1/7
The ordered fragments arrived in powder form and were prepared to reach a concentration of 20ng/µL.
The E. coli strain containing our entry vector, PYTK095*, was inoculated in LB media with supplemented chloramphenicol
in preparation for extraction.
2/7
MoClo assembly was done on the ordered fragments in accordance with PROTOCOL. The two constructs we had designed were
assembled in parallel, one tube for the CHI92 gene and one for the SP1 gene. The resulting plasmids were transformed
into competent E. coli using PROTOCOL, which were then plated on agar plates with LB and chloramphenicol.
The entry vector was extracted from the liquid culture using PROTOCOL.
3/7
The plates from the previous day were green-white screened and were shown to only contain white colonies.
This could suggest that the MoClo and transformation were both entirely successful, but it is more likely that
something went wrong. 3 colonies were harvested and placed in liquid culture to confirm.
4/7
(empty for now)
This week, we adjusted some of our methods
- The restriction enzymes BsmBI and BcuI were added to the MoClo reaction. Both the ordered vector and PYTK095*
have the same resistance gene. By letting the enzymes cut into the ordered vector we are reducing the likelihood
that cells without our gene of interest survive
- Instead of extracting plasmids outright, we do colony PCR.
- We scaled up the MoClo reactions to 30µL
7/7
A new MoClo assembly was done according tho PROTOCOL. Both of the genes were done in parallel once again. The assembled
plasmids were transformed into E. coli which were plated and set to incubate over night.
8/7
Colony PCR was done on the
E. coli colonies from yesterday using PROTOCOL. A 1% agarose gel was casted in
preparation for gel electrophoresis. The gel ran clear, indicating an error in our design. After investigation, it appears
like there is a BCuI cut site in the finished construct. Another one might be the primer design. This leaves us with three
options, and we decided to move ahead with all three.
- Order everything again, but this time as a complete construct ready to put into the backbone
- Inoculate colonies, extract the plasmids and hope for the best
- Re-do the MoClo without BcuI, but with BsmBI and BsaI
9/7
MoClo assembly and transformation
A new MoClo assembly was done according tho PROTOCOL. Both of the genes were done in parallel once again. The assembled
plasmids were transformed into E. coli which were set to incubate over night.
After the thermocycler finished the plasmids were transformed into E. coli according to PROTOCOL.Two plates for
each construct was made, one for a lower concentration of cells and one for a higher concentrations. The plates were left
to incubate overnight.
Plasmid extraction
The tubes that were inoculated yesterday were collected and their plasmids extracted in accordance with the GeneGet plasmid
miniprep kit. The resulting concentrations were high enough for five of the tubes, which were diluted to more usable
concentrations and placed in the freezer for storage.
10/7
Result of yesterday's MoClo
The colonies were screened with UV light. From the high concentration plate, one colony containing SP1 was white and 7 containing CHI92
were white. Of the colonies on the low concentration plates, none were visible.
Colony PCR
Colony PCR was done on the white E. coli colonies using PROTOCOL.
11/7
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14/7
The transformation from the previous week was not very successful. The specimen of E. coli containing
SP1 had some bacterial growth, but the specimen that was transformed with CHI92 showed no sign of growth and
no pellet was formed after centrifugation.
15/7
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16/7
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17/7
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16/7
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