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| {{:Team:British_Columbia/Templates/MainHeader}} | | {{:Team:British_Columbia/Templates/MainHeader}} |
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- | ==Team CRISPR== | + | =Notebook= |
- | ===July 3===
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- | List of BioBricks that are/will be on the way:
| + | <br> |
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- | Cas9:
| + | <br> |
- | *Cas9 - Joe and Anna
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- | *pTet -
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- | *const. promoter + Cas9 - Joe
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- | *promoter + Cas9 + Leader + R + spacer + R (on Kan)
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- | *L + R + spacer + R
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- | Target:
| + | <html> |
- | *tracerRNA
| + | <style> |
- | *repeat + spacer + repeat - Vanins*2 + underlings
| + | .icon { width: 180px; margin 5px;} |
- | *leader - Ray, Fisal, Dan
| + | </style> |
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- | Decoy:
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- | *PAM + spacer (on Amp)
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- | ==Team Cinnamaldehyde==
| + | The CRISPR, Flavouring, and Caffeine teams spent numerous hours in the lab to produce the results presented throughout the project. We tracked our progress through daily updates in our lab notebooks, which enabled the rest of the team to follow the work being done. This notebook is a compilation of the daily activities, procedure, and updates of these groups. <b>Follow the progress of each group in their notebook or look up the specific protocols they used and learn how to do it yourself by clicking the buttons below!</b> |
- | 4-coumarate-CoA ligase:
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- | *PCR worked
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- | ==Team Caffeine==
| + | <br> |
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- | ===June 20===
| + | <br> |
- | Heat-shock competent cells were made today - they can be found in the top compartment of the -80º Freezer #3 in the room full of freezers (thanks Cam!). We will be ordering primers to modify the TU Munich caffeine biosynthesis BioBricks we got in the Distribution Kit. These are found in wells 17H, 17J, and 17L on plate 2.
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- | We're making a start - what are the other groups up to?
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- | ===June 27=== | + | <div> |
- | Parts 17H/J/L were previously transformed into the above-mentioned competent cells and plated. Today, transformation efficiency was found to be very low - two colonies per plate after more than 18 hours at 37ºC. The three parts were miniprepped, with DNA concentration of 154.3ng/ul, 323.8ng/ul and 117.3ng/ul respectively.
| + | <center> |
| + | <a href="https://2013.igem.org/Team:British_Columbia/Notebook/CRISPR"><img class="icon" src="https://static.igem.org/mediawiki/2013/a/a9/Crispr2-01.png" |
| + | onmouseover="this.src='https://static.igem.org/mediawiki/2013/f/fa/Crispr_hover-01.png'" |
| + | onmouseout="this.src='https://static.igem.org/mediawiki/2013/a/a9/Crispr2-01.png'"/></a> |
| + | <a href="https://2013.igem.org/Team:British_Columbia/Notebook/Flavours"> |
| + | <img class="icon" src="https://static.igem.org/mediawiki/2013/9/96/UBCFlavourButton.png" |
| + | onmouseover="this.src='https://static.igem.org/mediawiki/2013/e/e1/UBCFlavoursButton2.png'" |
| + | onmouseout="this.src='https://static.igem.org/mediawiki/2013/9/96/UBCFlavourButton.png'"/></a> |
| + | <a href="https://2013.igem.org/Team:British_Columbia/Notebook/Caffeine"> |
| + | <img class="icon" src="https://static.igem.org/mediawiki/2013/d/d3/Caffeine2-01.png" |
| + | onmouseover="this.src='https://static.igem.org/mediawiki/2013/9/9c/Caffeine_hover-01.png'" |
| + | onmouseout="this.src='https://static.igem.org/mediawiki/2013/d/d3/Caffeine2-01.png'"/></a> |
| + | <a href="https://2013.igem.org/Team:British_Columbia/Notebook/Protocols"> |
| + | <img class="icon" src="https://static.igem.org/mediawiki/2013/3/3b/UBCProtocols2.png" |
| + | onmouseover="this.src='https://static.igem.org/mediawiki/2013/b/b5/UBCProtocols.png'" |
| + | onmouseout="this.src='https://static.igem.org/mediawiki/2013/3/3b/UBCProtocols2.png'"/></a> |
| + | </center> |
| + | </div> |
| | | |
- | ===July 4===
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- | We ran a confirmation PCR on the miniprepped plasmids with standard Biobrick primers, VF2 and VR, to confirm the presence of an insert in the backbone. Gel confirmation of the PCR products showed a band around 1kb, our expected gene size, for one of the genes only (17J). The other samples did not show any bands. Because the transformation efficiency was very low, unsuccessful transformation was thought to be causing the issues. We decided to retry transformation of the kit parts into "tried and true" cells, and to discard today's plasmids if the new transformation gave different results.
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- | ===July 5===
| + | </html> |
- | We acquired aliquots of "good" competent cells from Ray, our grad advisor, and re-tried the transformation of the distribution kit parts.
| + | |
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- | In order to confirm our parts of interest (17H/J/L) contained the desired insert, we retried PCR, this time with 1 uL of eluted DNA from the distribution kit as template. Even with this low concentration of template, amplification appeared successful. A bright band was seen for 17J and no bands for the other two, validating yesterday's PCR results.
| + | <br> |
| | | |
- | We designed and ordered primers to remove the "TCACA" yeast consensus sequence found in the TU Munich Biobrick parts, as our goal was to replace this sequence with a bacterial ribosome-binding site. This round of PCR was named "PCR 1" to indicate downstream PCR reactions that would use these products as template.
| + | <br> |
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- | ===July 8===
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- | Transformation of 17H/J/L with Ray's competent cells was found to be highly efficient, indicating our own heat-shock competent cells were faulty. We received the protocol used to prepare these cells, and a new stock was made today.
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- | ===July 9===
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- | Cultures of 17H/J/L were miniprepped, and with the products, we:
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- | *did a restriction digest with EcoRI and PstI to confirm the presence of an insert
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- | *prepared each plasmid for sequencing to confirm the sequence of the insert (and the presence of the yeast consensus sequence, which we hoped to remove)
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- | *PCR with primers designed to remove the yeast consensus sequence (TCACA)
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- | The confirmation gel for the restriction digest showed there was an insert slightly larger than 1kb for each of the genes, which was desired. However, PCR did not appear to work. Higher molecular weight smears were seen at around 2.5kb, indicating the template was not amplified, likely because there was too much of it. Around 200ng of plasmid template was used, annealing temperature was the lower primer Tm, and no DMSO was used. We will try this PCR again with 50ng of template, as per NEB's suggestions, and fewer cycles.
| + | <br> |
- | | + | |
- | ===July 11===
| + | |
- | PCR 1 was retried for all three parts, with the amount of template reduced to around 30 ng per 50uL reaction. Phusion HF (high fidelity) buffer was used, in the absense of DMSO. Thermocycling conditions were not changed, except the reduction of number of cycles from 30 to 20. Parts J and L were successfully amplified this time, with bands seen at around 1100-1200 bases on the agarose gel. However, nothing was seen for part H. One option is to try adding DMSO to a final concentration of 3% to hopefully allow amplification of part H.
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- | | + | |
- | ===July 12===
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- | With Ray, we designed and ordered the "stitching" primers that would allow us to assemble our Biobricks easily, by using restriction enzyme cut sites other than the four standard to avoid illegal cut sites. This follows the decision to combine the three available (from the kit) caffeine biosynthesis genes and Biobrick them behind a promoter and ribosome-binding site (RBS). We included a bacterial ribosome-binding site in each of the forward primers to allow transcription when each gene is also Biobricked individually. This round of PCR reactions was named "PCR 2."
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- | | + | |
- | PCR 2 would ideally yield the following products:
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- | *Part H: ---'''XbaI'''---gene H---'''SpeI'''---'''NcoI'''---
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- | *Part J: ---'''NcoI'''---'''XbaI'''---gene J---'''SpeI'''---'''BamHI'''---
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- | *Part L: ---'''BamHI'''---'''XbaI'''---gene L---'''SpeI'''---
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- | | + | |
- | Individually and ligated together, these products could be digested by XbaI and SpeI and inserted into a Biobrick vector cut with X and S.
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- | | + | |
- | ===July 15===
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- | | + | |
- | ===July 17===
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- | ===July 18===
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- | ===July 19===
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- | ===July 23===
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- | ===July 24===
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- | | + | |
- | ===August 12===
| + | |
- | ===August 13===
| + | |
The CRISPR, Flavouring, and Caffeine teams spent numerous hours in the lab to produce the results presented throughout the project. We tracked our progress through daily updates in our lab notebooks, which enabled the rest of the team to follow the work being done. This notebook is a compilation of the daily activities, procedure, and updates of these groups. Follow the progress of each group in their notebook or look up the specific protocols they used and learn how to do it yourself by clicking the buttons below!