Team:Brown/Project/Light pattern/Workflow

From 2010.igem.org

Light-Pattern Controlled Circuit

Workflow

Rack.jpg

Circuit Beginnings

Once we had designed our circuit, we went about obtaining all of the necessary parts for its construction. Because the registry did not contain the majority of parts crucial to our circuit, we collaborated with teams EPF-Lausanne, PKU, and Davidson-Missouri Western to obtain the relevant parts. From EPF-Lausanne, we received BBa_K191003 and BBa_K191005. We received from PKU Beijing AraC+pBad+T7ptag+Terminator, NahR+pSal+supD+Terminator, the Bistable switch, and other miscellaneous parts. From Davidson-Missouri Western, we received BBa_K091141 and BBa_K091147 to aid in the characterization of the pLac/Mnt hybrid promoter in the distribution. Thanks all for the assistance!

Experimental Procedure

Once we began receiving genetic material, we started our attempts at constructing each required module of our circuit. Clearly, our circuit has many components that, to build a complete example, would require an incredible amount of time. We decided that we could demonstrate our ideas in two ways:

  • Through extensive mathematical modeling, we can show the likely functionality of our circuit and the likely functionality of our circuit through extensive mathematical modeling, which is considerably less prone to error than laboratory work.
  • We can establish proof of concept by pairing our Conversion Module with the Memory Module. By testing this segment of the circuit together, we can show a progression across three states (as we move from light OFF->ON->OFF) by changing one input. Because the state of the circuit when the light is turned back off is different from that when the light was originally off, the crux of the circuit concept has been demonstrated and the ability to achieve more states is evident.


Mathematical Modeling Workflow

The following are the steps we took to arrive at our in silico model of the Light Pattern Controlled Circuit:

  1. We first had to assess what mathematical modeling entails, as none of our team members had previously been exposed to this kind of approach. We determined, largely by reviewing past team's modeling sections, that we had to generate differential equations for each product of the circuit.
  2. As we did not have any particular experience in this branch of applied math, we consulted with Dr. Sindi, an applied math professor at our University. This meeting helped get us on track for the creation of our many differential equations, as Dr. Sindi was an enormous help.
  3. Each differential equation we created is accompanied by a set of parameters. The differential equations themselves, without defined parameters, merely show the interrelation of the various parts of our genetic network. Defining parameters for each equation allows the equation to be linked specifically to the function of an individual promoter or factor. Thus, we conducted literature research and reviews past iGEM teams' work to compile the parameters for our equations.
  4. Given the wide range of sources for the parameters we collected, we made various assumptions to normalize the parameters to more realistic values. Given more time, in vitro characterization of each component of the circuit might have suggested the most appropriate values to use. Unfortunately, we were not able to get to this point, and so relied on literature and past work.
  5. We then created code in Matlab in order to solve our system of ordinary differential equations. This Matlab file collected all of the appropriate parameters, set initial conditions and events, and used built in numerical solvers to generate solutions to each differential equation. This gives equations for the various products of the circuit over time, and calling graphing methods enabled us to visualize, better understand, and display the functionality in silico of our circuit.


Proof of Concept Workflow

Before beginning our work towards our proof of concept for the light pattern controlled circuit, we first created our own frozen stock of competent cells, stocks of antibiotics, LB, etc.

Upon gathering material for the project, we first transformed BBa_K191003 and BBa_K191005, the LovTAP expression construct and the LovTAP-repressed reporter construct. After obtaining DNA from these samples, we double transformed these two parts into BL21 E. coli on Kan+Amp+ plates. See procedure section for more details. We noted that even without the LovTAP expression part (K191003), part BBa_K191005, the LovTAP reporter construct, expressed RFP. We attributed this to the presence of tryptophan in LB (which activates expression of RFP in K191005, and so we created and tested a Minimal Media solution that did not have tryptophan in it.

At this point, material from PKU arrived, and we transformed and made glycerol stock/miniprep of nearly all the parts they sent. We were unable, however, to transform a crucial component of our circuit - the memory module (with Kan resistance). This, at first, was because of a tricky kanamycin resistance in our competent cell stock! After much time spent resolving this contamination, we proceeded to re-transform the bistable part (into DH5a), but still were unable. Note: At the point of the wiki freeze, we have still been unable to transform this part, but will attempt to do so again before the Jamboree.

From the LovTAP-repressed reporter construct, which we termed WT LovTAP R2, we obtained solely the double-repressor component (TrpR-RBS-TetR-p(TetR)) by PCR, to which we added the rfc 21 standard restriction sites (as the TrpR promoter has illegal speI sites, making it incompatible with rfc 10). This new part, termed Lov2 was then ligated into pGEM, transformed and miniprepped.

We transformed and made stock of CI and AraC from the registry, and Mnt-TerTer from team Davidson-Missouri Western. By PCR, we added to the left hand of each CI, AraC, and Mnt-TerTer the bgl standard prefix + RBS, and to the right hand side bgl suffix. Once these products were ligated into pGEM, transformed and miniprepped, we were poised to perform a rolling assembly to create a composite part Lov2+RBS+CI+RBS+AraC+RBS+Mnt+TerTer.

As we were running short of time and the summer had concluded, we decided to add only CI to the LovTAP repressed double repressor. We double digested CI from the registry and a double terminator and ligated the two. PCR was performed to obtain bgl-prefix+RBS+CI+TerTer+bgl-suffix.

At the time of writing this wiki, this is as much as we had accomplished. However, we hope to complete our proof of concept construct by the time of jamboree, we hope to finish up this workflow: We will ligate the PCR product bgl-prefix+RBS+CI+TerTer+bgl-suffix with pGEM, transform, double digest both the transformed plasmid and Lov2, ligate the two together, and transform again. Finally, to complete the Conversion Module (see Logic Design), we will ligate the LovTAP expression construct upstream of our newly obtained product. Using One Shot Invitrogen cells, we hope to be able to successfully transform the bistable part, and will double transform with the Conversion Module to establish our proof of concept!