Self-service!

LovTAP

Mini-preps done and correct plasmids confirmed. Working on maxi-prep for transfection...

Make selection possible

Design primers to integrate puromycin into the LovTAP plasmid - Sander

Not done yet. Still need to decide in which direction the integration should be done (integrate puromycin into the LovTAP plasmid or integrate LovTAP into pMP-PB).

• Puromycin → LovTAP plasmid
  • No control over directionality (only a single available RS)
  • Less worries about mutations (mutated versions of puromycin will just die during the selection process)
• LovTAP → pMP-PB
  • Integration easy and controlled (lots of easy to use RS)
  • (Too?) Efficient expression thanks to strong promoter & intron.
  • Interference of non-used gene sequences?

Design primers to integrate puromycin into the RO plasmid - Sander

See LovTAP plasmid design. The RS availibility problem is bigger for this one...

Investigate use of different antibiotics in the plasmids

In general using the same antibiotics will result in a 50/50 split of intake of the plasmid. Alternatives are:

• Puromycin
• Zeocin
• Blasticidin
• Hygromycin B

Where Zeocin has a significantly different method of operation (Zeocin cuts up DNA while the other antibiotics interfere with the protein synthesis process).

Produce the modified plasmids

Using the previously designed primers:

• PCR → part we want to add surrounded by RS
• Digestion of the plasmid & PCR product
• Ligation
• Selection

Transfect

Transient transfection

Can be done as soon as the plasmid has been produced in high quantities (maxi-prep).

Stable transfection

Need to modify the plasmids for selection & perform max-prep.

Experiment

Basic tests

Test whether the LovTAP works (just test whether something is being expressed). Then check if there is a difference in expression level between the dark state and the light state. This difference should be a factor of about 5 (not sure, need to check again?).

Other tests

Try to optimize expression (amount of light, plasmid proportions, etc...).

Increase efficiency

The version of LovTAP we're using isn't the ultimate one, if we have enough time (and most of all, got the thing working in mammalian cells) we could try some form of site-directed mutagenesis to increase the efficiency.

Fussenegger

Nothing done yet! Come on, lots of work to do!

Get the main plasmid DNA sequence

Waiting for Fussenegger...

Create the RO plasmid

We have the backbone, just need to clone in the RO system -> design primers

Transfect into CHO

Experiment with HEK

Bioreactor

We talked to Dominique, who proposed a rather easy solution. Take an incubator and cover the window, add LEDs to a tubespin holder for light-state experiments and use a dark box (fixed to the shaker) for the dark-state experiments.

Requisition and modifiy an incubator

Get exclusive access to an incubator and modify it to not let any light pass in (stick something on the front window...).

Create LED strips

We need ~8 candella per tube (~8 lumen) of blue light. The LovTAP experiment used light at 468nm. The LEDs need to be ordered and the light chain+circuitry+power supply needs to be created.

Buy 20 of the following (2 packets): [http://www.ledstar.ch/10-Stueck-LowCost-LED-5mm-blau-13000mcd-ultrahell-unsortiert Blue LEDs]

Calculate the right resistances (and buy them).

Create the circuit (use a few AA batteries?).

Create a "dark box"

Modify the plexiglass box in the corner of the cell lab so it blocks all light (add paper/aluminium foil or paint). This is not needed for the first experiments where a bit of tape should suffice.

Biobricking

Nothing done yet! Use the primer design helper (in the dropbox primers folder) to quickly design the primers.

Make a list of all biobrickable stuff!

• TNFR:Fc
• LovTAP mammalian
• Melanopsin

Wiki - Sander

Made a basic templating system that can be extended later (shouldn't change the code of the pages themselves).

Start the designing!

Decided on the (slightly kitschy) lab-bench design. Comparable to the style of the 2010 EPFL iGEM team, but with more doodling and randomly placed objects in the background!

Fill it!

The templates should be complete enough to be able to work with the wiki now without needing to reenter all data later. In progress!

Others

✓ Competent cell preparation

Requisite for E. coli transformation. Done the 25th of May. The cells are in the -80ºC freezer.

Safety

All members followed safety course given by university. Need to meet with the [http://people.epfl.ch/cgi-bin/people?id=163673&lang=en Stéphane Karlen] to get a more detailed (more relevant) safety opinion. Email sent asking for an appointment (12-7 Diego).