Team:British Columbia/Notebook
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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. | 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. | ||
+ | |||
+ | 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. | ||
===July 8=== | ===July 8=== | ||
- | 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. | + | 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. |
===July 9=== | ===July 9=== | ||
Line 57: | Line 59: | ||
- | With Ray, we | + | ===July 15=== |
+ | 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 were named "PCR 2." | ||
- | PCR | + | PCR 2 would ideally yield the following products: |
*Part H: ---'''XbaI'''---gene H---'''SpeI'''---'''NcoI'''--- | *Part H: ---'''XbaI'''---gene H---'''SpeI'''---'''NcoI'''--- | ||
*Part J: ---'''NcoI'''---'''XbaI'''---gene J---'''SpeI'''---'''BamHI'''--- | *Part J: ---'''NcoI'''---'''XbaI'''---gene J---'''SpeI'''---'''BamHI'''--- | ||
*Part L: ---'''BamHI'''---'''XbaI'''---gene L---'''SpeI'''--- | *Part L: ---'''BamHI'''---'''XbaI'''---gene L---'''SpeI'''--- | ||
+ | |||
+ | 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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Contents |
Team CRISPR
July 3
List of BioBricks that are/will be on the way:
Cas9:
- Cas9 - Joe and Anna
- pTet -
- const. promoter + Cas9 - Joe
- promoter + Cas9 + Leader + R + spacer + R (on Kan)
- L + R + spacer + R
Target:
- tracerRNA
- repeat + spacer + repeat - Vanins*2 + underlings
- leader - Ray, Fisal, Dan
Decoy:
- PAM + spacer (on Amp)
Team Cinnamaldehyde
4-coumarate-CoA ligase:
- PCR worked
Team Caffeine
June 20
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.
We're making a start - what are the other groups up to?
June 27
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.
July 4
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.
July 5
We acquired aliquots of "good" competent cells from Ray, our grad advisor, and re-tried the transformation of the distribution kit parts.
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.
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.
July 8
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.
July 9
Cultures of 17H/J/L were miniprepped, and with the products, we:
- did a restriction digest with EcoRI and PstI to confirm the presence of an insert
- 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)
- PCR with primers designed to remove the yeast consensus sequence (TCACA)
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. Bands were found 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.
July 11
July 15
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 were named "PCR 2."
PCR 2 would ideally yield the following products:
- Part H: ---XbaI---gene H---SpeI---NcoI---
- Part J: ---NcoI---XbaI---gene J---SpeI---BamHI---
- Part L: ---BamHI---XbaI---gene L---SpeI---
Individually and ligated together, these products could be digested by XbaI and SpeI and inserted into a Biobrick vector cut with X and S.