne & Phillip
Aim Competent cell test
To test the competence of our cells, we performed a competent cell test according to the IGEM Competent Cell test kit protocol.
We dissolved the dried-down purified plasmid DNA from BBa_J04450 into 50 µl of water to obtain 100 pg/µl and 10 pg/µl concentrations. In short, 1 µl of each DNA concentration was added to 50 µl of competent cells in duplicates. We obtained plenty of colonies and the efficiency of transformation was determined sufficient. This batch of competent E. coli DH5ɑ cells was used for all of the following E. coli transformations.
- Saturday July 7th
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Aim
- Sunday July 8th
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Week 28
- Monday July 9th
Who:
Aim
- Tuesday July 10th
Who: Rianne & Phillip
Aim Transformation of competent E. coli DH5ɑ with the devices for the iGEM Interlab study
The next devices were provided to us by iGEM in the plates in the distribution kit. The following devices were resuspended into 10 µl sterilized deionized water.
Device Location on plate 7 Negative control 2D Positive control 2B Device 1 2F Device 2 2H Device 3 2J Device 4 2L Device 5 2N Device 6 2P 1 µl of this solution was transformed into 50 µl of competent DH5ɑ cells using the following protocol:
- LB plates with the appropriate antibiotics were prepared as described here (link to our protocol page LB agar plates)
- Eppendorf tubes (1.5 ml) were pre-chilled on ice and competent cells were thawed on ice
- 50 µl of competent cells was added to the pre-chilled eppendorf tubes, together with the DNA
- 30 min incubation on ice
- Heat shock for 45 sec in a 42℃ water bath
- Incubation on ice for 5 min
- 950 µl of LB broth was added to the cells
- Incubation for 1 hour, 37℃, 200 rpm
- Plating of the cells on the LB-agar plates. 100 µl one one plate, then the culture is centrifuged and the supernatant is partially discarded. The cell pellet is resuspended in approximately 100 µl and plated on a separate plate.
- Overnight incubation at 37℃ (agar side up)
- Wednesday July 11th
Who: Rianne
Aim Growth of the colonies used in the iGEM Interlab study
We obtained between 10-100 colonies per transformation. Two colonies were picked with a sterile pipette tip and transferred into 5 ml of LB with chloramphenicol. The cultures were grown in the appropriate broth overnight at 37℃ in a shaking incubator at 200 rpm.
Who: Rianne
Aim To purify the plasmids from Wen. et al out of E. coli for transformation into yeast
- 1882: PYD1 - CipA1 - EGII
- 1883: PYD1 - CipA3 - EGII
- CB: PRS425 - CBHII - BGLI
These three plasmids that we received from Wen. et al were kindly transformed by Vakhil Takhaveev into E. coli and plated onto LB agar plates. In order to purify the plasmids out of these cells, I grew three colonies of each strain into 10 ml of LB with the appropriate antibiotics. The next day, the plasmid was extracted from the full-grown cultures using a plasmid extraction kit. The following concentrations (ng/µl) were obtained:
Results
1882 1883 CB 1 278,15 572,80 443,80 2 664,10 98,0 361,70 3 321,25 652,0 439,95 The plasmids were stored at -20℃ until further use.
- Thursday July 12th
Who: Rianne
Aim Production of glycerol stocks for the strains used in the iGEM Interlab study
250 µl of culture was mixed with 250 µl of 50% glycerol and stored at -80℃. Simultaneously, a small amount of the glycerol stock was transferred into 5 ml of fresh medium supplemented with chloramphenicol and grown overnight at 37℃, 200 rpm.
- Friday July 13th
Who: Rianne & Phillip
Aim First cell measurement for the iGEM Interlab study
- 500 µl of the overnight culture was transferred into 4,5 ml of LB with chloramphenicol
- OD600 measurement of 1:10 dilutions
- Dilution of these 1:10 cultures to target OD600 0.2 in a final volume of 12 ml
- 1000 µl of the 0 hour time point culture was pipetted into an eppendorf tube and kept on ice until further use in a box with ice at 4℃.
- Six hours of incubation at 37℃, 200 rpm
- 1000 µl of the 6 hour time point culture was pipetted into an eppendorf tube and kept on ice until further use in a box with ice at 4℃.
- 100 µl of these cell cultures were transferred into wells of a 96-microtiter plate.
Colony 1 Colony 2 Negative control 0.191 0.192 Positive control 0.198 0.204 Device 1 0.152 0.149 Device 2 0.189 0.208 Device 3 0.19 0.191 Device 4 0.15 0.171 Device 5 0.094 0.109 Device 6 0.204 0.195 10 ml of culture was already pipetted into the 50 ml falcon tubes for all cultures, except for cultures of device 5, for which 9 ml was used. To these tubes, the following volumes (µl) were added:
Colony 1 Cell culture LB Colony 2 Cell culture LB Negative control 1257 743 Negative control 1249 751 Positive control 1212 788 Positive control 1176 824 Device 1 1579 421 Device 1 1611 389 Device 2 1269 731 Device 2 1154 846 Device 3 1263 737 Device 3 1257 743 Device 4 1599 401 Device 4 1404 596 Device 5 2553 447 Device 5 2202 798 Device 6 1176 824 Device 6 1231 769 However, while analyzing our results, we realized that we did not set the ‘gain’ setting for fluorescence measurement in the plate reader to a fixed number across the different measurements. Therefore, we need to repeat this measurement.
Aim Correlate OD600 measurements in our plate reader to colony forming units (CFU)
We first diluted the overnight cultures of the negative and positive control 1:8 and measured OD600 in our plate reader. We then further diluted these cultures to target OD600 0.1, confirmed by our plate reader and plated onto plates in several dilutions.
Aim Calibration experiments for the Interlab study
LUDOX, silica beads and fluorescein experiments as described on our interlab page were also performed. As described above, the fluorescein calibration needs to be repeated.
The exact protocol can be found here and the results of these experiments can be found on our Interlab page.
- Saturday July 14th
Who: Rianne & Phillip
Aim Counting colony forming units
We checked the colonies and counted them, to calculate how many cells were present in our culture of which the plate reader displayed the OD600 to be 0.1. The following numbers of colonies were found, where + and - stand for positive and negative control, respectively, and 1 and 2 stand for the two colonies that were picked at day 11-7-18. (4) and (5) stand for the dilutions plated as performed on 13-7-18.
+1(4) +1(5) +2(4) +2(5) -1(4) -1(5) -2(4) -2(5) 1 181 11 192 23 191 9 170 9 2 150 11 208 14 112 17 202 19 3 166 19 184 22 213 11 205 16 - Sunday July 15th
Who:
Aim Growth of yeast strain YGEN 0013 and the interlab strains for second measurement
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Week 29
- Monday July 16th
Who: Rianne
Aim Growth of yeast strain YGEN 0013 and the interlab strains for second measurement
YGEN 0013 colonies were picked and grown in Verduyn medium supplemented with the appropriate vitamins and trace elements, with addition of uracil, tryptophan and leucin. The strain was grown in 5 ml overnight at 30℃, 200 rpm.
To repeat the Interlab fluorescence measurements, the glycerol stocks of the interlab strains 12-7-18 were taken out of the -80℃, and grown in 5 ml of LB and the appropriate antibiotic overnight at 37℃, 220 rpm.
- Tuesday July 17th
Who: Rianne & Phillip
Aim Second Interlab measurement
The same protocol as described on 13-8-18 and in the Interlab study was used. The starting OD600 of the 1:10 diluted overnight cultures were:
Colony 1 Colony 2 Negative control 0.19 0.182 Positive control 0.167 0.168 Device 1 0.205 0.181 Device 2 0.17 0.175 Device 3 0.185 0.19 Device 4 0.168 0.184 Device 5 0.178 0.185 Device 6 0.195 0.174 Dilution of these 1:10 cultures to target OD600 0.2 in a final volume of 12 ml: 10 ml of culture was already pipetted into the 50 ml falcon tubes for all cultures. To these tubes, the following volumes (µl) were added:
Colony 1 Cell culture LB Colony 2 Cell culture LB Negative control 1263 737 Negative control 1319 681 Positive control 1437 563 Positive control 1429 571 Device 1 1171 829 Device 1 1326 674 Device 2 1412 588 Device 2 1371 629 Device 3 1297 703 Device 3 1263 737 Device 4 1429 571 Device 4 1304 696 Device 5 1348 652 Device 5 1297 703 Device 6 1231 769 Device 6 1379 621 The cell measurements and the fluorescein measurements were repeated, but now the gain settings were fixed between the measurements. You can find the results on our Interlab page.
- Wednesday July 18th
Who: Rianne & Ingeborg
Aim Purification of PhipZ
E. coli containing PhipZ (with both an ampicillin and zeocin marker) was grown in LB containing ampicillin and the plasmid was extracted using a plasmid extraction kit. Four parallel cultures were used to purify PhipZ and the following concentrations were obtained (ng/µl): 381.7, 319.9, 419.2 and 389.05. This will be sufficient for the rest of our project. The plasmids were stored at -20℃.
- Thursday July 19th
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- Friday July 20th
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- Saturday July 21st
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- Sunday July 22nd
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Week 30
- Monday July 23rd
Who: Rianne
Aim PCR on EGII and CBHI gene fragments
Primers for the gene fragments were diluted in MQ to 100 µM (a working stock of 50 µM was also prepared). Synthesized gene fragments were diluted in MQ to 5 ng/µl. For both EGII and CBHI, three PCR reactions were performed:
A B C PCR buffer (5X) 10 10 10 Primer A (50 µM) 1 1 1 Primer B (50 µM) 1 1 1 Polymerase 1 1 1 Template (5ng/µl) 1 1 1 Q buffer 10 Mg2+ 2 MQ 36 26 34 Primer melting temperatures:
EGII:
FW = 51,3
Rev = 49,2CBHI:
Fw = 51,3
Rev = 48,6PCR programme run in a thermocycler:
- 95℃ - 5 min
- 94℃ - 15 sec
- 44℃ - 1 min
- 72℃ - 1 min
- 72℃ - 10 min
- 4℃ on hold
Steps 2-4 were repeated 35 times
Who: Rianne
Aim Count colonies & pick colonies from yeast transformation
Negative control
-Leu, -Trp: 0 colonies
-Trp: at least 10 colonies
-Leu: 0 colonies1882+CB 1883+CB 1882 1883 CB 10% 1 2 61 55 21 90% 32 32 many many many Note: -trp plates look different than others. They are white-ish and the colonies are small.
Two colonies of each plate were used to inoculate 5 ml of Verduyn medium with the appropriate uracil, leucin or tryptophan supplementation. The cultures were grown overnight at 30℃ and 200 rpm
- Tuesday July 24th
Who: Rianne
Aim Agarose gel of 23-07-18 PCR products
3 µl of the ladder was loaded, 12 µl of the PCR products were mixed with 3 µl of sample buffer. NC = negative control (no template added). L = ladder.
- Upper lane: L - EGII(A)-NC(A)-EGII(B)-NC(B)-EGII(C)-EGII(C)-NC(C)-CBHII(A)-NC(A)
- Lower lane: L- CBHII(B)-NC(B)-CBHII(C)-NC(C)-ctrl EGII-ctrl CBHII
- Wednesday July 25th
Who: Rianne
Aim Sending plasmids pNZ8048 and pPMK4 to Leiden for collaboration
A small volume of purified plasmid pNZ8048 (obtained from Alisa Garaeva) and PMK4 (obtained from Buu Minh Tran) were transferred to screw-capped microtubes, and, accompanied by a dried drop on Whatman filter paper, sent to Leiden by mail.
The plasmids arrived in Leiden soon!
The Leiden Igem team with the eppendorf tubes containing the plasmids. - Thursday July 26th
Who: Rianne & Bram
Aim PCR of EGII
Because two bands appeared in the previous PCR reaction, we want to change the reaction conditions to see if we can obtain a single product with the right length. Two conditions were tested. The temperature was increased to prevent non-specific binding and DMSO was added.
A B PCR buffer (5X) 10 10 Primer A (50 µM) 1 1 Primer B (50 µM) 1 1 Polymerase 1 1 Template (5ng/µl) 2 2 DMSO 3 MQ 35 32 Primer melting temperatures:
EGII:
FW = 51,3
Rev = 49,2PCR programme run in a thermocycler:
- 95℃ - 5 min
- 94℃ - 15 sec
- 46℃ - 1 min
- 72℃ - 1 min
- 72℃ - 10 min
- 4℃ on hold
Steps 2-4 were repeated 35 times
Ladder - EGII A - EGII B Gel was kept in the fridge overnight before use, which is probably the reason why the bands are not as clear as desired
- Friday July 27th
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- Saturday July 28th
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- Sunday July 29th
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Week 31
- Monday July 30th
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- Tuesday July 31st
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- Wednesday August 1st
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- Thursday August 2nd
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- Friday August 3rd
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- Saturday August 4th
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- Sunday August 5th
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Week 32
- Monday August 6th
Who: Rianne
Aim Restrictions of cellulosome PCR products or gene fragments
Restriction reactions were performed in a 50 µl total reaction volume. (1µl of each restriction enzyme, 5 µl of 10X restriction buffer, 1 µg of DNA). For all reactions, NEB buffer 3.1 was used with enzymes BamH1 and Xho1.
Concentration stock (ng/µl) For ~1 µg (µl) MQ CBHI 225,5 5 38 EGII (PCR with DMSO, 26-7) 145 7,5 35,5 EGII (no DMSO, 26-7) 150 7,5 35,5 PhipZ 300 4 39 Reactions were incubated at 37℃ for 1,5 hours.
- PCR clean-up gave the following concentrations of restricted fragments:
- -CBHI31,35
- -EGII (DMSO)27,05
- -EGII 28,35
- -PhipZ32,55
The restricted clean fragments were stored at -20℃ until further use. The genes Scaffold part 1 and Scaffold part 2 were diluted to 5 ng/µl by addition of 200 µl MQ to the delivered dried genes. One third (~300 ng in 65,5 µl) was used for a 75 µl restriction reaction with the following additions:
Enzyme 1 (1 µl) Enzyme 2 (1 µl) Buffer (7,5 µl) Scaffold part 1 BamH1 Cla1 NEB 3 Scaffold part 2 Xho1 Cla1 Cutsmart The mixtures were incubated for 2,5 hrs at 37℃ and then kept at 4℃ overnight. The genes BGL part 1 and BGL part 2 were diluted to 10 ng/µl by addition of 100 µl MQ to the delivered dried genes. One third (~300 ng in 30 µl) was used for a 50 µl restriction reaction with the following additions:
Enzyme 1 (1 µl) Enzyme 2 (1 µl) Buffer (5 µl) BGL part 1 BamH1 Nru1 NEB 3 BGL part 2 Xho1 Nru1 NEB 3 The mixtures were filled up to 50 µl by addition of 13 µl MQ and incubated for 1,5 hrs at 37℃ and then kept at 4℃ overnight.
- Tuesday August 7th
Who: Rianne
Aim Restriction cleanup and ligation
PCR cleanup of the previous restriction reactions gave the following concentrations:
- -Scaffold part 13,2 ng/µl
- -Scaffold part 24,8 ng/µl
- -BGL part 12,55 ng/µl
- -BGL part 22,9 ng/µl
For a vector:insert(:insert) equimolar ratio of around 1:3(:3), the following ligation mixtures were prepared:
A, B: 100 ng plasmid. C, D: 50 ng plasmid reactions.
A (µl) B (µl) C (µl) D (µl) Control PhipZ 3.1 3.1 1,54 1,54 3,1 CBH 3 EGII 3.12 BGLI part 1 13.2 BGLI part 2 16.1 Scaffold part 1 11 Scaffold part 2 7 T4 ligase 1 1 2 2 1 Ligase buffer 1 1 3.5 2.5 1 MQ 1.9 1.78 1 4.9 Total volume 10 10 35 25 10 The ligation mixtures were incubated for 2 hours at 37℃, following 20 minutes at 80℃ to heat-kill the enzymes.
Who: Rianne
Aim Transformation
Ligated amount Into X competent cells (µl) A all 75 B all 75 C 35 200 D 25 150 V all 75 Following the same protocol as previously used. Cells were plated on LB-ampicillin plates (100µl, 250µl and rest) and incubated at 37℃ overnight.
- Wednesday August 8th
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- Thursday August 9th
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- Friday August 10th
Who: Rianne
Aim Grow the cellulosome-strains on cotton
Common cotton pads were purchased from a local supermarket and 20 mg cotton was weighed and transferred into glass tubes. The tubes were subsequently autoclaved.
Glass tubes with 2%, 0,2%, 0,02%, 0,002% and 0,0002% galactose were prepared. Both with and without the cotton. 10 ml of Verduyn medium (supplemented with the appropriate vitamins, trace elements and with uracil) was added to each tube. As a positive control, LB and E. coli bacteria were added to a glass tube containing cotton, to exclude non-growth because of chemicals in the cotton pads. Also, Verduyn medium containing 2% glucose was added to one of the glass tubes with cotton, to ensure viability of the yeast strains.
Colonies of CB1883 and CB1882 were taken from the plate and inoculated into the tubes. The cultures were incubated at 30℃, 150 rpm.
Results Growth in both positive controls, no growth in the remaining tubes.
- Saturday August 11th
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- Sunday August 12th
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Week 33
- Monday August 13th
Who: Jan Marten
Aim Negative controls for 10-08-2018
Colony PCR on e.coli pHIPZ7 as negative controls. Same primers and PCR settings as on 10-08-2018. A sample without primers is also made.
Gel was imaged on a UV lightbox, as the imaging PC was acting strange. No bands were visible on the negative controls.
- Tuesday August 14th
Who: Rianne
Aim Pick and grow colonies
Colonies 9 and 10 of both EGII and CBHI (of 10-08-18) were cultured, as well as 10 colonies from the plated with the transformed scaffold. The colonies were grown overnight in 5 ml LB supplemented with ampicillin.
Who: Matthijs
Aim Restriction digest of gBlock genes + restriction analysis on scaffold
Restriction digest of gBlock genes
Since the genes PAL2, CipA3 and BGLI were synthesized in two parts, they had to be ligated to use for further experiments.
- The CipA3 fragment contained a ClaI site
- BGLI contained ApaI and NruE, but since NruE cuts blunt ends, ApaI was used
- PAL2 contained NcoI
The fragments were hydrated to arrive at a final concentration of 10 ng/µl, the CipA3 fragments were already hydrated to a concentration of 5 ng/µl. The final reaction mixture contained the following ingredients:
Sample µl DNA µl Enzyme µl Buffer µl MQ µl Final PAL2 30 1 5 14 50 BGLI 30 1 5 14 50 CipA3 65 1.1 7.5 0 ~75 Where the enzyme used was corresponds to the enzyme as described above, and the buffer corresponds to the appropriate buffer for the enzyme. Both fragments for each gene are here already mixed.
Restriction digest was incubated at 37℃ for 2 hours and inactivated by heating to 80℃ for 20 minutes. The mixture was finally held at 4℃
Restriction analysis on scaffold
Colonies were picked previously by Rianne. 10 of these were selected for restriction analysis to verify the transformation. For the plasmid containing CipA3, the enzyme SmaI was used. SmaI has two recognition sites on the empty plasmid, pHIPZ7, producing one fragment of 4.6kb and one of 900b. When the gene is successfully inserted it will remove one of the recognition sites, producing only one large fragment of roughly 8.2kb.
The following scheme was used to prepare the reaction mixtures for all restriction digests of the CipA3 containing plasmids.
Sample µl DNA µl Enzyme µl Buffer µl MQ µl Final CipA3 1 1.3 0.5 2.5 15.7 20 CipA3 2 1.7 0.5 2.5 15.3 20 CipA3 3 1.43 0.5 2.5 15.6 20 CipA3 4 2.93 0.5 2.5 14 20 CipA3 5 2.96 0.5 2.5 14 20 The amount of DNA to add was calculated such that a final content of 250 ng of DNA would be reached. The restriction digest was incubated at 37℃ for 2 hours, after which the enzymes were inactivated by heating at 80℃ for 20 minutes.
Who: Matthijs
Aim Ligation of gBlock fragments + Restriction analysis on gel
Ligation of gBlock fragments
After the restriction digest, the fragments had to be ligated together. For this we use T4 ligase. The reaction was mixed as follows:
Sample µl DNA µl T4 µl Buffer µl MQ µl Final PAL2 6 0.5 1 2.5 10 BGLI 6 0.5 1 2.5 10 CipA3 8.5 0.5 1 1 10 The amount of DNA to add was calculated such that a total of 25 ng for each fragment was present. The ligation reaction was incubated at 16℃ for 16 hours overnight, after which the enzyme was heat inactivated by heating to 80℃ for 20 minutes.
Restriction analysis on gel
The restriction analysis of the CipA3 containing plasmids was run on an agarose gel. 1% of agarose was used in TAE buffer. 5 µl of SYBR green was added to 50 ml of agarose in TAE as prescribed by the manual. The gel was run at 100V for about 30 minutes after which it was imaged using a UV light source. As a standard DNA ladder, 1 kb generuler was added to the well in the middle.
CipA3 1-CipA3 2-CipA3 3-CipA3 4-CipA3 5-Ladder-CipA3 6-CipA3 7-CipA3 8-CipA3 9-CipA3 10 Who:
Aim
- Wednesday August 15th
Who: Rianne
Aim Glycerol stocks and plasmid minipreps
The cultures of the transformed EGII, CBHI and the scaffold were taken out of the incubator and glycerol stocks were prepared and stored. A plasmid miniprep of the cultures resulted in the following concentrations (ng/µl) of plasmid for restriction analysis and stored at -20℃:
- EGII 9 - 144,50
- EGII 10 - 160,05
- CBHI 9 - 115,20
- CBHI 10 - 207,40
- Scaffold 1 - 193,59
- Scaffold 2 - 146,95
- Scaffold 3 - 175,05
- Scaffold 4 - 85,35
- Scaffold 5 - 84,50
- Scaffold 6 - 96,75
- Scaffold 7 - 103,95
- Scaffold 8 - 95,65
- Scaffold 9 - 105,05
- Scaffold 10 - 108,0
- Thursday August 16th
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- Friday August 17th
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- Saturday August 18th
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- Sunday August 19th
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Week 34
- Monday August 20th
Who: Matthijs
Aim HF PCR on gblocks fragments
High fidelity PCR was used to amplify the ligated gblock fragments. The following primers were used:
Sample FW RV PAL2 PAL2.1-FW PAL2.2-REV BGLI BGLI-FW BGLI-RV CIPA3 CIP3A-FW CIP3F-REV Primers were diluted to 30 pmol/μl. The pipetting scheme was as follows:
Sample DNA µl FW µl RV µl Buffer µl HF µl H2O µl PAL2 2 1,7 1,7 10 2 32,6 BGLI 2 1,7 1,7 10 2 32,6 CIPA3 2 1,7 1,7 10 2 32,6 Control 0 1,7 1,7 10 2 34,6 The PCR protocol was as follows:
- activation - 95C - 5m
- denaturing - 94C - 15s,30x
- annealing - 50C - 1m, 30x
- extension - 68C - 2m, 30x
- Final extension - 72C - 10m
HF PCR on PAL2, BGLI and CIPA3 The BGLI fragment seems to have a size of about 3kb which is as expected. The size of PAL2 is too small and is most likely not correct. CIPA3 did not give any signal. BGLI concentration after PCR: 69.2 ng/µl
- Tuesday August 21st
Who: Matthijs
Aim Ligation of BGLI into pHIPZ7
Since BGLI was the only sample with a proper PCR signal, it was attempted to ligate into pHIPZ7, our standard yeast expression plasmid.
First, double restriction with BamHI and XhoI was done using the following scheme:sample DNA Buffer BamHI XhoI H2O BGLI 3.6 µl 2.5 µl 0.5 µl 0.5 µl 12.9 µl The reaction was performed at 37℃ for two hours after which the enzymes were inactivated by heating the sample at 80℃ for 20 minutes.
This product was purified by using a PCR purification column to remove the enzymes and buffer components.
Next the product was mixed with linearized plasmid at a 1:3 weight ratio and T4 ligase was added to ligate the product into the plasmid.Sample Insert DNA pHIPZ7 T4 buffer T4 H2O BGLI 6 µl 5 µl 1.5 µl 1 µl 1.75 µl The reaction was performed overnight at 16℃ after which the enzyme was inactivated by heat treatment at 80℃ for 20 minutes.
The product was again cleaned up and Taq polymerase was performed to verify insertion. The general sequence primers were used as these should give a large product when a sequence has inserted at the restriction site and a small product otherwise.
PCR conditions were standard Taq conditions with an annealing temperature of 50℃ as the melting temperature of the primers was quite low.
Who: Ingeborg
Aim Restriction digest of EGII and CBHII
Restriction digest
For the plasmids containing EGII and CBHII, the enzyme NCOI was used.
NCOI has one recognition site on the empty plasmid, pHIPZ7, producing one fragment of 5.8kb. When the EGII gene is successfully inserted it will add one recognition site, producing one large fragment of roughly 6.4kb and a smaller fragment of 967b.
When the CBHII gene is successfully inserted it will add two recognition sites, producing one large fragment of roughly 5.5kb and two smaller fragments, a fragment of 1186b and one of 766b.The following scheme was used to prepare the reaction mixtures for all restriction digests of the EGII and CBHII containing plasmids.
Sample µl DNA µl Enzyme µl Buffer µl MQ µl Final EGII - E9 2.18 0.5 2.5 14.82 20 EGII - E10 1.56 0.5 2.5 15.44 20 CBHII - C9 2.17 0.5 2.5 14.83 20 CBHII - C10 1.2 0.5 2.5 15.8 20 The amount of DNA to add was calculated such that a final content of 250 ng of DNA would be reached.
The restriction digest was incubated at 37℃ for 2 hours, after which the enzymes were inactivated by heating at 80C for 20 minutes.
Restriction analysis on gel
The restriction analysis of the EGII and CBHII containing plasmids was run on an agarose gel. 1% of agarose was used in TAE buffer. 5 µl of SYBR green was added to 50 ml of agarose in TAE as prescribed by the manual. The gel was run at 100V for about 55 minutes after which it was imaged using a UV light source. As a standard DNA ladder, 1 kb generuler was added to the well in the middle.
E9-E10-C9-C10-Ladder - Wednesday August 22nd
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Aim
- Thursday August 23rd
Who: Ingeborg
Aim Transformation of BGL1 and PAL2
Cells were plated on LB-ampicillin plates (250µl and 100µl concentrated) and incubated at 37℃ overnight.
- Friday August 24th
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Aim
- Saturday August 25th
Who: Jan Marten
Aim Restriction analysis on BGL1 and PAL2
5 colonies of BGL1 and 5 colonies of the PAL2 transformations are inoculated into LB ampicillin medium, and incubated overnight with agitation at 37℃.
- Sunday August 26th
Who: Jan Marten
Aim Restriction analysis of BGL1 and PAL2
The colonies have grown overnight and are miniprepped to isolate the plasmids. The BGL1 plasmids, along with a pHIPZ7 control plasmid are restricted with XbaI. The PAL2 plasmids are not processed, as it turns out the PCR reaction didn’t add the needed restriction sites. Therefore these plasmids cannot be correct.
The restriction tubes are placed in a PCR machine. 2 hours at 37℃, then infinite time at 4℃.
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Week 35
- Monday August 27th
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Aim
- Tuesday August 28th
Who: Ingeborg
Aim Transformation of BGL1 (ligation product made by Matthijs)
Cells were plated on LB-ampicillin plates (250µl and 100µl concentrated) and incubated at 37℃ overnight.
Who: Rianne
Aim Agarose gel of Matthijs’ ligation products of BGLI fragments
4µl ladder, 8µl of the samples
Ladder-B1-B2-Control Who: Rianne
Aim Grow colonies EGII and BGLI 9
EGII and BGLI colonies 9 were take from the glycerol stocks and inoculated into 5 ml LB with ampicillin and grown overnight in a 37℃ incubator, whilst shaking at 220 rpm.
- Wednesday August 29th
Who: Rianne
Aim Taq colony PCR of EGII, BGLI and CBHI colonies/cultures
20µl reaction volume, taq polymerase (according to manual manufacturer)
Different sets of repair fragment primers:
A) CasR2 Fw & Rv
B) CasR3 Fw & Rv
C) CasR5 Fw & Rv
D) CasR4 Fw & CasR1 RvExpected sizes:
Insert + promotor, terminator, docking module (2150 bp)
BGLI = 3150 bp + 2150 = 5300
EGII = 1640 bp + 2150 = 3790
CBHI = 1820 bp + 2150 = 3970All primer sets are tested on the empty PhipZ plasmid as a control reaction.
For the CBHI colony 9 primer set B was used and EGII was tested with primer set A. A small amount of culture was added to the reaction mix to provide the template DNA. 30 colonies of the BGLI plates were tested with primer set A.Cycling programme:
94℃ 5 min15 cycles:
94℃ 45 sec
64-57℃ 45 sec
72℃ 5:30 min15 cycles:
94℃ 45 sec
57℃ 45 sec
72℃ 5:30 min72℃ 10 min
12℃ indefinite - Thursday August 30th
Who: Rianne
Aim Agarose gel of 29-8 pcr products
APhipZ - BPhipZ - CPhipZ - DPhipZ - Ladder - CBHI - EGII 
BGLI: Ladder - 1 - 2 - 3 - 4 - 5 - 6/10 - 11/15 - 16/20 - 21/25 - 26-30 Who: Jan Marten
Aim PCR on the PAL2 and CIPA3 fragments
PCR is performed on the PAL2 and CIPA3 gene fragments.
Primers: For PAL2, PAL2f-fw and pal2f-rv are used to attach a C-terminal His-tag, and pal2g-fw and pal2g-rv are used to attach a N-terminal His-tag.
For CIPA3, primers cipa3a-fw and cipa3c-rv are used.
Master mix: 5 ul buffer, 1 ul dntp’s, 0.2 ul of each primer, 1 ul template, 0,5 ul Qiagen high fidelity polymerase, 42 ul MQ water.
9 mixes are made:- CIPA3
- CIPA3
- CIPA3
- PAL2 primer set F
- PAL2 primer set F
- PAL2 primer set F
- PAL2 primer set G
- PAL2 primer set G
- PAL2 primer set G
Machine settings:
94 degrees, 3 minutes initial denaturation
94 degrees, 45 seconds denaturation
52 degrees, 45 seconds, annealing
72 degrees, 2 minutes, extension
Repeat denaturation, annealing, extension 35 times
72 degrees, 10 minutes, final extension
- Friday August 31st
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- Saturday September 1st
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- Sunday September 2nd
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Week 36
- Monday September 3rd
Who: Jan Marten
Aim measuring OD of cultures, and galactose induction
The OD of the cultures are measured:
Strain fructose raffinose 1882 1 0.05 0.02 1882 2 0.16 0.09 1882 3 0.52 0.22 1883 1 0.02 0.02 1883 2 0.2 0.07 1883 2 0.55 0.25 At 13.10, 0.5% final concentration galactose is added to 1882.3 and 1883.3 cultured on fructose.
At 15.10, 0.5% final concentration galactose is added to 1882.3 and 1883.3 cultured on raffinose.
After 2 hours of incubation, the cells are spun down at 6000 rcf, 5 minutes, and resuspended in 10 ml of Verduyn medium. Spun down again, and resuspended in 20 ml Verduyn medium.
10 ml of this is transferred to a culture flask, more Verduyn medium is added, to a final volume of 20 ml. 2% cellobiose final concentration is added, along with uracil.At 17.10, the OD of the cultures is measured:
fructose raffinose 1882.3 0.44 0.108 1883.3 0.456 0.134 The positive control had an OD of 0.432, the negative control was 0.514
- Tuesday September 4th
Who: Ingeborg
Aim Transformation of the PAL2 plasmid (obtained from Shreyans)
Cells were plated on LB-kanamycin plates (250µl and 250µl concentrated) and incubated at 37℃ overnight.
Who: Rianne
Aim PCR PAL2 on pAL2 syn gene in pUC57-kan obtained from Shreyans (Roelfes group)
Phusion polymerase with primer sets:
PAL2C Fw & PAL2B Rv
PAL2B Fw & PAL2A RvMix (50 µl):
PCR buffer 10 µl
Primer A 5 µl (final concentration 1µM)
Primer B 5 µl
Polymerase 1,5 µl
Template 1 µl (from 51,3 ng/µl stock)
MQ 27,5 µl- 95℃ - 5 min
- 94℃ - 15 sec
- 65℃ - 1 min
- 72℃ - 4.5 min
- 2-4 - 35X
- 72℃ - 10 min
- 4℃ - indefinite
Who: Rianne
Aim Culture BGLI colony number 2
Colony number 2 (see 30-8-18) was grown overnight in 5 ml LB with ampicillin, at 37℃, 200 rpm.
Who: Rianne
Aim Prepare yeast extract samples for SDS-PAGE
Samples prepared by Jan Marten were taken from the -20℃ storage and thawed. The samples include the 1883 pellet and 1883 supernatant, and 1882 pellet and 1882 supernatant.
1ml of the supernatant was added to 667 µl of 12,5% TCA. After washing, the pellet was resuspended into 400 µl of 12,5% TCA. The samples were stored at -20℃ overnight.
Who: Jan Marten
Aim Measure OD of yesterday’s cultures, and to start new cultures of the 1882 and 1883 strains on Verduyn medium with fructose
At 14.00 hrs the OD of yesterday’s cultures are measured.
fructose raffinose 1882.3 3.9 3.6 1883.3 3.5 4.3 The positive control had an OD of 4.5, the negative control was 4.4. Since the negative control is not supposed to grow, the experiment is performed again.
20 ml of Verduyn medium with uracil and 1% fructose is used as a growth medium. Inoculate 2 cultures of 1882 and 1883 each. (1882.1, 1882.2, 1883.1, 1883.2) - Wednesday September 5th
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Aim
- Thursday September 6th
Who: Jan Marten
Aim Transfer cultures to fresh medium
100 ul of each culture started on 04-09-2018 is transferred to 20 ml of fresh Verduyn medium with uracil and fructose. Incubate the cultures at 30 degrees celsius.
- Friday September 7th
Who: Rianne
Aim PCR purification of the restricted product and ligation into PhipZ
Vector (5800 bp) 25 ng: insert (2200 bp) 28,5 ng
- 2 µl T4 ligation buffer
- 2 µl T4 ligase
- 2.4 µl restricted PhipZ (21.2 ng/µl)
- 6.1 µl restricted PAL2 (9.4 ng/µl)
- 7.5 µl MQ
Incubation for 3 hours at 37℃
Transformation: 5 µl of the ligation product into 50 µl of competent E. coli cells.
As a control, 1.5 µl of the restricted PhipZ plasmid was transformed in parallel.
The transformed cells were plated 100 µl, 200 µl, 350 µl and Rest. Of the control 200 µl was plated only. The plates were incubated at 37℃ overnight.Who: Jan Marten
Aim Measure OD and galactose induction
At 12.00 hrs, the OD of the cultures is measured.
1882.1: OD 1.35
1882.2: OD 1,26
1883.1: OD 1,53
1883.2: OD 1,29Induce with 0.25 ml 40% galactose solution. This leads to a concentration of 0.5% in the medium.
Dilute: 10 ml culture + 10 ml fresh medium. Incubate the cultures at 30 degrees celsius.At 18.00 hrs, 1 sample of each strain is spinned down, washed 2x with Verduyn medium without carbon source, and inoculated in Verduyn medium with uracil and cellobiose (0.5% final concentration).
OD at T=0:
1882.1: 0,59
1882.2: 0,63
1883.1: 0,54
1883.2: 0,36 - Saturday September 8th
Who: Rianne
Aim Taq colony PCR on PAL2-PhipZ colonies
Primer set A was used (see 29-08-18). Taq reaction mixture following manufacturers manual. 50 colonies were screened. The first 5 were transferred to a single reaction tube (A-E), subsequently 5 colonies per reaction tube (F-N), resulting in 14 reactions, 20 µl each.
Expected size: approximately 4400 bp
Cycling programme:
94℃ 5 min15 cycles:
94℃ 45 sec
64-57℃ 45 sec
72℃ 5 min15 cycles:
94℃ 45 sec
57℃ 45 sec
72℃ 5 min72℃ 10 min
12℃ indefiniteWho: Jan Marten
Aim measure OD and inoculate cellobiose cultures
At 12.00 hrs the 24 hrs galactose induction cultures are washed with Verduyn medium without carbon source, and inoculated into Verduyn medium with 0.5% cellobiose. As described yesterday.
Some of the leftover cultures is pelleted and plated onto ReCell plates.At 13.00 hrs the OD of all cultures is measured
Strain Evening culture (T=18 hrs) Morning culture (T=0 hrs) 1882.1 1.64 0.85 1882.2 1.47 1.04 1883.1 1.65 0.81 1883.2 1.22 0.81 - Sunday September 9th
Who: Rianne
Aim Gel of the PCR samples of 8-9-18 PAL2
Ladder: 4µl, samples: 10µl
Gel 1: Ladder - A - B - C - D - E - F - G 
Gel 2: Ladder - H - I - J - K - L - M - N All colonies are negative.
Who: Rianne
Aim BGL1 another colony PCR and agarose gel
Another colony PCR on the BGL1 colonies was performed similar to the PCR on 29-8-18. Samples A-E contain 1 colony (31-35), samples F-N contain 5 each (36-80). Primer set A was used.
Gel 1: Ladder - A - B - C - D - E - F- G 
Gel 2: Ladder - H - I - J - K - L - M - N Who: Jan Marten
Aim Measure OD of cellobiose grown cultures
At 13.00 hrs the OD of the cultures is measured.
Strain Evening (T=40 hrs) Morning (T=24 hrs) 1882.1 2 2.04 1882.2 1.84 2.12 1883.1 2 1.85 1883.2 1.62 1.94
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Week 37
- Monday September 10th
Who: Rianne
Aim Prepare yeast extract samples for SDS-PAGE
Jan Marten prepared samples and stored them at -20℃. The samples included 1882 1 and 2, 1883 1 and 2, both 0 hours and 24 hours induction. For each sample, pellet and supernatant were stored. The pellets were transferred into 400 µl 12.5% TCA. 1 ml of the supernatant was added to 667 µl 12.5% TCA. Except for the 24 hours samples, where only approximately 800 µl of supernatant was available but added to the same amount of TCA. The samples were stored at -20℃ overnight.
- Tuesday September 11th
Who: Rianne
Aim SDS-PAGE and Western blot of protein samples 10-09-18
The samples were taken from the -20℃ storage and processed further according to the protocol. The samples were loaded onto two SDS-PAGE gels. One of the gels was stained, the other was used for Western blotting. The samples were loaded in the following order: Ladder - 1882(1)(Pellet)(0 hours), 1882(2)(P)(0), 1883(1)(P)(0), 1883(2)(P)(0), 1882(1)(P)(24), 1882(2)(P)(24), 1883(1)(P)(24), 1883(2)(P)(24) - empty well - 1µM OsmY positive control (obtained from A. Iyer).
- Wednesday September 12th
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- Thursday September 13th
Who: Rianne
Aim Colony PCR BGLI
The same protocol as described on 29-8-18 was used. Reactions A-J contained 1 single colony, reactions K-N five.
- Friday September 14th
Who: Rianne
Aim Colony PCR BGLI agarose gel
4 µl ladder, 10 µl sample loaded
Gel 1: Ladder - A - B - C - D - E - F - G 
Gel 2: Ladder - H - I - J - K - L - M - N - Saturday September 15th
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- Sunday September 16th
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Week 38
- Monday September 17th
Who: Jan Marten
Aim Start cultures to make a growth curve for strains 1882 and 1883 in a plate reader
11 cultures are started, including induced and uninduced cultures, positive and negative controls.
1883.1 2 hrs galactose induction, to be cultured on cellobiose
1883.2 2 hrs galactose induction, to be cultured on cellobiose
1883.1 6 hrs galactose induction, to be cultured on cellobiose
1883.2 6 hrs galactose induction, to be cultured on cellobiose
1883.1 not induced, to be cultured on cellobiose
1883.2 not induced, to be cultured on cellobiose
1883.1 6 hrs galactose induction, to be cultured without carbon source
1883.2 6 hrs galactose induction, to be cultured without carbon source
1883.1 positive control, 6 hrs galactose induction, to be cultured with 1.25% glucose
1883.2 positive control, 6 hrs galactose induction, to be cultured with 1.25% glucose
negative control (wildtype s. cerevisiae, 6 hrs galactose induction, cultured on cellobiose)All cultures are grown on 20 ml Verduyn medium with 0.5% fructose and uracil, and leucine and tryptophan added for the negative control. Culture at 30 degrees Celsius with agitation.
- Tuesday September 18th
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- Wednesday September 19th
Who: Jan Marten
Aim to induce, wash, and distribute the cultures from 17-09-2018
At 10.30 hrs the cultures to be induced for 6 hrs are induced by adding 2.0% final concentration of galactose.
At 14.30 hrs the cultures to be induced for 2 hrs are induced by adding 2.0% final concentration of galactose.
At 16.30 hrs the cultures are spinned down in falcon tubes at 4000 rpm in a large centrifuge. Samples are washed 2x with Verduyn medium without a carbon source.
The cultures are mixed with supplements as described on 17-09-2018 and 200 ul of each culture are loaded into a 96 well plate, in duplo, with starting OD’s of 0.2, 0.1, and 0.05. 3 Verduyn cellobiose blanks and 2 Verduyn cellobiose glucose blanks are loaded as well.The plate reader is set to measure, for 48 hrs, once every 20 minutes. The plate is agitated for 30 seconds before every measurement, and the interior is kept at 30 degrees Celsius over the duration of the experiment.
The plate reader is a Tecan Geneos. - Thursday September 20th
Who: Rianne
Aim Colony PCR BGLI
The same protocol as described on 29-8-18 was used. Reactions A-O contained 1 single colony, reactions P-Y 2. The reaction volume was 15 µl. The colonies were taken from the plates of 28.08.18. Gels were run by Owen.
Samples A-R 
Samples S-Y - Friday September 21st
Who: Rianne
Aim Colony PCR BGLI
PCR was performed on the plates of 11-9-18. 50 colonies were picked in 15 µl reactions. The agarose gels were run by Owen.
Samples 1-36 
Samples 37-50 - Saturday September 22nd
Who: Jan Marten
Aim To miniprep and restrict Puc57kan Pal2
1.5 ml of e.coli containing Puc57kan Pal2 (induced yesterday) is miniprepped. Eluate in 50 ul.
1) 25 ul is mixed with 10 ul Jena 10x universal buffer, 1 ul BamHI and 1 ul PstI, and 63 ul MQ
2) 12.5 ul is mixed with 5 ul Jena 10x universal buffer, 1 ul BamHI, and 31.5 ul MQ
3) 12.5 ul is mixed with 5 ul Jena 10x universal buffer, 1 ul PstI, and 31.5 ul MQ
Incubate 2 hrs at 37 degrees, and store overnight at 4 degrees.E.coli pHIPZ7 is inoculated into LB ampicillin and grown overnight at 37 degrees.
- Sunday September 23rd
Who: Jan Marten
Aim To miniprep e.coli pHIPZ7, restrict, and load and run an agarose gel
E. coli pHIPZ7 is miniprepped.
A gel is loaded:
Slot 1: Ladder (Jena 1kb ladder). Load 3 ul.
Slot 2: Unrestricted pHIPZ7 plasmid
Slot 3: Sample 1 from yesterday, 5 ul, mixed with 1 ul 5x loading dye
Slot 4: Sample 2 from yesterday, 5 ul, mixed with 1 ul 5x loading dye
Slot 5: Sample 3 from yesterday, 5 ul, mixed with 1 ul 5x loading dye
Run for 30 minutes at 90 mA.
It appears that 1) from yesterday has cut only once, the same goes for 2) and 3).
Add 2 ul of PstI to 1) and 2), and add 1 ul BamHI to 3).
Incubate 2 hrs at 37 degrees Celsius.Mix 5 ul of each sample with 1 ul 5x loading dye, load in the same order as above, and run for 30 minutes at 90 mA.
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Week 39
- Monday September 24th
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- Tuesday September 25th
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- Wednesday September 26th
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- Thursday September 27th
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- Friday September 28th
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- Saturday September 29th
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- Sunday September 30th
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Week 40
- Monday Oktober 1st
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- Tuesday Oktober 2nd
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- Wednesday Oktober 3rd
Who: Jan Marten
Aim Transform E. coli BL21 DE3 Star with Pal2 and His-EGII
E. coli BL21 DE3 Star is transformed with pSB1C3 T7 promotor + Pal2 colonies 1 and 7, pSB1C3 T7 promotor + His-EGII colonies 1 and 2, and Bba_K1692003 (pSB1C3 T7 promotor + Pal2).
Thaw cells on ice, make 100 ul aliquots, add 100 ng of DNA to each aliquot, incubate for 30 minutes. Heat shock 1 minute at 42 degrees celsius, and incubate on ice for 2 minutes. Add 900 ul LB medium, and incubate at 37 degrees celsius for 1 hour. Plate on LB chloramphenicol plates, in 10% and 90% fractions. Incubate overnight at 37 degrees celsius. - Thursday Oktober 4th
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- Friday Oktober 5th
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- Saturday Oktober 6th
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- Sunday Oktober 7th
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Week 41
- Monday Oktober 8th
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- Tuesday Oktober 9th
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- Wednesday Oktober 10th
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- Thursday Oktober 11th
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- Friday Oktober 12th
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- Saturday Oktober 13th
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- Sunday Oktober 15th
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