IGEM:IMPERIAL/2009/Feedback & Debriefs/Feedback 18 8

M0 - Phase 1
When presenting M0 give an overview (of M0 and how it links the other modules) - difficult to understand if we jump straight in - We’re using AI to go from M1 to M2.

This assay shows the growth curve for cells using glucose, measuring how much glucose left at OD = 0.7 and finding when to add IPTG. Too much IPTG kills the cell - careful.

In protocols, specify all parts and strains being tested.

How do we link data from M0 to M1 - not clear.

M1 must be done before M0. Need to know IPTG concentration from M1 to do M0 assays.

State/describe how protein production changes/is affected by variations in M0. (Have to link how OD and glucose affects protein production - this is not clear from the description).


 * Keep focus in mind. Won’t have time to do everything.   Need to select as we agreed we need to focus on encapsulation.  Dry lab in there to support wet lab.  Every endeavour must support the good of the project.

In the first growth phase (using glucose) - grow up cells so they are ready for protein production. We probably won’t get secondary carbon source exponential phase after glucose.


 * Make clear statements to explain to an outsider what is being done and why. Very important so everyone can follow.

Need a timeline of all the check points that are exact. All measurements we need to check status of the system at various time points.

M0	IPTG	M1	M2	Temp change	M3 Checks OD600	Reaches 0.7 RFP			RFP produced GFP				Produce GFP Glucose	Initial amount- vary time delay		Glucose decreases to switch point to trigger M2

K: We need to use different glucose concentrations with different IPTG concentrations - K: How are you going to find your exact glucose and IPTG concentrations?

Need to be clear on how it all links - want experiment by experiment, what we pick and carry forward.

At IPTG insert, need cells to be in exponential phase and enough of them.

V: Will 0.7 OD change? No, is a fixed value. So we need to define the amount of glucose at the start. Always do induction at same OD = reproducible. At 0.7 should have same amount of glucose that has been taken up in that phase.

K: We still need to test how much glucose to add at beginning to get to OD = 0.7 in a certain time. So we can add glucose once, so you know how much you have at OD = 0.7. We must make sure there is enough glucose left to be able to go through M1 and M2.

We want an assay showing time it takes to get to stationary phase, as a function of initial glucose and IPTG. Track OD all the time and we therefore have 2 variables. With an injector make it automatically add IPTG at OD = 0.7.

If you have a construct of RFP and GFP can do it all in one. Measure initial glucose concentration in phase 1 of the experiment. Measure it at 0D = 0.7 and measure IPTG to show which gives you the best output.

May want to do it without PP?

M0 - Phase 2
In the promoter testing assay, for the control, need to not put in the antibiotic. Use a plasmid which is exactly the same but without the promoter. So find GFP production without promoter.

We can cut down on the number of secondary carbon sources that we will test. Perhaps test 4 or 5 as this should be sufficient.

How do you work out the concentrations between different carbon sources? Need to make it relative.

Whatever carbon source we use, it must not be the limiting factor when measurements are taken. Or else we cannot compare.

Note that steady state will be in between the middle of the exponential growth curve.

M0 - Phase 3
We need brackets for certain molarity as concentration is more reliable.

Do we need 10 different concentrations? Can cut down on these.

V: The plateau is reached in stationary phase of growth, surely its better to be in the middle for exponential growth?

We get most amount of GFP at the end. K: We probably won’t get a 2nd exponential growth curve with the secondary carbon source.

V: In papers that show this media induced system - what do they do? Look at the growth curve.

Can we use RFP in another DNA construct to track what is happening to protein production when CRP kicks in? So, use RFP for protein production and GFP for colanic acid formation. By using 2 colours we can demonstrate how things are switching off and on. We can then add all the graphs together to keep track of the system. Don’t always go to E0240.

MODULE 1
The standard curve gives you activity of the enzyme, not concentration of protein. How do we relate [protein] to activity? How can we measure [protein]?

It is a nice added feature to know the concentration of enzyme, but with promoter characterisation we can get PoPs, we want how much activity we can get with ‘x amount’ of cells.

In the current assay, the enzyme is in excess. This only gives you the linear region of the activity graph.

M: When limiting substrate amount instead, you get substrate and enzyme of the same order, and you can see how they affect - get a non linear form. This gives you more information. Will give you an order of magnitude at least (if you know the stochiometry of the equation).

M: However, is this feasible? Do we have time? Importance compared to encapsulation?...

Which fluorescence filters are required?! Send to James C.

PAH ASSAY

Proposing to do it on crude extract at 450nm, things may interfere but that’s the only assay we can find. K: We are just going to have to do the experiments and see what happens.

M2
Cloning is bottleneck so adapt constructs to have essential gene next to terminators- reduces ligation steps.

Trehalose

Overlapping wavelengths - will cause a problem for the molecule of interest to be measured.

How are we simulating dessication? How are we inducing those stresses?

There must be stuff in the methods part of the literature - do we know what they did with respect to dessication?

M3
Make sure strain doesn’t naturally express cI.

Have to prioritise. Key is triggering with heat. Tuning is secondary to this. Isn’t a PoPS input. Is a PoPS generator with level of temperature. Use strongest constitutive promoter - this will drain resources but there isn’t anything we can do about this.

Need to list priority constructs. Then a subset of useful constructs. May need to cut corners - must get into all desired details.

We need final system with parts tested in context of final system.

Fluorescence is very sensitive to temperature. Re-characterise F2620 for PoPS output using conditions of our system? Instead use LacI and characterise this? - Info can be found in the practical from the SB course. Therefore no longer use F2620.

RELEASE
Ensure protein is released. Do we need to test the degradation of the capsule to ensure release of protein in the gut? Simulate conditions of the stomach and intestines. - Jeremy Nicholson - discuss gut conditions and how to simulate. See IGEM:IMPERIAL/2009/Degradation