Difference between revisions of "Team:Technion Israel/Color"
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Revision as of 14:09, 19 October 2016
Introduction
Chromogenic proteins usually serve as a useful reporter in determining gene expression levels without the need of a fluorescent microscope. However, the FlashLab technology implements these chromogenic proteins for a different purpose. Due to the chips structure, when the bacteria moves towards or away from substance, a cluster is formed and the presence of chromogenic proteins allows the user to spot it in the naked eye without the need for a complex device (for more information about our chip click here).
Implementation
Three chromogenic proteins were tested for the S.Tar system, all which were provided and extracted from the iGEM 2016 kit.
These proteins were:
K1357008 expressing purple color.
K1357009 expressing blue color.
K1357010 expressing red color with fluorescent capabilities
To test these proteins, a strong promoter (J23100) was added, with the help of restriction enzymes to each of them. In parallel, two of these proteins were added downstream of the Tar on the plasmid to test the expression level of both under the same promoter (figure 1).
a.
b.
Fig. 1: a. High expression biological circuit ;
B0034 RBS, chromoprotein and
terminator
b.High expression biological circuit ;
B0034 RBS,
K777001 Tar chemoreceptor with strong promotor, chromoprotein and
terminator.
Results
At the beginning we looked for chromogenic proteins in the iGEM Kit 2016. We found a list
of chromogenic protein and start to work with two chromogenic protein, K1357008 ( purple)
& K1357009 (blue) , and one protein that have both, chromogenic and fluorescent abilities,
K1357010. We decided to test three different color hoping that one of them will suit our system.
The first step was adding a strong promoter, J23100, to each color plasmid. For that we used restriction enzymes (see: notebook). In parallel we designed two plasmids that contain Tar and one of the chromogenic protein under the same promoter.
After transformation we could see colored bacteria on agar plats but when we tried to make an overnight culture for the next step we could not see the color in the Tar+color strians. We centrifuge the culture and discovered that we have colored bacteria but they not dense enough for us to see in the naked eye (fig 2).
Fig. 2: E.coli Top 10 with (from right) K1357010 (RFP), K1357008 (purple) and K1357009 (blue) after centrifuge.
We didn't give up on the idea. Instead we tried to play we the condition in which our overnight culture is growing. Our basic chemotaxis experiment, swarming assay, require a minimal growth medium, TB, and an optimal temperature of 30 degree. Optimal growth of E.coli is 37 degree (makor safrot kan on the Tem.). So we played in that range hoping to discover an optimal condition for both, chemotaxis and color formation.
It was easy to get and see the color in 37C (fig 3) with LB medium, just by letting the culture enough time. For the same temperature and time in TB we couldn’t see the color. Initially we thought that the TB medium is not rich enough for colored protein formation. We check the O.D and saw a significant difference between the LB and the TB medium. After almost 24 hours of incubation we could finally see the color in the TB tubs.
Fig. 3: overnight starters 37C, in LB. In the tubs: Top 10 RFP, UU1250 with the purple plasmid and UU1250 with blue plasmid.
Next we played with the temperature. As mention above the optimal temperature for chemotaxis is 30 degree in this temperature bacteria growth is slow and color formation even slower. We tried to grow the bacteria in 37 degree and then move it to 30 degree for few generation. Due we could see chemotaxis after that process the bacteria didn't always make enough protein for as to see in logical time period. That will affect our final product so we have to find another solution (table 1).
Table. 1: color formation test results. N.D-no data, N.C- no color.
We didn’t want to play with the order of the parts as we were afraid we will harm the chemotaxise ability of the bacteria so we decided to separate and work with two plasmids, one with the color protein and second with our receptor. In this approach we got the desirable color in the overnight culture whiteout farther treatment.
Outlook
We succeed to get colored bacteria that are comfortable to work with in the optimal condition to our assay. At that point both, the color and the receptors, cloned on high copied plasmid. Every time the culture grow, the bacteria copied one of the plasmid as a high copy and the second one as low copy. By moving the receptor to low copy plasmid, we could achieve constant color bacteria and enough receptors for bacterial movement.
References:
1. MAEDA, Kayo, et al. Effect of temperature on motility and chemotaxis of Escherichia coli. Journal of bacteriology, 1976, 127.3: 1039-1046.
