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Revision as of 13:26, 8 September 2016
Notebook
Workspace
Our lab
We have our own iGEM TU Delft lab in the new Applied Science building on the edge of the TU Delft campus. It is classified as an ML-1 lab, the lowest safety level to work with modified organisms, which is enough for our experiments. Apart from this lab, we are also working in an optical lab, which is also ML-1 classified. In here built our own laser set-up.
Our office
Our office is our homebase for when we're not working in the lab. Here we work on things like the safety tool, the wiki, the modeling, and processing our results. This summer the Bionanoscience department moved to a new building, so it took some time before we had our own office. When we finally got our office, we quickly made it our home. Next to our office, there is a meeting room, where we have a weekly meeting with our TA’s and PI’s to keep everyone up to date and discuss problems we might encounter.
Lab safety
Our lab is classified the lowest safety level (level 1), meaning that our experiments involve low to no risk. All the members of the team have successfully completed the following safety tests: Lab safety test General safety test of the building we currently work in Biological safety test for ML-1 lab General safety test of the building we worked in before June All the members of the team have received safety training, including: Introduction to sterile working General lab training (using a PCR machine, making gels, etc.) General safety information, regarding contact persons and locations The safety of our experiments was supervised by Erwin van Rijn (Safety Manager of the lab) and Jeremie Capoulade (Safety Manager of the lasers). The supplies we needed in the lab were provided with the help of our instructor Esengül Yildirim. The research has been conducted with respect to the regulations of biosafety for The Netherlands, that can be found here.
Day Notes
29th June 2016
María
Prepare 10mL of antibiotic stocks of both antibiotics that are going to be used during the project: Chloramphenicol (Cm) and Ampicillin (Amp). Make 500µL and 1mL aliquots.
Prepare 1L of LB medium and 3 400mL bottles of LB Agar. Send to autoclave.
30th June 2016
María
Add antibiotics to the LB Agar bottles prepared the previous day and pour plates to have them ready for when we start transforming.
4th July 2016
Lycka
Digestion gBlocks mVenus and mKate with EcoRI and PstI.
Made liquid culture of strain containing pSB4A5 bakcbone with RFP.
María
Digestion of pSB1C3 linearized backbone from the distribution kit with EcoRI and PstI.
5th July 2016
María
Dissolution of necessary parts from distribution kit: GFP BioBrick BBa_E0840 and promoters BBa_J23100, BBa_J23105, BBa_J23108, BBa_J23113 and BBa_J23117.
Dissolution of K1149051 (a kind gift of Imperial College), which was sent dry on filter paper.
Lycka
Ligation of mKate into backbone pSB1C3.
Transformation of ligation product mKate and the BioBricks dissolved by María into Escherichia coli Top10 cells. These were plated on LB medium supplemented with Cm.
6th July 2016
Lycka
Stocked primers VF2 and VR: storage stock (100µM) and working stock (10µM).
Colony PCR of transformants from yesterday with primers VF2 and VR followed by gel electrophoresis. Gel picture yielded no bands. This might be attributed to a fault in the transformation.
7th July 2016
María
Transformation of same BioBricks as on 5th July using a different transformation protocol since we believed that the negative result was due to a mistake during transformation.
8th July 2016
María
Finally, the plasmids containing K1149051 and BBa_E0840 yielded a few colonies. However, the ones containing the promoters did not. Thus, we believe that the distribution kit plate containing the promoters is defective and the mKate ligation also featured some problems.
11th July 2016
Lycka
To test whether the distribution kit might be the problem, transformation of BBa_J23113 in OneShot® TOP10 chemically competent cells (Invitrogen) to make sure the transformation process is not the cause of the negative results. Also, a plasmid containing TU Delft 2015 csgA Biobrick was used as a positive control.
For the mKate construct we will obtain the backbone from another construct instead of the linearized backbones from the iGem distribution kit.
12th July 2016
Lycka
Measure DNA concentration of biobricks from registry by nanodrop.
Nanodrop
| Product | Concentration (ng/µl) |
|---|---|
| J23113 | 89.3 |
| J23117 | 91.7 |
| J23105 | 89.3 |
| J23108 | 92.7 |
| J23100 | 93.4 |
| E0840 | 81.9 |
As can be seen from the table, there is DNA presence in the samples. Therefore, something else should be causing the transformations not to work. Since last year's distribution kit plates are still available in the lab and those plasmids were not taken we will try to get these parts from 2015 distribution kit.
María
Transfer colonies for K1149051 and BBa_E0840 to overnight LB+Cm culture. A colony containing CsgA was transfered as well to be able to isolate and use that plasmid as standard backbone for all synthesized parts.
13th July 2016
María
Plasmid isolation of overnight cultures prepared yesterday and preparation of K1149051 and BBa_E0840 samples for sequencing.
Lycka
Cryostocks of K1149051 and BBa_E0840 overnight culures. Stored at -80 degrees.
Digestion of backbones pSB1C3, pSB4A5 and all gBlocks with EcorI and PstI.
14th July 2016
Lycka
Gel purification of digested backbones. Dissolved in nuclease free water, stored at -20 degrees.
Ligation of synthesized fragments into backbones.
María
After all the problems transforming in the past weeks, we tested if we could use our homemade chemically competent cells for future transformations by transforming a couple of positive controls, which were taken from last year's plasmids. The transformations worked so these cells can still be used.
15th July 2016
María
Transformation of Lycka's ligation products from yesterday and the BioBricks from the distribution kit were given one last chance. Transformed cells were plated after doubling their concentration.
Lycka
Dissolution of promoter BioBricks from 2015 iGEM distribution kit.
18th July 2016
Lycka
Restriction of backbones pSB1C3 and pSB4A5 with EcorI and PstI. Gel electrophoresis of digested fragments. Gel picture yielded no bands.
19th July 2016
Lycka
Transformation of BioBricks from the distribution kit of 2015, since the ones from 2016 yielded no colonies. Positive control: csgA. Negative control: water. Biobricks: J23100, J23108, J23105, J23117, J23113. Overnight cultivation yielded no result, we decide to drop this and add the promoters to the parts that need it by PCR.
María
Restriction of pSB1C3 and pSB4A5 backbones with EcoRI and PstI
Ligation of synthesized parts that could not be ligated on 14th of July: INP_Sil_Sdom in pSB1C3 and BolA_ind and BolA_con in both pSB1C3 and pSB4A5.
20th July 2016
Lycka and Tessa
Transformation of plasmids ligated by Maria (19th of July) together with the ones ligated on 14th of July. After overnight culture the following plates contained colonies. pSB1C3: INP_Sil_Sdom, OmpA_Sil_Taur, Sil_Sdom, SulA, BolA_ind, BolA_con, phaP. pSB4A5: BolA_con. The ones with a negative result were ligated again the next day.
21st July 2016
María
Colony PCR of yesterday's transformation results from Lycka and Tessa. However, not all transformations yielded colonies.
Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.
Lycka
Ligation of mKate, mVenus, mCerulean, OmpA_Sil_Sdom and LacI into pSB1C3. Ligation of OmpA_Sil_Taur, OmpA_Sil_Sdom, Sil_Sdom, SulA and BolA_ind into pSB4A5. Left at room temperature overnight.
22nd July 2016
María
Continue with colony PCR of 20th of July transformation results from Lycka and Tessa.
Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.
Lycka
Transformation of ligation products from yesterday. Cells containing backbones pSB1C3 or pSB4A5 were plated on plates supplemented with chloramphenicol or ampicilin, respectively. After overnight cultivation the following plates contained colonies: mKate, mVenus, mCerulean, OmpA_Sil_Sdom, Sil_Sdom, SulA, BolA_ind.
23rd July 2016
María
Continue with colony PCR of yesterday's transformation results from Lycka.
Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.
Pick all colonies selected by colony PCR on 21st, 22nd and 23rd of July and transfer to selective LB according to the resistance expressed by each plasmid.
24th July 2016
Lycka
Cryostock and plasmid isolation of overnight cultures prepared yesterday by Maria. Cryostocks stored at -80 degrees, isolated plasmids stored at -20 degrees.
25th July 2016
Lycka
Nanodrop and prepare for sequencing plasmids isolated yesterday.
Sequencing resulted in the following. Correct sequence: Sil_Sdom (pSB1C3), OmpA_Sil_Taur (pSB1C3), Sil_Sdom (pSB4A5), SulA (pSB4A5). Sequence with mutations: SulA (pSB1C3), BolA_ind (pSB1C3), J23108 (pSB1C3), BolA_con (pSB4A5), BolA_ind (pSB4A5). Different sequence: BolA_con (pSB1C3), INP_sil_Sdom (pSB1C3), OmpA_Sil_Sdom (pSB4A5). Mutated sequences can be repaired by PCR and blunt end ligation.
María
Prepare new digested pSB1C3 and pSB4A5 backbones by restricting with EcoRI and PstI and gel isolating the restricted product.
New ligation of those synthesized fragments that have not yielded any positive results (transformation or sequence confirmation) in the past week in their respective backbones.
27th July 2016
Lycka
Colony PCR of plates transformed yesterday.
Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated the same day in selective LB overnight.
María and Tessa
Make new chemically competent cells. They were tested for transformation capacity with a positive control plasmid (RFP in pSB4A5). Also, untransformed cells were plated in plates supplemented with Cm or Amp to test whether they had developed a resistance during the process.
28th July 2016
María
Cryostock, plasmid isolation and preparation of samples for sequencing of the overnight cultures prepared yesterday by Lycka. Only INP_Sil_Sdom in pSB4A5 was sequenced confirmed with a mutation to be fixed by PCR. The other sequences aligned to a fragment of GFP unknown to us, we assumed it either came from a contamination of the old restriction enzymes we were using or from the pSB1C3 backbone that was taken from last year's team plasmid stock and probably was poorly labeled. Thus, we decided to use another backbone and the new enzymes sponsored to all iGEM teams by New England Biolabs.
29th July 2016
Lycka and Célina
Repeat of the colony PCR of plates from the 26th of July that yielded a negative result.
Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in LB and the applicable antibiotic overnight.
Transformation of OmpA_Sil_Sdom in both different backbones.
30th July 2016
Lycka
Colony PCR of OmpA_Sil_Sdom in both backbones from the 29th of July. The PCR machine was filled with colonies from older plates which had not yielded any good colonies yet: mKate (26th of July), mKate (mKate 30th of June), INP_Sil_Sdom (20th of July).
Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in LB and the applicable antibiotic overnight.
Cryostocking and plasmid isolation of colonies picked by on 29th of July. Cryostocks stored at -80 degrees, isolated plasmid stored at -20 degrees.
31st July 2016
María
Cryostock and plasmid isolation of the overnight cultures prepared yesterday by Lycka and preparation of samples for sequencing of plasmids isolated yesterday and today. Sequence of mCerulean was confirmed (although it had a silent mutation). OmpA_Sil_Sdom in pSB4A5 and INP_Sil_Sdom in pSB1C3 had a deletion to be fixed by PCR. The rest either of sequences contained the random GFP sequenced mentioned before (since they are old ligation products) or multiple mutations that could not be fixed by PCR.
2nd August 2016
Tessa
Amplified E0840 (GFP) out of a pSB1C3-GFP plasmid using Phusion PCR. Four reactions of 50µl.
Nanodropped PCR products.
Nanodrop
| Product | Concentration (ng/µl) |
|---|---|
| E0840 | 339.6 |
| E0840 | 554.1 |
| E0840 | 596.9 |
| E0840 | 567.6 |
Ran a 1% agarose gel of the PCR product to verify product size.
Stored product 2 and 4 in the fridge for later use.
10th August 2016
Tessa
Restricted PCR product of 3rd August, J23100 E0840, J23113 E0840 and J23117 E0840, with EcoRI-HF and PstI.
Purified restriction product.
Nanodropped purified product.
Nanodrop
| Product | Concentration (ng/µl) |
|---|---|
| J23100 E0840 | 14.7 |
| J23113 E0840 | 21.3 |
| J23117 E0840 | 23.3 |
Ligated purified product into pSB1C3.
Transformed ligation product into TOP10 strain. Using RFP as positive control and sterile MiliQ as negative control. Plated on plates with LB agar and CM.
11th August 2016
Tessa
Colony PCR'd colonies from yesterdays transformation using Q5® mastermix.
Ran a 1% agarose gel of the PCR product.
L is the Promega 1kb ladder, 1-6 is J23100 E0840, 7-14 is J23113 E0840 and 15-22 is J23117 E0840.
Transferred colony 14 (J23113 E0840 pSB1C3) and 21 (J23117 E0840 pSB1C3) into liquid LB.
12th August 2016
Tessa
Miniprepped colony 14 and 21 of yesterdays overnight culture.
Nanodropped miniprep product.
Nanodrop
| Product | Concentration (ng/µl) |
|---|---|
| J23113 E0840 in pSB1C3 | 140.6 |
| J23117 E0840 in pSB1C3 | 325.6 |
Cryostocked colony 14 and 21.
Send miniprep product for sequencing. [Sequence Confirmed]
18th August 2016
Tessa
Transformation of the InterLab Study plasmids into BL21.
19th August 2016
Tessa
Only Test Device 2 and 3 and positive control have yielded (very little) colonies. Decided to try and tranform them into Top10.
22nd August 2016
Tessa
Transformation of the InterLab Study plasmids into Top10.
Colony PCR of J23100 E0840 pSB1C3 using GoTaq®. Plate from 11th August.
Ran a 1% agarose gel to verify part size
L is the Promega 1kb ladder, 1-9 are J23100 E0840.
Colony 1,2 and 3 were put in liquid LB+CM and incubated overnight.
23rd August 2016
Tessa
Colony PCR of the InterLab Study plates using DreamTaq®. Picked eight colonies of each plate.
Cryostocked J23100 E0840 pSB1C3 in Top10.
Ran a 1% agarose gel of the InterLab Study colony PCR products.
L is the Promega 1kb ladder, 1-8 is Test Device 1, 9-16 is Test Device 2, 17-24 is Test Device 3, 25-32 is Negative Control and 33-40 is Positive control.
Colony 1, 2, 4, 9-11, 17-19, 26, 27, 29 and 33-36 were picked for overnight culture in liquid LB+CM.
24th August 2016
Tessa
Put InterLab Study cultures in fridge for later use.
25th August 2016
Tessa
Resuspended FITC according to InterLab protocol.
29th August 2016
Tessa
Transformed J23100-, J23105-, J23108-, J23113- and J23117 E0840 pSB1C3, and BolA_con pSB4A5 into BL21.
30th August 2016
Lycka
PCR lineralization of vectors and inserts for gibson assembly pha operon (BBa_K1149051) to fluorophores (GFP, mVenus, mKate, mCerulean). Phusion polymerase. Annealing temperature 60 degrees, elongation time 2 minutes.
Tessa
Did the InterLab Study plate reader measurements according to the InterLab plate reader protocol.
Streaked J23105- and J23117 E0840 pSB1C3 out on new plates, because they had too many colonies.
Colony PCR'd six colonies of BolA_con, J23100 E0840, J323108 E0840 and J23113 E0840 using GoTaq®.
Ran a 1% agarose gel of PCR products to verify part size.
L is the Promega 1kb ladder, 1-6 is BolA_con, 7-12 is J23100 E0840, 12-18 is J23108 E0840 and 19-24 is J23113 E0840.
Picked colony 1, 6, 11, 13, 18, 20 and 21 for overnight culture.
31st August 2016
Lycka
Electrophoresis of PCR fragments yesterday.
Picture on the left shows simulated agarose gel. Picture on the right shows the actual agarose gel. 1 - 4: vectors for mCerulean, mKate, mVenus and GFP, respectively. 5 - 6 inserts for mVenus and GFP. Gel did not yield bands at the right height. PCR is repeated at an annealing temperature of 57 degrees.
Second gel also did not yield the right bands. PCR repeated overnights by María at Tm 64 degrees and elongation time 3.5 minutes in the presence of DMSO.
Transfer from cryostock to plate BL21 strains with fluorophores expressed. On LB agar supplemented with chloramphenicol.
Tessa
Cryostocked J23100-, J23108- and J23117 E0840 pSB1C3 in BL21. BolA_con didn't grow.
Cryostocked K1149051 pSB4A5 in BL21.
1st September 2016
María
PCR with new primers of those parts that need to be expressed under the lac promoter. These primers will allow cloning on Addgene plasmids from the pBb series. The plasmids chosen (pBbS5a and pBbA5c) express lacI and the cloning site is placed downstream of a lac promoter. The parts to be amplified are:
1. BolA
2. INP_Sil_Sdom
3. OmpA_Sil_Sdom
4. OmpA_Sill_Taur
5. Sil_Sdom
6. SulA
Lycka
Electrophoresis of overnight PCR for lineralization.
Picture on the left shows simulated agarose gel. Picture on the right shows the actual agarose gel. 1 - 4: vectors for mCerulean, mKate, mVenus and GFP, respectively. 5 - 6 inserts for mVenus and GFP. For all lanes except lane 5, the right bands are present. Also many other bands were present so the product was gel purified.
Transfered one colony of BL21 on each plate from yesterday in a 50mL falcon tube with 10mL filter sterilized M9 medium with glucose supplemented with chloramphenicol.
Tessa
Put three colonies of J23100-, J23105- and J23117 E0840 pSB1C3 in BL21 into liquid LB+CM.
2nd September 2016
Tessa
Cryostocked J23100-, J23105- and J23117 E0840 pSB1C3 in BL21.
Lycka
Nanodrop gel purified PCR fragments from yesterday. Concentrations are too low to work with. Repeat PCR under the same conditions, but with double volume (100µL). Done by María.
Measure OD600 of cultivated M9 tubes.
OD600 measurements
| Genotype | OD600 |
|---|---|
| GFP J23108 | 1.24 |
| mVenus | 0.04 |
| mKate | 0.20 |
| mCerulean | 0.03 |
The strains containing GFP and mKate grew really well, the ones containing mVenus and mCerulean did not. All of the tubes were put back in the incubator. The one with GFP was diluted 10 times in M9 medium, the one with mKate was diluted 2 times. Aliquots of 150µL were loeaded in an 96 well plate and measured in a plate reader at 37 degrees for 7 hours.
María
PCR of same samples as Lycka in a reaction volume of 100µL. After gel isolation and purification only for mCerulean and mKate good concentrations were reached (160 and 260 ng/µL, respectively), for the rest of samples either no bands were seen in the agarose gel or the concentration was too low again.
Protocols
During our lab work we used the following protocols.
- Competent Cells
- PCR (Phusion)
- Gel Isolation (From Promega Wizard™ Kit)
- Restriction (NEB enzymes)
- Ligation
- Transformation (TOP10)
- Transformation (BL21)
- Transformation (OneShot® TOP10)
- Colony PCR (Q5® High-Fidelity 2X Master Mix)
- Colony PCR (GoTaq)
- Colony PCR (DreamTaq)
- DNA Electrophoresis
- Overnight Cell Cultures
- Cell Cryostock
- Plasmid Isolation (From Promega PureYield™ Plasmid Miniprep Kit)
- Restriction Product Purification (From Promega Wizard™ Kit)
- Grow overnight culture from single colony or competent cells aliquot.
- Inoculate 1:100 in the desired volume of LB.
- Grow to OD 600 ~0.35 (important that it is close to 0.35, 0.3-0.4 is good).
- Chill on ice for 20 min (it can chill for a 1-2 hours).
- Centrifuge at 8000g for 8 minutes in Falcon tubes, decant supernatant.
- Resuspend by pipetting gently in 1/10 (of the volume of LB from step 2) of ice-cold TSS.
NOTE: It can take a while to break up the pellet (you can use a 25 mL pipette and scrape the pellet off the side of the tube before pipetting).
Recipe for TSS (100 mL):
- 10 g PEG 3350
- 5 mL DMSO
- 2 mL 1 M MgCL2
- LB to 100 mL
- Freeze 100 µL aliquots in liquid nitrogen & store at -80 degrees C in 1,5mL Eppendorf tubes.
- ALWAYS do a transformation positive control using a plasmid that you know is efficiently taken up by competent cells to confirm competency.
- Just as important: ALWAYS do a negative control transformation (add untransformed cells to a plate with antibiotics) to confirm that you don't have an antibiotic-resistant contaminant strain in your competent cell stock. This happens from time to time and can cause lots of agony!
- Prepare the mix for all the samples in a single 1.5mL tube. For one sample:
NOTE: The 250ng of template DNA are approximate, choose a volume that works fine for all your samples and fill it in the excel sheet so you are able to prepare a mix for all samples at the same time.Component Volume (µL) for 20µL reaction Volume (µL) for 50µL reaction 5X Phusion HF Buffer 4 10 10mM dNTPs 0.4 1 FW Primer 1 2.5 RV Primer 1 2.5 Phusion polymerase 0.2 0.5 Template DNA (<250ng) Variable Variable Nuclease-free H2O Up to 20µL Up to 50µL - Add the total volume per sample (without DNA) to each PCR tube.
- Add the template DNA to each tube.
- Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):
Step Temperature (ºC) Time Initial denaturation 98 30sec Denaturation 98 10sec x30 cycles Annealing Tm-3 20sec Extension 72 15-30sec/1kb Final extension 72 5min 4 Hold - Run a DNA electrophoresis following the DNA electrophoresis protocol
- Run a DNA electrophoresis (following the DNA electrophoresis protocol), loading the full volume of PCR or restriction product into the agarose gel. If necessary, use multiple wells.
- Cut the band (or bands) corresponding to the fragment of interest with a razor blade and put it in a pre-weighted 1.5mL tube, then calculate the weight of the gel fragment.
- Add 10µL of Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50-65ºC until gel slice is completely dissolved.
- Pre-warm an aliquot of Nuclease-free water at 50-65ºC.
- Insert SV Minicolumn into Collection Tube and label both of them according to the labelling of your samples.
- Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute.
NOTE: When pipetting into the column, aim the pipette to the wall not the membrane to avoid damaging it. - Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
- Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.
- Add 700µL of Membrane Wash Solution (if it is the first use, dilute it with 95% ethanol following the bottle’s instructions).
- Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
- Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.
- Repeat steps 9-11 with 500µL of Membrane Wash Solution and centrifuging for 5 minutes.
- Once the Collection Tube is empty, centrifuge the Minicolumn assembly at maximum speed for 1 minute with the microcentrifuge lid open to allow ethanol full evaporation.
- Transfer the SV Minicolumn to an empty 1.5mL tube carefully labelled.
- Add 500µL of the pre-warmed water (300µL for higher concentrations or when small amounts of DNA are suspected) directly to the centre of the SV Minicolumn, without touching the membrane with the pipette tip.
- Incubate at room temperature for 5 minutes.
- Centrifuge at maximum speed for 1 minute.
- Discard the SV Minicolumn, cap the tube containing the eluted DNA and keep at 4ºC (for immediate use) or -20ºC (for storage).
- Get the detailed protocol for restriction from the following website, according to the enzymes used. When high restriction product concentrations are needed (generally for backbones) the amount of DNA can be increased to 3-5g. The volume of DNA necessary can be obtained from the concentration measured at NANODROP.
- Mix all the components stated by this restriction tool in a 1.5mL Eppendorf tube and incubate at 37ºC for 1-2h.
- Make your own ligation protocol.Add ligation tool!!
- Mix all the components in a tube (always add the T4 ligase at the end).
- Incubate for at least 3 hours at 16ºC.
