Difference between revisions of "Part:BBa K1172915"

Latest revision as of 14:06, 29 October 2013

Biosafety-System TetOR alive (TetR)

The Biosafety-System TetOR alive BBa_K1172915 is an improvement of the BioBrick BBa_K914014 by replacing the first promoter into the rhamnose promoter P_Rha, integration of the alanine racemase BBa_K1172901 and utilization of the repressor TetR to regulate the transcription of the Barnase behind the tetO promoter. Because this system is known for a tight repression and a fast activation, it is expected that bacteria containing this Biosafety-System are dead or alive...

Sequence and Features

Biosafety system TetOR alive

Combining the genes described above with the Biosafety-Strain K-12 ∆alr ∆dadX results in a powerful device, allowing us to control the bacterial cell division. The control of the bacterial growth is possible either active or passive. Active by inducing the tetO operator with tetracycline and passive by the induction of L-rhamnose. The passive control makes it possible to control the bacterial cell division in a defined closed environment, like the MFC, by continuously adding L-rhamnose to the medium. As shown in Figure 1 below, this leads to an expression of the essential alanine racemase (alr) and the TetR repressor, so that the expression of the RNase Ba is repressed.

Figure 1:Biosafety-System TetOR alive in the presence of L-rhamnose. The essential alanine racemase (Alr) and the repressor TetR are expressed, resulting in a repression of the expression of the RNAse Ba. Consequently the bacteria show normal growth behavior.

In the event that bacteria exit the defined environment of the MFC or L-rhamnose is not added to the medium any more, both the expression of the alanine racemase (Alr) and the TetR repressor decrease, so that the expression of the toxic RNase Ba (Barnase) begins. The cleavage of the intracellular RNA by the Barnase and the lack of synthesized D-alanine, caused by the repressed alanine racemase inhibit the cell division. Through this it can be secured that the bacteria can only grow in the defined area or the device of choice respectively.

Figure 2:Active Biosafety-System TetOR alive outside of a defined environment lacking L-rhamnose. Both the expression of the alanine racemase (Alr) and TetR repressor are reduced and ideally completely shut down. In contrast, the expression of the RNase Ba (Barnase) is turned on, leading to cell death by RNA cleavage.

Results

Characterization of the tetracycline tetO operator

First, the tetO operator was characterized to get a first impression of its basal transcription rate. Therefore, the bacterial growth was investigated using the unrepressed tetO operator on different carbon source using M9 minimal medium with either glucose or glycerol. The transcription rate was identified by fluorescence measurement of GFP BBa_E0040 behind the tetO operator BBa_R0040 using the BioBrick BBa_K1172914.
As shown in Figure 9 below, the bacteria grew slightly better on glucose then on glycerol. This is due to glucose being the better energy source of these two, because glycerol enters glycolysis at a later step and therefore delivers less energy. Moreover an additional ATP consumption is needed to drive glycerol uptake. For the investigation of the basal transcription the fluorescence measurements, shown in Figure 10, is more interesting.
In contrast to the other promoters characterized, like [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#Characterization_of_the_arabinose_promoter_PBAD P_BAD] or [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L#Characterization_of_the_lactose_promoter_Plac P_lac], the fluorescence does not differ between the carbon sources used. This was expected in this case, because this operator is not enhanced by intracellular cAMP like the arabinoe or lactose promoter.

Figure 9: Characterization of the bacterial growth of the Biosafety-Strain K-12 ∆alr ∆dadX containing the plasmid BBa_K1172914 with GFP (BBa_E0040) under the control of the tetO operator. The M9 medium was supplemented with 5 mM D-alanine. It could be demonstrated, that the bacteria grow faster on M9 minimal medium containing glucose than on M9 minimal medium with glycerol.

Figure 10: Characterization of the fluorescence of the Biosafety-Strain K-12 ∆alr ∆dadX containing the plasmid BBa_K1172914 with GFP (BBa_E0040) under control of the tetO operator. The Biosafety-Strain was cultivated on M9 minimal medium supplemented with 5 mM D-alanine..

As for the other characterizations, the specific production rate was calculated to demonstrate in this case, that the carbon source does not influence the basal transcription, as shown in Figure 11. The specific production rates were calculated via equation (1) :

With the calculation of the specific production rate of GFP it can be demonstrated that the GFP synthesis rates does not differ between the cultivation on glucose and glycerol. The specific production rate show fluctuation on both cultivation, resulting in an up and down, so that there is can no obviously obvious be seen. This demonstrates that tetO operator is not enhanced by cAMP and confirms the results of the lactose and arabinose promoter, as they can be enhanced by cAMP and their basal transcription differs on glucose and glycerol.
Moreover the specific production rate was calculated between every single measurement point, so the curve in Figure 11 is not smoothed and the fluctuations have to be ignored, as they do not stand for real fluctuations in the expression of GFP. They are caused by measurement variations in the growth curve and the fluorescence curve. And as neither of this curves are ideal, the fluctuations are the result. Nevertheless this graph shows the difference between the two carbon sources.

Figure 11: Specific production rate of GFP expressed via the tetO operator in dependence of different carbon sources.

Characterization of the Biosafety-System TetOR alive

The Biosafety-System TetOR alive was characterized on M9 minimal medium using glycerol as carbon source. As for the characterization of the pure tetO operator above, the bacterial growth and the fluorescence of GFP BBa_E0040 was measured. Therefore, the wild type and the Biosafety-Strain E. coli K-12 ∆alr ∆dadX both containing the Biosafety-Plasmid BBa_K1172915 were cultivated once with the induction of 1% L-rhamnose and once only on glycerol.
It becomes obvious (Figure 12) that the bacteria, induced with 1 % L-rhamnose (red and black curve), grow obviously slower, than on pure glycerol (orange and blue curve). This might be attributed to the high metabolic burden encountered by the induced bacteria. The expression of the repressor TetR and the alanine racemase (Alr) simultaneously causes a high stress on the cells, so that they grow slower than the uninduced cells which express only GFP.
Comparing the bacterial growth with the fluorescence in Figure 15, it can be seen that the fluorescence of the Biosafety-Strain is much higher than the fluorescence of the wild type. This was also observed for the Biosafety-Strain [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L#Characterization_of_the_Biosafety-System_Lac_of_Growth Lac of Growth] and therefore the wild tpye is taken for further analysis of the Biosafety-Plasmid. While the Biosafety-Strain shows an decreasing fluorescence, the wild type shows an expected increase during cultivation. As observed for the other Biosafety-Systems, the fluorescence seems to follow the same trend than the bacterial growth. The uninduced cells show approximately an exponential rise of fluorescence, while in comparison the fluorescence of the induced bacteria increases only slowly.

Figure 12: Characterization of the bacterial growth of the Biosafety-System TetOR alive on M9 minimal medium with glycerol. The Figure compares the wild type K-12 and the Biosafety-Strain K-12 ∆alr ∆dadX containing the Biosafety-Plasmid BBa_K1172915 and the induction by 1% L-rhamnose to pure glycerol.

Figure 13: Characterization of the fluorescence of the Biosafety-System TetOR alive. The Figure compares the wild type K-12 and the Biosafety-Strain K-12 ∆alr ∆dadX containing the Biosafety-Plasmid BBa_K1172915 and the induction by 1% L-rhamnose to pure glycerol.

From the data presented above, it cannot be determined if the expression of the repressor TetR does affect the transcription of GFP or not. Considering the wild type containing the Biosafety-System, the slower growth is a first indication that the repressor TetR and the alanine racemase (Alr) are highly expressed, but the growth of the bacteria shows nearly the same kinetics as the fluorescence. So it could be possible that the repressor does not affect the expression level of GFP under the control of the tetO operator. This becomes more clear by the calculation of the specific production rate of GFP by equation (1) . As shown in Figure 14 below the specific production rate does not really differ between the uninduced Biosafety-System and the Biosafety-System induced by 1% L-rhamnose.
At the beginning the production of GFP in the presence of L-rhamnose (red curve) is lower than in its absence (orange curve), so that the expression of GFP seems to be repressed in the presence of L-rhamnose, but later one this changes and the specific production rate is constantly higher in the induced Biosafety-Strain. The fluctuation can be ignored, because the specific production rate of GFP was calculated between every single measurement point. The curve in Figure 14 is not smoothed , as they do not stand for real fluctuations in the expression of GFP. They are caused by measurement variations in the growth curve and the fluorescence curve. But this unexpected tendency that the production of GFP is lower when the bacteria are uninduced is obvious, so the Biosafety-System TetOR alive unfortunatly seems not to work. As the Biosafety-Systems [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L#Conclusions Lac of Growth] and [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#Conclusions AraCtive] work, there must be a problem in the interaction of the TetR repressor with the tetO operator. This is discussed in the next section.

Figure 14: Specific production rate of GFP for the Biosafety-System TetOR alive, calculated via equation (1). The production rate of GFP of the uninduced bacteria fluctuates so that the Biosafety-System might not working as expected.

Conclusions

As mentioned above it seems that the Biosafety-System TetOR alive does not work as expected. This is also confirmed by Figure 15 showing the specific production rates of GFP after 7,5 hours of the induced Biosafety-System TetOR alive (red bar), the uninduced Biosafety-System (orange bar) and the pure tetO operator (BBa_K1172914). While induction does indeed results in a repression, the much lower expression from the pure tetO promoter/operator was not expected.

File:Team-Bielefeld-Biosafety-System-TetORalive-Overview.jpg

As the Biosafety-Systems [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L#Conclusions Lac of Growth] and [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#Conclusions AraCtive] show the same genetic construction, but differ only in the repressor and promoter, this cannot be an error of the construction. Also the sequence analysis show the expected sequence, so that this system should actually work. In the Partsregistry similar problems were reported, so this is might be due to an error in either of the parts, the repressor TetR BBa_C0040 and/or the tetO operator BBa_R0040. So the Biosafety-Systems [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L Lac of Growth] and [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S AraCtive] work, while the Biosafety-System TetOR alive needs vigorous testing to identify and remove its problems. Nevertheless it would be worth to improve this part due to the tight shutdown and high activation of the Tet system which would be optimal for a biosafety system.