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Team:Cambridge/Diary/Week 11
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A gel was run to check the control fragments. (5ul of each 10ul elution). No significant differences observed between fragments, all were visible and correctly sized. | A gel was run to check the control fragments. (5ul of each 10ul elution). No significant differences observed between fragments, all were visible and correctly sized. | ||
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| + | We quite urgently need to get some biobricks submitted, so Oli began attempting to make the fragments we'll need to Gibson together to make biobricks of the riboswitches we've been trying to test. Hopefully we can make some successfully transformed colonies quite quickly... | ||
=== Thursday === | === Thursday === | ||
Revision as of 23:12, 8 September 2012
| Week: | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
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Contents |
Monday
We continue trying to debug the Gibson reaction, with incremental success. The Gibson control plates from yesterday showed that there is no significant difference between the competency of our cells, or our master mixes. However PJ obtained a 10x greater efficiency.
We decided to rule out a problem with the plates. Prewarming does not appear to be a factor in the success of the plating step.
Paul's been working on developing his germination protocols. Germination medium has been determined and the protocols for imaging the sporulation and germination of B.subtilis have been ironed out.
Tuesday
Gibson control plates from yesterday showed no difference between PJs plates and ours.
Paul Grant, another member of the Haseloff lab, suggested that he observe Tom assemble and transform, but failed to detect any gross incompetency. We thought that the problem might be the PCR machine - it takes a little while to load ours, whereas the Haseloff lab's is a hotblock that can be preheated, minimizing the time the T5 exo has to digest our fragments.
New control fragments were obtained, some with different "minelute" gel extraction kits. One assembly was set up with mineluted control, the other with normally extracted control. Additionally, a reaction was set up to assemble our 4-part ratiometric construct. We used the Haseloff hotblock PCR machine to assemble. Transformation was done with our competent cells, and they were plated.
Also, Paul began designing his first Gibson primers! He's aiming to make a biobrick out of a plasmid containing the spoVAA gene under control of the PssB "fast" promoter. Peter Setlow's team from the University of Connecticut have kindly offered to send us the B. Subtilis strain containing the modified chromosone and two E.coli cultures containing plasmids used to facilitate the single crossover recombination event that placed spoVAA under the control of PssB. Designing Gibson primers may prove a little tricky though as we don't have access to the full sequence.
Lastly, the results from the platereader came through. B.Subtilis needs more magnesium to grow than e.coli, as expected, but there is still this weird peak at around 50mM MgCl2 in the growth rate. Oli will
Wednesday
Only one of the control plates from yesterday (the minelute control) has colonies on it, and still a low number. The other has none on it at all. The ratiometric construct plate has a couple of colonies on it, but their lack of fluorescence implies that they are not what we want.
A gel was run to check the control fragments. (5ul of each 10ul elution). No significant differences observed between fragments, all were visible and correctly sized.
We quite urgently need to get some biobricks submitted, so Oli began attempting to make the fragments we'll need to Gibson together to make biobricks of the riboswitches we've been trying to test. Hopefully we can make some successfully transformed colonies quite quickly...
Thursday
Paul and Tom had a large handover session before Tom left on holiday. The fate of the project may now lie in the hands of an engineer!
Friday
The strains for sporulation arrived and have been plated. the long range pcr of the flu and lux 9kb fragments was run again but no gel has been run yet.
