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Team:Grenoble-EMSE-LSU/Documentation/Notebook/May
From 2013.igem.org
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<h1>May</h1> | <h1>May</h1> | ||
| - | <h2>Week | + | <h2>Week 4 (20-24)</h2> |
| + | <h3>Monday</h3><br><br> | ||
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| + | We begin settling in and setting ourselves up in the lab. The adventure starts now! Plenty of paperwork to do and people to check in with before beginning. Safety comes first, and with it safety courses to teach us what to watch out for when working in the synthetic biology lab.<br><br> | ||
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| + | <h3>Tuesday</h3><br><br> | ||
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| + | We spend some time discovering the equipment at our disposal, how it works and what it can and can't do. We have access to a microplate reader (absorbance and fluorescence), a spectrophotometer (absorbance), an inverted fluorescence microscope, a PCR machine an, multiple centrifuges and electrophoresis machines, and plenty of pipetmans, plastic disposables and gloves. I'm sure there's a couple of things I've forgotten on the list, but at least we know we have what's necessary to start on great footing.<br><br> | ||
| + | We have transformed fresh cells with a GFP gene controlled by a PBad promoter using <b>INSERT PROTOCOL NAME HERE</b> | ||
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| + | <h3>Wednesday</h3><br><br> | ||
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| + | Experimental work begins in earnest!<br><br> | ||
| + | We have a GFP gene controlled by a PBad promoter, and we want to test this promoter to see if it will be useful in our later experiments with KillerRed, one of the key proteins in our project. We can use the GFP Tristar microplate reader. Production is induced with arabinose, a kind of sugar.<br><br> | ||
| + | Qualitatively, fluorescence starts appearing three hours after induction with arabinose. Originally we didn't think it would take this long. A microscope picture was taken, where we can clearly see GFP fluorescence. Our advisors say that we didn't have enough controls to make sure this was actually GFP expression, though. THis is true as we didn't compare our results to wild-type cells, and it means we will have to be more rigorous from now on. Examples would be wild-type cell autofluorescence, LB autofluorescence, and an estimation of the measurement errors incurred by our use of devices such as the microplate reader.<br><br> | ||
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| + | <h3>Thursday</h3><br><br> | ||
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| + | As we know the state of the cells is often determined by the amount of cells in a culture when a fresh culture is started, we want to obtain OD600 measurements at the same time as fluorescence measurements on the microplate reader. Unfortunately we can't do this with our microplate reader, since the photomultiplier has a certain position for fluorescence readings and another for absorbance readings. (the user has to manually change the position of the detector every time one wants to change the mode). We could also have problems if we used clear plastic microplates to do fluorescence measurements as well as absorbance measurements since there can be fluorescence crosstalk between adjacent wells. | ||
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| + | We thus have to use fluorescence only. A characterization curve has to be made in order to link actual OD600 measurements to their equivalents in fluorescence. The instrument would measure the amount of light scattered by the samples, which is a function of OD, by using the same emission and excitation filters. | ||
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| + | The trend seems to be decreasing fluorescence with increasing OD600, contrary to our expectations, as intuition would have it that scattered light increases with the amount of cells. The trend seems linear but this isn't sure because of measurement noise: we would need additional points to make sure. Another experiment has to be done. | ||
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| + | <h3>Friday</h3> | ||
| + | <h2>Week 5 (27-31)</h2> | ||
<h3>Monday</h3> | <h3>Monday</h3> | ||
<h3>Tuesday</h3> | <h3>Tuesday</h3> | ||
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<h3>Thursday</h3> | <h3>Thursday</h3> | ||
<h3>Friday</h3> | <h3>Friday</h3> | ||
| - | |||
<h2>Week 3</h2> | <h2>Week 3</h2> | ||
<h2>Week 4</h2> | <h2>Week 4</h2> | ||
Revision as of 13:48, 14 September 2013
