Here we show the final structure of our improved bistable. As well as the two operons with their protein fusions and the promoter with the asRNA, we show the different restriction sites we have introduced and the enzymes that target each site. Finally, the total length of our construction is indicated in the figure.
Here we show the final structure of our improved bistable. As well as the two operons with their protein fusions and the promoter with the asRNA, we show the different restriction sites we have introduced and the enzymes that target each site. Finally, the total length of our construction is indicated in the figure.
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==42ºC state induction speed==
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Although the development of the improved flip-flop is focused to enhance bistability and not toogle swith speed, we compared the change speed for the “IPTG state” to the “42ºC state” transition (see the note at the end of the page) with the basic flip-flop. This assay was performed using our previously constructed X90 (SspB, RybB double deletion) E. coli strain expressing the improved flip-flop (module I and II) and the basic flip-flop with an empty plasmid (as control). The cultures were harvesting at 37ºC overnight and the switch was force by changing the temperature to 42ºC. Green and red fluorescence levels were measured by fluorometry. The results of this assay are shown in Figure 1.
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[[Image:Temp change.jpg|800px|center]]
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'''Figure 1. Fluorescence/O.D. of basic and improved flip-flop during a 42ºC continuous induction.''' We had to adjust the fluorescence level of the basic flip-flop, dividing the green fluorescence intensity by 7, as in this system the green fluorescence intensity is 7-fold higher than the red fluorescence intensity. This readjustment was not necessary for the improved flip-flop due to the fact that the basal intensity levels of the fluorescent proteins are similar. It can be seen that the state switch in the improved flip-flop occurs before than in the basic flip-flop. Furthermore, the graphics show a bigger green-red fluorescence difference in the improved flip-flop, compared with the basic flip-flop.
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==Induction time to achieve 42ºC state stability==
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We wanted to determine the 42ºC induction time necessary to make the state change from "IPTG state" to "42ºC state" stable (see the note at the end of the page). For this purpose we induced the state change of a culture previously established in the IPTG state by heat shock at 42ºC during different times: 2h, 3h, 4h and the whole assay (7h aprox.). After heat shocking, bacteria were grown at 30ºC. Red and green fluorescence levels were measured during 7 hours, including the heat shock time. The results are shown in figure 2. This assay was performed using a ''MC4100 E. coli'' strain.
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[[Image:Temperature induction.jpg|800px|center]]
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'''Figure 2. Relative fluorescent level of the improved flip flop with different temperature inductions time lapses.''' These heat shocks start at minute zero and finish at the point indicated by the arrows. In figure 1D, this heat shock is maintained during the whole experiment. It can be seen that 2 and 3 hours heatshock is not enough to maintain the 42ºC state stability, as predicted by the mathematic models. However, we can see that a 4-hour induction shows a higher stability of this state.
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==42ºC state stability==
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In order to compare the 42ºC state stability of the basic and improved flip-flop, this state was induced by harvesting bacteria cultures at 42ºC during 11 hours. This long incubation period was used to effectively achieve the cultures in the 42ºC state at the beginning of the measurements. At the “Induction Stop” point, of the graphics below (Figure 6), the cultures of basic and improved flip-flop were set at 30ºC (without IPTG) and fluorometry data was taking during six and a half hours. In these graphics it is also shown how the bacteria reach the 42ºC state after change their state. This assay was performed using our previously constructed ''X90'' (SspB, RybB double deletion) ''E. coli'' strain expressing the improved flip-flop (module I and II) and the basic flip-flop with an empty plasmid (as control).
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[[Image:Stability flipflop.jpg|800px|center]]
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'''Figure 3. Fluorescence/O.D. of basic and improved flip-flop after 42ºC induction during 11 hours (before “Induction Stop”) and without any induction during 6,5 hours (after “Induction Stop”).''' We had to adjust the fluorescence level of the basic flip-flop, dividing the green fluorescence intensity by 7, as in this system the green fluorescence intensity is 7-fold higher than the red fluorescence intensity. This readjustment was not necessary for the improved flip-flop due to the fact that the basal intensity levels of the fluorescent proteins are similar. In the graphics we can see that the 42ºC state stability is markedly higher in the Improved Flip-Flop compared to the Basic Flip-Flop, and that the difference between the fluorescence levels of both states is also much more pronounced.
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We have showed by fluorometry measurements that our improved flip-flop increases the difference between the fluorescence levels of opposite bistable states, lowering the repressed state levels to almost zero . We have also demonstrated that the stability of the improved flip-flop is much higher than the basic flip-flop under this stimulus.
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'''Note:''' For an easier understanding of the bistable system we denominate "IPTG state" to the transcriptional state caused by IPTG induction; and "42ºC state" to the transcriptional state caused by 42ºC heat shock induction. In the improved flip-flop the 42ºC state causes GFP expression and the IPTG state causes RFP expression. The Basic Flip-Flop works in the opposite way: the 42ºC state causes RFP expression and the IPTG state causes GFP expression. The 42ºC/IPTG denomination will be very useful for the comparison of basic and improved flip-flops.
Proteolysis is a very common system to regulate gene expression in all organisms. In bacteria, proteins can be targeted for proteolysis by adding a special degradation tag in their coding sequences. That’s how the Sspb-Clpx system works. In E.coli wild type, ClpX recognize specific degradation tags – ssrA tags -, unfolds the attached protein and translocates this denatured polypeptide into ClpP for its degradation, which is very fast. The action of this ClpXP protease is enhanced by the SspB adaptator protein.
The idea is to add the DAS+4 tag to the proteins of one of the states of the flip-flop and express the Sspb under the promoter of the opposed state. Specifically, it's added the DAS+4 tag to the GFP and the LacI proteins, expressed under the cI promoter, and under the Lac promoter Sspb is expressed.
Furthermore, asRNAs are short non-coding RNAs that can inhibit mRNA translation by binding to the 5’ untranslated region (UTR) of that mRNA. That is the case of the Salmonella OmpN gene, whose translation is inhibited by the conserved RybB asRNA, which forms a short duplex with the ompN CDS.
This device has a fusion of OmpN start sequence with those proteins whose translation we want to regulate. The RybB asRNA binds to nucleotides from 5th to 20th relative to AUG. Consequently, a shorter fusion in which it's fused the first 7 codons of the OmpN gene CDS upstream of the RFP and Sspb CDS. These fusions do include the OmpN RBS.
Here we show the final structure of our improved bistable. As well as the two operons with their protein fusions and the promoter with the asRNA, we show the different restriction sites we have introduced and the enzymes that target each site. Finally, the total length of our construction is indicated in the figure.
IMPORTANT: This biobrick complements the Improved Flip Flop (Module I), the module II is necessary for the correct running. You can find it in Part:BBa_K510019
For further information please vistit our [http://2011.igem.org/Team:UPO-Sevilla wiki] or contact with us at igem@upo.es
