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Team:Harvard/fences/notebook
From 2010.igem.org
notebook calendar
06-14-2010 [ top ]
LacI Transformation
Performed bacterial transformation according to Silver:_Bacterial_Transformation, however we used 1μL in step 3, and used entire tube of TOP10 competent cells instead of only 10-15μL.
Transformed the following, each into its own tube of TOP10 E.Coli:
1) LacI with a rapid degredation tail attached, biobrick BBa_C0012, located on plate 1, well 2O, plasmid pSB1A2
2) LacI wildtype, biobrick BBa_I732100, located on plate 2, well 10E, plasmid pSB1A3
Streaked 100μL each of 1 and 2 onto separate LB + Amp dishes, along with a control dish of 100μL untransformed TOP10 E.Coli cells.
Plates incubated overnight, colonies observed in all plates except the control.
06-15-2010 [ top ]
Oligonucleotides for LacI
the following sequences for LacI and a Nuclear Localization Signal (NLS) were ordered for PCR:
LacIn.BB.Rev
5'-AAG GCT GCA GCG GCC GCT ACT AGT TCA AAC CTT TCT CTT CTT CTT AGG ATG AAC AAC AGA AGA CTG CCC GCT TTC CAG TCG GGA AA - 3'
LacIn.BB.Fwd
5'- CCT TGA ATT CGC GGC CGC ATC TAG AAT GAA ACC AGT ACC GTT ATA CGA TGT C -3'
NLS.BB.Rev
5'-GCG GCC GCT ACT AGT TCA AAC CTT TCT CTT CTTCTT AGG ATG AAC AAC AGA AGA -3'
NLS.BB.Fwd
5'-CTA GAT CTT CTG TTG TTC ATC CTA AGA AGA AGA GAA AGG TTT GAA CTA GTA GCG GCC GCT GCA -3'
VP16 transcription activating domain bacterial transformation
Performed bacterial transformation according to Silver:_Bacterial_Transformation, however we used 1μL in step 3, and used entire tube of TOP10 competent cells instead of only 10-15μL.
Transformed the following into its own tube of TOP10 E.Coli:
VP16 Eukaryotic Activating Domain, biobrick BBa_K105001, located on plate 3, well 13C, plasmid pSB1A2.
In the course of reconsitituting the the biobrick plasmid from its well, too little DH2O was added (1μL), so an additional 10μL were added to the well.
The first tube of TOP10+biobrick plasmid was erroneously heatshocked before being chilled for 30 mins on ice, so a second tube of TOP10 cells was introduced, and the correct proceedure followed for the second tube. Additionally, the first tube was then allowed to chill and heat shock again, ultimately producing two tubes of transformed TOP10 cells.
100μL of both tubes were streaked onto an LB + Amp dish, and incubated at 37°C on the plates overnight. Colonies the following morning, colonies were observed on both plates, although fewer on the plate containing the E.Coli which were erroneously heat-shocked too soon.
LacI Miniprep Preparations
Prepared 6 overnight cell cultures, consisting of three colonies each from the LacI wt and LacI+rapid degradation tail E.Coli cells transformed previously.
Each culture consisted of 5mL of LB+Amp, and a scraping of 1 E.Coli colony, in a 100cc tube.
These overnight cell cultures were set to shake at 37°C overnight.
06-16-2010 [ top ]
LacI Miniprep
Performed miniprep of overnight cell cultures of 3 colonies each of wildtype LacI transformed bacteria and LacI+rapid degradation tail bacteria.
Minipreps performed according to pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook].
LacI Nanodrop
Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.
| Plasmid | Quantity (ng/μL) | 260/280 |
|---|---|---|
| O2 #1 | 77.1 | 1.81 |
| O2 #2 | 90.1 | 1.9 |
| O2 #3 | 50.4 | 1.95 |
| E10 #1 | 99.6 | 1.93 |
| E10 #2 | 104.6 | 1.92 |
| E10 #3 | 58.6 | 1.98 |
LacI Plasmid Digest
We digested 15μL of each of the 6 samples with 1μLH2O, 1μL EcoR1, and 1μL Pst1 (high efficiency restriction enzymes).
Ran an E-gel of the digest results.
| Lane # | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
|---|---|---|---|---|---|---|---|---|---|---|
| O2 #1 | ladder | O2 #1 | O2 #2 | O2 #3 | E10 #1 | E10 #2 | E10 #3 |
VP16 Colony Inoculation
Prepared 3 10cc tubes each containing .5μL LB+amp and cells from one of the three colonies of the VP16 cell culture of the previous night (colonies from the second-tube plate, the cells in which were heatshocked in the correct order).
Tubes were set to shake at 37°C overnight.
Preparing Glycerol Stocks of LacI wt and LacI+LVA
Prepared 2 cryo tubes, with the following labeling:
Tube 1) contains BBa_C0012, from plate 1, well 2O
On top: '#1'
On side: '6-16-10 MP pSB1A2 LacI+LVA'
Tube 2) contains BBa_I732100, from plate 2, well 10E
On top: '#2'
On side: '6-16-10 MP pSB1A3 LacI Wildtype'
Each cryo tube contains .5mL of the respective overnight cell culture (O2 colony #1 and E10 colony #1), and .mL of 80% glycerol.
Both Cryo tubes were placed in the box labeled 'Genetic Fence iGEM 2010' in the -80°C freezer.
06-17-2010 [ top ]
VP16 Miniprep
Following QIAgen Plasmid DNA Purification Protocol Using the QIAprep Spin Miniprep Kit and a Microcentrifuge the following plasmid was purified:
- VP16
Protocol
- 4ml from each overnight cell culture of VP16 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm
- remainder of overnight cell cultures were placed in the fridge for transfer to microcentrifuge tubes at a later time
- excess LP+amp decanted, the pellet resuspended in 250μL P1
- contents transferred from each 15ml conical to a new epindorf tube
- 250 μL P2 added to each tube, mixed by inverting gently 4-6 times
- 350 μL N3 added to each tube, mixed by inverting as before
- tubes centrifuged for 10 minutes at 13,000 rpm
- supernatants pipetted into new QIAprep sin columns
- columns centrifuged for 30-60 seconds, flow through discarded
- QIAprep columns washed with 0.5 ml buffer PB each and centrifuged for 30-60 seconds
- columns washed with 0.75 ml buffer PE and again centrifuged for 30-60 seconds
- flow through was discarded and tubes centrifuged for an additional minute
- column was placed in a clean 1.5 ml microcentrifuge tube
- 50 μL buffer EB was added, tubes were let stand for 1 minute and centrifuged for one minute after
- flow through kept and purity tested
Nanodrop Values for VP16
- VP16 #1
- 95.7 ng/μL
- 260/280 = 1.92
- VP16 #2
- 102.4 nm/μL
- 260/280 = 1.92
- VP16 #3
- 90.7 ng/μL
- 260/280 = 1.92
Digestion
Quantities per double digestion reaction
- 10μL sample
- 1μL EcoRI
- 1μL PSD1
- 2μL buffer
- 6μL H20
Single digest reaction
- 10μL sample
- 1μL EcoRI
- 2μL buffer
- 7μL H20
ingredients combined and all tubes were put in 37°C bath for 10 minutes
Digest with SPE1 and XBA1
- 10μL sample
- 1μL SPE1
- 1μL XBA1
- 2μL buffer
- 6μL H20
- All 3 epindorfs of VP16 were combined for a total volume of 90 μL or 9μg
- 1 unit enzyme cuts 1 μg DNA in 30-60 minutes
Digestion Gel
Ingredients
- digested enzymes from above reaction
- 1 eppendorf containing 2μL uncut VP16 and 18μL H2O
- kb ladder
- IMAGE TO BE ADDED + details
Gel Recipe: 1 X TAE
- 900 ml dH2O
- 100 ml TAE
1 gel
- 150 ml buffer TAE
- add ~2.8g agarose
- mix in beaker
- microwave until completely dissolved
- add 5 μL ethedium bromide per 100 ml gel after solution has cooled (no longer steaming)
- pour gel and let sit til solidified
Procedure
- run products on gel for ~45 minutes
- cut out band containing DNA fragment
- follow extraction protocol in QIAquick spin handbook for QIAquick gel extraction kit protocol
Nanodrop values for resulting DNA
- 5.6ng/μL
- 260/280 = 2.24
Tube placed in -20 freezer labeled VP1 fragment XBA SPE1
Glycerol Stocks of VP16
- 0.5 ml 80% glycerol stock combined with 0.5 ml overnight culture of colony #2 of V{16 TP{10 cells
- final product placed in the team fence box after mixing in the -80°C freezer
Bacterial transformation of GAL4 DNA binding domain
BBa_K105007, plate 3 well 9I, psB1A2
Procedure
- removed 1 tube TOP10 chemically competent E.Coli and placed on ice
- cleaned biobrick depository w/ ethanol
- pipetted 1 μL of plasmid into the TOP10 cell tube
- placed TOP10 cell tube on ice for 30 minutes
- heat shocked at 42°C for 30 seconds
- placed on ice for 2 minutes
- added 170 μL SOC medium
- streaked on LB + amp paltes, incubated at 37°C overnight
06-18-2010 [ top ]
LacI+NLS PCR
LacI+NLS primers arrived in mail
spun at 14,000 rpm for 10 minutes diluted stocks to 100 μM
LacIn.BB.Rev (1.7nM) diluted with 17μL DH2O LacIn.BB.Fwd (1.5nM) diluted with 1011μL DH2O NLS.BB.Rev (4.2nM) diluted with 42μL DH2O LacIn.BB.Fwd (101.1nM) diluted with 15μL DH2O
PCR recipe
- 20μL per reaction
- 1μL template plasmid
- 1μL R primer
- 1μL F primer
- 4μL HF buffer 5X
- 2μL DNTPs
- 0.5μL pFU polymerase
- rest H2O to make the volume per tube 20μL
used LACIN program in phusion PCR machine to perform PCR
06-21-2010 [ top ]
Barnase and Barstar
Barnase and Barstar Plasmids from ADDGENE arrived in mail
pMT316 - Barstar. [http://www.addgene.org/pgvec1?f=c&identifier=8608&atqx=pMT316&cmd=findpl Info from ADDGENE]
pMT413 - Barnase, Barstar. [http://www.addgene.org/pgvec1?f=c&identifier=8606&atqx=pMT413&cmd=findpl Info from ADDGENE]
pMT1002 - Barnase, Barstar. [http://www.addgene.org/pgvec1?f=c&identifier=8621&atqx=pMT413&cmd=findpl Info from ADDGENE]
Notes from lab meeting
Karmella suggested we try adding an LVA tail to the Cre we use for the crelox fence system.
GAL4 DBD Innoculation
Pipetted 5mL of LB+Amp each into capped cc tubes, and mixed bacterial scrapings from 5 separate colonies of transformed GAL4 DNA binding domain E.Coli. Set to shake at 37 degrees C overnight.
06-22-2010 [ top ]
GAL4DBD Miniprep
- pipetted 4 ml from each overnight cell culture from colonies 1-5 into a 15 ml conical centrifuged at 4400 rpm for 6 minute
- remaining overnight cell cultures were placed in fridge
- decanted LB+amp, resuspended cells in 250 μL P1 buffer
- contents transferred to eppendorfs
- 250 μL of P2 buffer was added per tube, the tubes were inverted 4-6 times
- 350 μL N3 buffer added to each, tubes inverted 4-6 times
- centrifuged for 10 min at 13,000 rpm, supernatant collected ad transferred to QIAprep spin columns
- centrifuged for 30-60 seconds, flow through discarded
- 0.5 ml buffer PB added and centrifuged for 30-60 seconds, flow through discarded, spun columns for an additional minute
- QIAprep columns put into new eppendorfs
- 50 μL buffer EB added to columns, let stand for 1 minute
- centrifuged for 1 minute at 13,000 rpm
- tubes labeled "GAL4" plus the specific colony number
PCR
PCR program for LacIN modified after Monday's unsuccessful O2 and E10 PCR (no product)
1=95°C for 10:00
2=95°C for 00:15
3=50°C for 00:30
4=72°C for 01:30
5=steps 2-4 X 29
6=72°C for 10:00
7=4°C for ∞
PCR for E10 LacI
- above PCR was repeated without O2 so as to finalize our E10 biobrick for use
- PCR was performed using the standard proportions of reagents
- The above settings on the PCR machine were used
To Make Agarose Gel
- 150 mL TAE buffer combined with 1.5 g agarose in beaker
- solution microwaved until agarose disolved
- 7.5 μL Ethidium Bromide added after beaker was no longer steaming
- solution poured into clean gel mold and gel was left to solidify
- E10 PCR product from the morning was consolidated into one tube,
- final volume =90μL PCR product + 18μL 5X loading dye =110μL
DNA was extracted from gel by slicing out the bands and DNA was purified using the QIAquick Gel Extraction Kit, following protocol specific to using a centrifuge
see [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx#Tabs=t2 QIAquick spin handbook]
Innoculations
Colonies from the following cultures were innoculated and set to shake at 37°C overnight:
- PMT413
- PMT316
- firefly luciferase
- GFP (mut)
- cre recombinase
- lox66
- lox71
06-23-2010 [ top ]
Minipreps
The following colonies were minipreped after passing the night in the shaking incubator (37°C)
- PMT413
- PMT316
- firefly luciferase
- GFP (mut)
- cre recombinase
- lox66
- lox71
Nanodrops for above minipreps
Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.
If two numbers are given, separated by a slash, then two measurements were taken, both are presented.
| Plasmid | Quantity (ng/μL) | 260/280 |
|---|---|---|
| Lox 71 #1 | 41.4 | 1.82 |
| Lox 71 #2 | 59.6 | 1.89 |
| Lox 71 #3 | 17.4/80.2 | 1.98 |
| GFP #1 | 21.2 | 1.83 |
| GFP #2 | 110.0 | 1.90 |
| GFP #3 | 60.9/124 | 1.88 |
| pMT316 #1 | 11.6 | 1.76 |
| pMT316 #3 | 16.5 | 1.81 |
| Cre #1 | 84.9 | 1.93 |
| Cre #2 | 107.7 | 1.88 |
| Lox66 #2 | 51.3 | 1.87 |
| Firefly Luciferase #2 | 309.3 | 1.91 |
| Firefly Luciferase #3 | 325 | 1.9 |
| pMT413 #1 | 461.7 | 1.89 |
| pMT413 #2 | 362.2 | 1.91 |
| pMT413 #3 | 381.8 | 1.92 |
06-24-2010 [ top ]
Primers for Barstar, Barnase, and Gal4DBD arrive
Primers:
BB.Barstar.Fwd - 36.1 nM, diluted with 361μL H2O
5'- CCT TTC TAG AAT GAA AAA AGC AGT CAT TAA C -3'
BB.Barstar.Rev - 30.0 nM, diluted with 300μL H2O
5'- AAG GCT GCA GCGGCC GCT ACT AGT TTA AGA AAG TAT GAT GGT GAT GTC -3'
BB.Barnase.Fwd - 38.5 nM, diluted with 385μL H2O
5'- CCT TTC TAG AAT GAA AAA ACG ATT ATC ATG G -3'
BB.Barnase.Rev - 13.2 nM, diluted with 132μL H2O
5'- AAG GCT GCA GCG GCC GCT ACT AGT TTA TCT GAT TTT TGT AAA GGT CTG -3'
BB.Gal4DBD.Fwd - 38.5 nM, diluted with 385μL H2O
5'- CCT TTC TAG AAT GAA GCT ACT GTC TTC TAT -3'
BB.Gal4DBD.Rev - 32.9 nM diluted with 329μL H2O
5'- AAG GCT GCA GCG GCC GCT ACT AGT CGA TAC AGT CAA CTG TCT TTG -3'
Digestion
Our first digestion was unsuccessful except for the confirmation of the PMT316 fragment; this can be attributed to an incorrect combination of restriction enzymes. The digestion was performed again in the afternoon using EcoRI + SpeI for the biobricked plasmids (lox71, GFP mut, lox66, cre recombinase, and firefly luciferase) and EcoRI + HindIII for PMT413.
Gel Image, first digestion: 350px
PMT316 digests in lanes 8 and 9 show bands of approximately 439 bp. (above image)
The length of the shortest bands were confirmed to be roughly the same length of the desired excerpt with the exception of Lox66 and Lox71 where it was determined that the inserts being only 34 bp had run off the gel, therefore the single band seen was the remaining plasmid.
Yeast Growth Assay
To assay the effect of Methoxyfenozide on yeast, three flasks containing 25μL DMSO (control), 2.5μL of 100mM Methoxyfenozide stock in 25mL DMSO to form the 1μM condition, and 25μL of 100mM Methoxyfenozide stock in 25mL DMSO to from the 10μM condition. Each beaker also contained y190 yeast strains and YPD medium.
To construct a growth curve of the yeast under the 0μM, 1μM and 10μM conditions, 1mL from each flask would be pipetted into a cuvette and measured in a spectrophotometer ever hour to assess yeast density.
Unfortunately in this first attempt, no growth was observed after 4 hours, in the control and experimental groups, so the test was called off, to be repeated tomorrow.
| Time | 0μM Methoxyfenozide | 1μM Methoxyfenozide | 10μM Methoxyfenozide |
|---|---|---|---|
| 11:00 AM | .045 | .042 | .036 |
| 12:00 PM | .036 | .040 | .041 |
| 3:00 PM | .035 | .036 | .039 |
06-25-2010 [ top ]
B21 Innoculation
Inoculated 3 colonies of B21 transformed E.Coli and set them to shake for 8 hours. We will maxiprep the B21 plasmid for use as a vector backbone.
PCR of Gal4DBD and Barnase, attempt 2
Ran 3 tubes of each for a total of 6. 7x Mastermix:
- 14μL DNTP
- 3.5μL Polymerase
- 28μL 5x Buffer
- 73.5μL DH2O
Each tube contained:
- 1μL Fwd primer
- 1μL Rev Primer
- 1μL Minipreped sample
- 17μL Mastermix
Reduced annealing temp to 50°C from 56°C which was the last attempt, ran otherwise identical LACIN program
Gel of Gal4 DBD and Barnase from second PCR attempt
Ran 1.2% E-gel of the 6 PCR product tubes.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Ladder | Gal4 DBD 1 | Gal4 DBD 2 | Gal4 DBD 3 | Barnase 1 | Barnase 2 | Barnase 3 | Ladder |
QIAQuick Purification of PCR Product
Because colonies 1 and 2 of Barstar worked properly on last week's gel, the PCR products in those two tubes were combined and purified using the Qiagen PCR purification protocol.
- as there was about 25μL of PCR product in the two together, 125μL of PB buffer was added (5x the concentration of the PCR product)
- pipetted the mix into a QIAquick spin column, spun for 30 seconds at 13,000 rpm
- discarded flow-through
- added .75mL PE buffer to the column, spun for 30 seconds
- discarded flow-through, and spun again for 1 minute
- moved the column to a fresh eppendorf tube
- added 50 μL EB buffer to the column, spun for 1 minute
- labeled the eppendorf tube 'Barstar PCR purified'
06-28-2010 [ top ]
B21 plasmid maxiprep
colony inoculated overnight and medium was centrifuged to obtain a pellet.
followed instructions as described in the QIAgen Plasmid Purification Handbook pg. 19-23. (2005 edition)
06-29-2010 [ top ]
pMT1002 Miniprep
- 4ml from each overnight cell culture of pMT1002 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm
- remainder of overnight cell cultures were placed in the fridge for transfer to microcentrifuge tubes at a later time
- excess LP+amp decanted, the pellet resuspended in 250μL P1
- contents transferred from each 15ml conical to a new epindorf tube
- 250 μL P2 added to each tube, mixed by inverting gently 4-6 times
- 350 μL N3 added to each tube, mixed by inverting as before
- tubes centrifuged for 10 minutes at 13,000 rpm
- supernatants pipetted into new QIAprep sin columns
- columns centrifuged for 30-60 seconds, flow through discarded
- QIAprep columns washed with 0.5 ml buffer PB each and centrifuged for 30-60 seconds
- columns washed with 0.75 ml buffer PE and again centrifuged for 30-60 seconds
- flow through was discarded and tubes centrifuged for an additional minute
- column was placed in a clean 1.5 ml microcentrifuge tube
- 50 μL buffer EB was added, tubes were let stand for 1 minute and centrifuged for one minute after
- flow through kept, eppendorfs labeled pMT1002 plus their colony number
Also see pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook] for miniprep instructions.
pMT1002 Miniprep Nanodrop values
| Plasmid | Quantity (ng/μL) | 260/280 |
|---|---|---|
| pMT1002 #1 | 68.2 | 1.84 |
| pMT1002 #2 | 138.7 | 1.84 |
| pMT1002 #3 | 127.2 | 1.88 |
PCR of miniprepped Barnase
20μL per tube
In each tube:
- 1μL Forward primer
- 1μL Reverse primer
- 0.5μL polymerase
- 4μL 5X buffer
- 2μL DNTPs
- 1μL DNA sample
- 10.5μL water
3 samples were made for the Barnase
pMT1002 Barnase PCR
B21 Plasmid Digest
put 10x green FD buffer on ice to thaw.
Tube A:
- 1μL Xba1
- 1μL Pst1
- 2μL green 10x FD buffer
- 3.4μL colony A plasmid (the amount used to get .5μg of plasmid)
- 12.6μL H2O (to bring the total to 20μL)
Tube B:
- 1μL Xba1
- 1μL Pst1
- 2μL green 10x FD buffer
- 1.7μL colony B plasmid (the amount used to get .5μg of plasmid)
- 14.3μL H2O (to bring the total to 20μL)
Pst1 and Xba1 digest of B21 plasmid, lane 4 and 5 cut, 7 and 8 uncut
Second Attempt
Tube A:
- 1μL Xba1
- 1μL Pst1
- 5μL green 10x FD buffer
- 12.5μL colony A plasmid (the amount used to get .5μg of plasmid)
- 30.5μL H2O (to bring the total to 50μL)
Tube B:
- 1μL Xba1
- 1μL Pst1
- 5μL green 10x FD buffer
- 6.6μL colony B plasmid (the amount used to get .5μg of plasmid)
- 36.4μL H2O (to bring the total to 50μL)
Ran on a 1% agarose gel, 100 Volts for 30-40 mins.
Lane 2 buffer, lane 8 and 9 Colony B, lanes 15 and 16 colony A
Cut out the backbone (upper band, and placed into four eppendorf tubes, labeled 'B21 backbone' and with their respective weight before the gel was added. Placed all four in the iGEM team fence box in the -4°C freezer to be purified tomorrow.
Other
- Designed primer sequences for ACC synthase degradation site, linker sequence connecting Barnase to the degradation site.
- Designed primers to amplify germination/embryogenesis promoter and AtArp2 promoter.
