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Shipment & videoconference
Hello Claudia and Mexico Team!
Thank you for the email and sorry for the late response. We had small celebrations yesterday, as this was the last day when we are all together- people going on holidays etc.
First, About the conference- is Thursday ok for you? Around 2pm is good for you?
Now, there might be a problem with the parts:
1) we don't have read out system 2, just 1- we can send that
2) we can send you cph8 pcr
3) lux CDE- still not working, at this stage it is the same as yours- we haven't made a progress
4) we don't have pcb prodcing strain working either
5) as for mutated S284T strain- see the message from Will belowe, it turned out that it wasn't mutated after all...
About LovTap- we are doing crude assay tomorrow, with blue 470 nm leds (2200 mcd/B) and Vs=4.5 V. We plan to induce our double mutants ( with read out system and LovTap) in a shaker i na 37C room, covered with a box with blue light on the sides (attached LEDs) and 'dark' state under aluminium foil in the same conditions. We'll have control: without IPTG (less induction of LoVTap), medium with antibiotics and not transformed cells. I'm reading now about the time of lexposure to the lgiht and growing condition, we'll probably keep it for much longer as we just want to know now if there is any response to blue light, we'll reapeat the experiment with different light wavelengths. Do you have any ideas about rough conditions from it? Our light intesity was estimated from original paper (Strickland)- I can ask Sarah (our engeneer) to send you more details if you like. Thank you for the updates about the LovTap - that's really useful.
this is from Will:
Our luciferases glow but the sequencing showed that two of them weren't mutants. We think this is cause we selected the brightest ones, which are more likely to be green.
To check for luminescence we spun 1ml down (8000rpm, 4 min) then resuspended in citrate buffer (pH 4.8). The sequencing showed we have a 356K mutant but the 356R and S284T weren't mutated. We are trying some other mutants.
One problem is that the red mutants are less bright than the wild type. We are going to try growing a larger batch then adding the luciferase, which will hopefully help us identify red ones by eye. We haven't deleted the 6 extra bases yet, so our cells should make less luciferase than yours, you might see more light.
The device we use to measure luminescence is a Modulus multi mode reader from Turner biosystems (http://www.luminometer.com/instruments/modulus-fluorometer-luminometer-photometer.php).
Haven't gotten hold of the one that measures the spectral output yet though.
All the best- Marta and Illuminati team.