Sensitivity analysis: Models... have feelings too!

Vicki and Chinee has put quite an amout of time into tracking down those sensitive ones in our system. Vicki is currently working with the Sensitivity analysis tool in Matlab commandline in order to find the sensitive parameters that influence the GFP output the most. The following graph is the result her sensitivity analysis model has generated so far: (click to enlarge)



The closer the graph gets to 0, the lower the influence this parameter has on the GFP output, and vice versa. According to this graph, the kForward (reaction rates)values 1, 3, and 4 seems the most influential parameters relative to 2 and 5; thus these parameters will be looked at further.

From these results, Vicki performed parameter optimization on them. In parameter optimization function, she was able to play with the individual parameter values in order to come up with a best fit line model to the predicted levels of GFP output. The following is the graphical result of the above function.



Here, the circles represent the made up predicted data, and the colored lines represent each optimized parameters. The optimized rate constants for kForward1, 3, and 4 fit nicely to the pattern of the predicted data; however, the optimized rate constants for kForward 2 and 5 do not, meaning that these parameters do not play a big role in determining the GFP output values.

From these two functions, Vicki would be able to, once we get some lab results, optimize our significant rate constants to fit the behavior of our AI-2 system.

Along with Vicki, Chinee was also working on the parameter sensitivity; however, with a different tool. In Simbiology, Chinee was able to outline the key difference between the initial amount and the reaction rates of the parameter. The following is the graph when all the parameters have a reaction rate value of 1:



The following is the graph when all the parameters have a reaction rate value of 10:



The only difference between the above two graphs are that the lines in the second graph are much more steep than the lines in the first graph. This means that the reaction rate only influences how immediate the reactions happen.

And finally, the graph when all the parameters have an initial amount of 10:



The above graph is very different from the ones above. From this, one can see how initial amounts of parameter play a much bigger role than the rate constants of each parameters.

I, Kevin, and Carol attempted to fix some biological misunderstandings within the reactions, and produce a graph that displays the pattern of each parameters. The following is the graph:



The patterns observed are reasonable and match what was expected. The lab data is needed to get an accurate model of our system; however, from it we can still test the sensitivity of each parameters by changing them one by one and simulating the results.

We hope to get some lab data by this week.

Building Circuits: A Few Successful Moves to the End, but too many Failures...

Hi! You heard from me last week in my third-person commentary and now I’m back in the first-person flesh. My name is Vicki and I recently graduated from the Engineering Science program at the University of Toronto, with a major in biomedical engineering. I grew up in Calgary and am very excited to help my hometown kick some synthetic biology butt. When I am not indulging in the satisfaction that only a properly-sequenced synthetic BioBricked circuit can provide, I am usually either swimming, skating, biking, learning German or a combination of any of the above (ever try riding a bicycle whilst on skates? It’s as perilous as it sounds!).

Over the last few weeks, I have been deeply entrenched in converting a mutant protein sequence in a TOPO plasmid into a fully functional and biobricked sequence in a pSB1AK3 plasmid, complete with the appropriate and properly-integrated promoter, RBS and terminator sequences. Indeed, it has been a most gruelling month of gradient PCRs, colony PCRs, plasmid isolations, restriction digests, ligations and – craziest of all – understanding what I’m doing and explaining it to the lab group in coherent sentences! Because even though the principles of synthetic biology and biobricking are supposed to make genetic engineering so easy that even an engineer like me can work with it, it’s really quite challenging when you cannot see what is really happening in terms of molecular interactions on the nano-scale and smaller. Although many parts of the project have flummoxed me, I am developing a better appreciation of what I am doing as I gain more experience in the lab.

So, here is my work to date.

Battle 1: Vicki vs gradient PCR of LuxOD47A in TOPO. The purpose of this step was to use biobrick gene-specific primers to make copies of a biobricked version of LuxOD47A.

Winner: Vicki

Battle 2: Vicki vs ligation of LuxOD47A into the pSB1AC3 plasmid. This is so that the gene sequence can be integrated into competent TOP 10 bacteria. Prefix-end cuts were made with EcoRI and XbaI (in separate samples).

Winner (VK vs EcoRI cuts): EcoRI cuts L. The restriction digests of these look like someone painted white-out in the 12 kb range just to spite me.

Winner (VK vs XbaI cuts): Vicki. The EcoRI enzyme sample that wasn’t up to par is spending its days in solitary confinement at room temperature. Indeed, things are so much cleaner when enzymes decide to cooperate.

Battle 3: Vicki vs the sequencing machine, part I. After a successful restriction digest of the XbaI-cut samples, I sent them down for confirmation that my PCR and restriction digest results weren’t just lying to me.

Winner: Vicki

Battle 4: Vicki vs integration of promoter and terminator sequences, attempt I. I tried both to see which would work best.

Winner (VK vs J13002 promoter): J13002 promoter L. That one didn’t integrate in any of the colonies that I PCR’d and RD’d

Winner (VK vs B0015 terminator): Vicki. We’ll move forward with a newly-made LuxOD47A-B0015 construct, while the attempted J13002-LuxOD47A samples can go roast in the autoclave.

Battle 5: Vicki vs the sequencing machine, part II. Yea, I know I skipped a lot of steps here. It wouldn’t tell you anything interesting beyond what you’ve already read. After good restriction digests and colony PCR results that would have been fine if not for a superfluous negative control band that didn’t show up anywhere else, we sequenced anyway. We had a scare when a simple [Crtl+F _ (B0015 sequence)] didn’t yield any results on the text file, but were reassured when we blasted the sequences against each other as that algorithm accounts for the reverse sequence given by the reverse primers that I used.

Winner: Vicki, with credit to BLAST

Battle 6: Vicki vs the integration of the nefarious promoter sequence. Lest the J13002 samples go the same way as our uncooperative EcoRI enzyme, we decided to attempt this integration thing one last time. Two colonies behaved appropriately, and the J-part lived to see another day.

Winner: Vicki

Battle 7: Vicki vs the sequencing machine, part III. Although I had to wait four days for my results, they were what we expected.

Winner: Vicki

This takes us to now. I need to make more of the biobricked sequence in question by letting it grow in bacteria, which I’ll do later today so that it can grow overnight. Results to come!

Some musings from mutant-circuit-world

The last few days have been flavoured by a plethora of successes, not-quite-successes, and other incidences of interest and amusement.

THE GOOD: Jamie has usurped the throne of AI-2 Circuitland. With a complete and properly-sequenced LuxOU component of the circuit, complete with the necessary promoter and terminator sequences, Jamie gets to sit back, chillax and admire his work as he helps the rest of the lab group complete their work. Congrats, Jamie, and B0015-R0040-LuxOU-B0015 FTW!

THE BAD: Negative controls FTL! Emily and Vicki have both been plagued by unsightly bands in the negative control lane, despite using new and purportedly uncontaminated equipment in their PCRs and restriction digests every time. Of particular bamboozlement, Vicki’s negative control in her latest colony PCR had a band at 1kb that did not appear anywhere else in the gel. Nevertheless, the two will continue to move forward with their experiments. The sequencing results of Vicki’s LuxOD47A BBk circuit showed that she does indeed have the proper product present. And Emily is set to conquer the colony PCR, so hopefully we’ll see the awesome results of that at the end of the day.

THE UGLY: Vicki spat into a K-laced plate and placed it in the incubator to see what would happen. She was most disappointed to see that the plate was cleaner when she returned than when she first left it there. She’ll try again with a plate free of antibiotics next week. Results to come!