Team:Toronto/Notebook

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May 2012

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June 2012

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July 2012

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August 2012

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September 2012

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May 17

16 plates each of LB agar, ampicillin, tetracycline, and chloramphenicol were made.
To test the effectiveness of our antibiotic stock, five vials were created, and were kept in the shaker.
Vial one contained LB control; vial two contained LB and DH5α; vial three contained LB, DH5α, and ampicillin; vial four contained LB, DH5α, and chloramphenicol; vial five contained LB, DH5α, and tetracycline.

May 18

Placed all the agar plates in the fridge, as no contamination was visible. Vial two was cloudy, as expected, indicating DH5α growth. The large block of ice formed over ten years' neglect in the 4°C fridge below our lab bench was cleaned with a chisel and mallet.
Four plates were prepared with DH5α: LB agar; LB ampicillin plate; LB tetracycline plate; LB chloramphenicol plate.
Vial Two was disposed of after bleaching. Stocks of 70% and 100% EtOH were obtained free from the Bendeck lab upstairs, replenishing our meagre stock.

May 22

Made 1L of LB, as well as autoclaved some glassware. Only plate one from May 18 showed any bacterial growth after incubation at 4°C overnight, indicating good sterile technique during the training exercises yesterday.
SNC Lavalin, Monsanto, and New England BioLabs were contacted for funding enquiries. Approached Rob Reedijk about consolidation of financial accounts. Obtained A. thaliana seed from the McCourt lab at EEB, along with their recommended growth protocol.

May 23

For demonstration purposes, we transformed cyan fluorescent protein from the BioBrick distribution plates with ampicillin resistance into DH5αC (competent DH5α).

May 24

Checked the cyan fluorescent transformation, which failed. To check whether the BioBrick plasmids were compromised, or whether our technique was to blame, we will retry the transformation with another BioBrick. Ordered supplies from MedStore.

May 25

Made kanamycin plates, and checked on the May 24 transformations, which proved to be successful. Aimed to finalise the phytase construct.

May 28

Various sponsorship opportunities were pursued for the lab for much of the day.
2 test transformations were performed as another tutorial. Kanamycin's efficiency was tested with the following three treatments: LB; LB and DH5αC; LB, DH5αC, and K.

May 29

Tested a 2µL/mL kanamycin overnight culture versus an LB blank on the spectrophotometer. Placed two different projects into the shaker: Project One (10mL LB; 10µL C; 20µL DH5α): LB control; LB, DH5α, and C; LB, DH5α, C, and BioBrick K414007. Project Two (10mL LB; 20µL DH5α): LB control; LB and DH5α; LB, DH5α, and 20µL K; LB, DH5α, and 25µL K; LB, DH5α, and 30µL K.

May 30

The LB controls failed from yesterday, so the 500mL container of LB from which we took yesterday's samples was thrown out. The K414007 promoter was miniprepped using a Qiagen Plasmid Extraction Kit. Using the nanodrop, we found our extraction sample to have a density of 20ng/µL – good miniprep. Performed a transformation using K414007, so as to keep plates in the fridge with this part handy.

June 1

Competent E. coli were made using the CaCl2 protocol. The transformation from yesterday was successful. We put K414007 into the -20°C freezer. The primers for the following BioBricks we intend to make as part of our constructs were designed: Kozak, HSP18.2 terminator, PHT1;2 promoter, Extensin signal, AtPAP15, and PhyA1 (which has two exons, and therefore, four primers needed). We also miniprepped the K382024 terminator, and made a long-term stock as well as a lab stock.

June 4

The BioBrick K414007 was grown up so that it was possible to make a long-term stock. We did so by inoculating a colony on an LB and chloramphenicol plate. Our competent cells from June 1 were tested by transforming K414007. A restriction digest was run on K382024 using EcoR1 (E) and Spe1 (S).

June 5

K414007 was miniprepped and a long-term stock was made by suspension in 1000µL 40% glycerol and placed in the -80°C. The nanodrop showed densities for K414007 of 78 and 100 ng/µL. As a test for competency, the newly-made competent DH5α was transformed successfully. We now have a stock of 49 aliquots in the -80°C. The chemical cupboard in the lab was also organised.

June 7

A 20% SDS solution was made for DNA isolation next week, and PCR primer designs were finalised.

June 8

The PCR primers were ordered – desalted, 25 nmole, with no 5' or 3' modifications. The following four strains of Aspergillus from Sporometrics: (i) SCCM# 11-B5 A. niger (Sporometrics Internal QC); (ii) SCCM# 15-E9 A. brasiliensis (Environmentally acquired); (iii) SCCM# 17-B6 A. niger (Environmentally acquired); and (iv) SCCM# 11-B6 A. niger (Sporometrics Internal QC).
Made LB agar, into which we added kanamycin from old stock we found in the chemical storage cupboard and was more effective than the stock we bought last month. (Remember that the antibiotic solution is added to the LB agar after it is autoclaved, but before it has cooled to solidification) Two plates of each Aspergillus strain were prepared in PD agar in order to make a long-term stock. The plates were left to culture over the weekend at 37°C. MS medium was made, and A. thaliana seeds were inoculated on the plates, which were stored in the cold room at 4°C.

June 11

Made a long-term stock of all Aspergillus strains by suspending in 40% glycerol and storing in the -80°C. Attempted a Chelex prep on the A. niger strain 11-B5, which failed. Transformed K652001 using DH5alphaC.

June 12

Made 500mL CTAB extraction buffer for D. carota DNA extraction, and 500mL TENS buffer for A. niger DNA extraction. The fume hood was cleaned, and all the chemicals were organised therein. The K652001 transformation was done with two plasmids (since the BioBrick kit came with two plasmids): one failed, the other did not. As a result, we must send this DNA for sequencing before continuing with PCR on this BioBrick. The D. carota DNA extraction was attempted, but had to be postponed, because the proteinase K stock exploded in the -20°C.

June 13

Digested and ligated the Kozak sequence and did a transformation. The D. carota DNA extraction was attempted, and failed because regular 15mL Falcon tubes containing the DNA samples broke in the refrigerated centrifuge.

June 14

The first portion of the D. carota DNA extraction was completed, and the result was left to incubate in the -20°C overnight, as per the extraction protocol. The June 13 Kozak transformations failed, so another attempt shall be made to remedy this situation.

June 15

UTSU was contacted in order to begin the process of cheque re-issue for monies owed to iGEM. The lab bench was re-organised in order to make everything easier to access. The D. carota genomic DNA extraction was completed. A grow-box was made with red and blue LEDs such that the A. thaliana could grow properly under regulated lab conditions. The following solutions were made in the lab: 1L 5M NaCl, 500mL 20% SDS, 500mL 3M NaOAc pH 5.3, 500mL 25mM MgCl2, and 500mL 7.5M NH4OAc.

June 18

Another ligation and transformation of the Kozak sequence was attempted. We tried making an agarose gel, but it did not solidify in time, so the gel was postponed until June 19. The 1% agarose gel was to run the carrot genomic DNA. The two carrot gDNA samples were nanodropped: sample one (1) has a density of 68.4 ng/µL, and sample two (2) has a density of 91.4 ng/µL.

June 19

Got $300 contribution from the Skule Alumni Fund. Went on an outing to Queen St. W. to get an amber bottle, electrical wiring, and a scalpel. Made a 1% agarose gel, which broke (again!) and made 400mL of 24:1 chloroform: isoamyl alcohol, as well as 1L Tris-HCl, pH 8.0.

June 20

Made 1L each of Super Optical Both with Catabolite repression (SOC) and Terrific Broth. Made a 1% agarose gel, which broke...again. Made yet another gel, using 1.2g agarose, 120mL 1x TBE buffer, and a voltage of 60V. This procedure worked, and the carrot DNA samples were run, and DNA presence in the samples was confirmed. The gels were accidentally thrown out after imaging. A gDNA prep of the A. niger was also done, with some modifications from the original protocol. Some A. thaliana were planted in pots from the MS-K plates they were cultured on. They were excised by scalpel and transferred to pots filled with potting soil to an approximate 4" height. The plants were watered with milli-Q.

June 21

The shaker was observed to see if its malfunction one week ago was purely by chance; it appeared to be so, and thus, the following five treatments were placed into the shaker for overnight incubation: (i) LB control; (ii) LB, DH5alpha, ampicillin; (iii) LB, DH5alpha, chloramphenicol; (iv) LB chloramphenicol and K652001; and (v) LB ampicillin, and psb1A3.
A 1% agarose gel (made using the 1.2g agarose recipe discussed previously) was made, and the A. niger DNA prep was run. The gel was inconclusive, and must be run again tomorrow. The DNA primer melting temperatures were determined and posted for the team on Dropbox. A 100x Antibiotic-antimycotic solution, composed of amphotericin B, streptomycin, and penicillin was bought for use on the potted plants. The solution was aliquoted into 15mL Sarstedt tubes, placed in the -20°C. A few tubes were thawed, diluted ten-fold, and transferred to a spray bottle. The solution will be sprayed on twice weekly henceforth.

June 22

The K652001 and psb1A3 treatments from the shaker were miniprepped. The samples were stored in elution buffer (10mM Tris-HCl, pH 8.0) in the 4°C. A shortage of QIAprep Mini Columns was discovered in the lab, rendering these minipreps more time-consuming than usual. The first successful (out of seven previous attempted) gel was run on the a. niger gDNA.

June 25

The day was devoted to running a PCR cycle in order to isolate the carrot extensin gene. In a 50µL reaction, the following volumes of reagents were used: 5µL 10X Standard Taq Reaction Buffer; 1µL 10mM dNTP mix; 0.5µL 20mM upstream primer; 0.5µL 20mM downstream primer; 7.31µL DNA template at 68.4ng/µL; 0.25µL Taq DNA Polymerase; and 35.44µL DEPC-treated water. Initial denaturation of 5 minutes at 95°C, followed by 30 cycles of 95°C (30 seconds), 73°C (60 seconds), and 68°C for 60 seconds, and a final extension at 68°C for 5 minutes. The PCR was unsuccessful, as our phosphorimage shows.

June 26

A second PCR cycle in order to isolate the carrot extensin gene was attempted; a gel showed that the PCR was, again, unsuccessful. The following five treatments were placed in the 37°C as an O/N culture: LB control; LB, DH5α, ampicillin; LB, DH5α, chloramphenicol; LB, DH5α, chloramphenicol, K652001; LB DH5α, ampicillin, pSB1A3; and Terrific Broth, DH5α, ampicillin, and pSB1A3.The A. thaliana DNA was lyophilized in preparation for the plant gDNA miniprep.

June 27

Ligation of the Kozak sequence to pSB1A3, and research into the melting temperatures of the primers for HSP18.2, AtPAP15, and PHT1;2.

June 28

Isolated A. thaliana DNA-3.6 ng/ul and 3/7 ng/ul (double the value of yesterday); precut before freezing + centrifuged to prevent viscous lysates from shearing DNA apart
Transformed psb1A3 + kozak ligations into competent cells, plated and left to grow overnight

June 29

PCR of HSP18.2 performed with A.Thaliana DNA to isolate gene with restriction sites.
Gel run with PCR products indicates that the PCR has failed, no visible bands.
Selected 4 white colonies from PSB1A3 + kozak transformation, growing up with ampicillin.

June 30

Miniprepped 4 kozak colonies (names koz 1-4) from overnight culture. Koz 2 was found to be red after spinning down, and was discarded. Other 3 samples were miniprepped and nanodropped. Koz 1: 110.9 ng/ul; Koz3: 118.8 ng/ul; Koz4: 47.6 ng/ul;

July 3

Digested Koz 1,3,4 with E and P, ran a gel to see if there were any visible bands at approx 40 BP, indicating successful ligation Gel results ambiguous. koz 4 may be successful, will run again later.

July 4

Gel ran with koz 1, 3, 4 again, along with synthesized oligo to get an idea of where the correct band should be Results show that all 3 ligations failed. Kozak sequence did not ligate successfully.
PCR performed with A. thaliana gDNA to isolate PHT1; 2 promoter, as well as with extracted carrot DNA to isolate carrot extension gene
Gel ran with PCR products of PHT1; 2 isolation attempt show no visible bands, indicating failure
Gel ran with PCR products of carrot extensin isolation attempt show no visible bands, indicating failure.

July 12

Due to continuously failed attempts at DNA isolation and ligation, the team has decided to weigh our options as to whether to continue our current attempts and methods, or to opt for the synthesis of our constructs. Research is being done into pricing and time requirements.

July 15

We have decided to have part of our constructs synthesized to save time. We are synthesizing the following parts: 1.Kozak-RIAFP-HSP18.2; 2.AT1g73160 (Root specific Promoter); 3.Kozak-Carrot Extensin-PhyA- K382024; These will take 2-3 weeks to be delivered. We will ligate the promoters onto Construct 1 and 3.

July 30

RIAFP Construct has arrived. Plasmid was re-suspended and transformed into competent cells, and streaked into ampicillin plates, and grown overnight.

July 31

Yesterday’s transformation was successful, large number of colonies observed on plate. 2 colonies were selected and grown in the shaker overnight with LB, and appropriate controls

August 1

Prof Yoshioka has allowed her PHD student to help us with floral dips, as well as agrobacterium transformations. A. thaliana seeds were pre-soaked to induce germination

August 2

The RIAFP plasmid miniprepped from yesterday was digested, ligated, and transformed into PSB1C3 plasmid with the K414007 promoter. A. Thaliana seeds were sown after being pre-soaked on Aug. 1

August 3

Colonies were selected from the K414007-RIAFP-PSB1C3 plate, and grown overnight

August 4

Colonies from overnight culture were miniprepped, and gel was run to see if ligation was success. Band seen were 500 BP shorter than expected, indicating that the promoter did not ligate successfully.

August 6

We have decided to switch to standard assembly instead of 3 antibiotic assemblies. A gel was ran that contained K414007 digested with E and S, RIAFP digested with X and P, and PSB1C3 that was digested with E and P. the resulting bands were extracted, ligated together, and transformed into competent cells. Cells were streaked onto a C plate and left to grow overnight.

August 7

An account was set up with the TCAG sequencing server, and the VF2 primer was ordered. A single colony appeared on the C plate from yesterday’s transformation. Colony was grown up overnight . PhyA construct has arrived. Plasmid was re-suspended and transformed onto A plates, and left to grown overnight.

August 8

PhyA transformation was successful. Colonies were selected to grow overnight in ampicillin-K414007+RIAFP overnight culture was miniprepped. Plasmids was digested with E and P. Gel was ran that confirmed correct insert length for successful ligation. Root Specific Promoter construct has arrived. Plasmid was re-suspended and transformed onto A plates, and left to grown overnight.

August 9

Primers for sequencing picked up. Will send off K414007+RIAFP plasmid tomorrow for sequencing. Phya A construct from Overnight culture was miniprepped and nanodropped. Roots Specific Promoter transformation was successful. Colonies were selected to grow overnight in ampicillin

August 10

K414007+RIAFP construct was sent off for sequencing under name LTC to see if successful ligation can be confirmed Roots Specific Promoter overnight culture was miniprepped.

August 13

Sequencing results returned. LTC ligation was successful. One construct has been finished. Roots Specific Promoter plasmid digested with E and S. PhyA plasmid digested with X and P. PSB1C3 digested with E and P. Digested plasmids were ligated together, and transformed. Cells were streaked into C plates and grown overnight.

August 14

Multiple colonies seen on C plate from last night’s transformation 4 Colonies selected to be grown overnight with C.

August 15

4 colonies from overnight culture were miniprepped, and digested with E and P. Gel was run, and results were ambiguous, due to large black stain in center of gel. All 4 miniprepped plasmids were sent off for sequencing 4 more colonies were selected from the PHYA+Root specific promoter plate and grown overnight

August 17

Results from sequencing returned. Results show that the Puc57 and PSB1C backbone had ligated to each other without the inserts. Will use gel extraction to avoid this from happening in the future. 4 new colonies were miniprepped and sent off for sequencing

August 18

Sequencing results show no success in ligation. Fused backbones are still prevalent. Long term stock of PhyA and Root Specific Promoter were grown up overnight in A.

August 19

Phy A and Root specific promoter overnight culture was miniprepped and nanodropped. Agrobacterium were transformed with the LTC construct and streaked with C and Rif plates.

August 20

PhyA Plasmid extracted yesterday was digested with X and P. Root Specific Promoter Plasmid extracted yesterday was digested with E and S. Gel was ran, in which the inserts were extracted and ligated into PSB1C3. Ligation was transformed into competent cells and streaked on C plates. PCR was performed to isolate the carrot extensin gene using PhyA plasmid. Gel of PCR products was ran, and results showed a 150 BP band, approximately that of the carrot extensin gene with biobrick overhang.

August 21

Transformation from yesterday was successful. 2 colonies were selected and were grown up overnight in C. 4 PCR tubes of HSP18.2 were made to isolate insert with biobrick overhang. Gel ran indicates failure. No bands visible. PCR tubes also prepared for isolation of PHYA. Gel ran on PCR product indicates preliminary success.

August 22

4 Colonies selected from Aug 19 Agrobacterium transformations to grow in shaker over 2 days. 2 overnight cultures from yesterday, known as PINE1, AND PINE 2, were miniprepped and nanodropped.

August 22

4 Colonies selected from Aug 19 Agrobacterium transformations to grow in shaker over 2 days. 2 overnight cultures from yesterday, known as PINE1, AND PINE 2, were miniprepped and nanodropped.

August 23

Digested all PCR products we have (2 carrot extensions, and two PhyA’S) and ligated them to PSB1C3 backbone. All 4 ligations were transformed into competent cells and grown overnight on C plates.

August 24

Obtained grown agrobacterium cultures and performed whole cell PCR in attempt to extract HSP18.2. Gel was run indicating that PCR failed.

August 27

Pine1 and Pine 2 were sent off for sequencing

August 28

Ran PCR for HSP18.2 and PhyA Transformation of Yesterdays PCR products for carrot extensin was successful. Colonies were selected and grown overnight in C. Root specific promoter was ligated to PSC1C3, and transformed. Cells were streaked onto C plates and grown overnight. PhyA PCR product was digested, ligated and transformed. Cells streaked into C plates and grown overnight. Began sub culture of Agrobacterium: Added 250 ul Agro glycerol stock to a Lb-Agar-Rif-C plate. Grown at 28C for 2 days.

August 29

Sequencing Results returned show Pine1 and Pine 2 have correct sequence. All genetic constructs are now complete. PhyA Transformation was successful. Colonies selected and grown overnight in C. Carrot extensin +PSB1C3 overnight culture was miniprepped and nanodropped. Roots specific promoter+PSB1C3 transformation from yesterday was successful. One colony was selected and grown up overnight. Agrobacterium was transformed with PINE Construct, and streaked into RIF-C plates. Cells were streaked and left to grow for 2 days. PCR of HSP 18.2 attempted with LTC plasmid. Gel of PCR products indicates that it was not successful.

August 30

Roots Specific Promoter was miniprepped and nanodropped. PHYA in PSB 1C3 miniprepped and nanodropped.

August 31

PINE Agrobacterium colonies were selected and transferred to a liquid culture for 2 days. Made new Rif-C plates. Prepped PhyA for sequencing . Ran PCR of HSP18.2. Gel results show that PCR failed.

September 2

PINE culture and TLC subculture re-grown in our lab, as Earth Sci was inaccessible over the weekend. LTC glycerol stock streaked in LB-RIF-GENT-C plates. Grow to Tuesday @ 28C.

September 4

Incubated PINE agrobacterium in liquid culture over 2 days.

September 5

LTC floral Dip medium was prepared.

September 5

Pine Overnight culture was made into long term stock. Part of it was used to start subculture 1, LTC floral dip was performed with flowering A. thaliana.

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 <h2><a href="#acc4">August 2012</a></h2>
 <p>Click on a highlighted day to view the notebook items for that day.</p>
+<div id="calendar" style="margin-left:50px;">
+   <ul id="days">
+    <li>
+     <span title="Sunday">SU</span> <span class="sep">|</span>
+     <span title="Monday">MO</span> <span class="sep">|</span>
+     <span title="Tuesday">TU</span> <span class="sep">|</span>
+     <span title="Wednesday">WE</span> <span class="sep">|</span>
+     <span title="Thursday">TH</span> <span class="sep">|</span>
+     <span title="Friday">FR</span> <span class="sep">|</span>
+     <span title="Saturday">SA</span>
+    </li>
+   </ul>
+<!-- On links Below: NU = Not Used; NA = Not Active; AL = Active Link -->
+   <ul class="weeks">
+    <li>
+     <a class="nu" href="#" title="">--</a> <span class="sep">|</span>
+     <a class="nu" href="#" title="">--</a> <span class="sep">|</span>
+     <a class="nu" href="#" title="">--</a> <span class="sep">|</span>
+     <a class="al" href="#august1" title="Active Date Link">01</a> <span class="sep">|</span>
+     <a class="al" href="#august2" title="Active Date Link">02</a> <span class="sep">|</span>
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+     <a class="al" href="#august4" title="Active Date Link">04</a> <span class="sep">|</span>
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+     <a class="al" href="#august27" title="Active Date Link">27</a> <span class="sep">|</span>
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+     <a class="nu" href="#" title="">--</a> <span class="sep">|</span>
+    </li>
+   </ul>
+</div>
 </section>
 <section id="acc5">
 <h2><a href="#acc5">September 2012</a></h2>