Biosafety System AraCtive

Overview

Figure 1: Biosafety-System AraCtive.

The Biosafety-System araCtive <bbpart>BBa_K1172909</bbpart> is an improvement of the Biobrick <bbpart>BBa_K914014</bbpart> by changing the first promoter into the rhamnose promoter P_Rha, integration of the alanine racemase <bbpart>BBa_K1172901</bbpart> and usage the repressor AraC to regulate the transcription of the Barnase behind the P_BAD promoter. Because of the tight repression of this promoter, this System has the lowest basal transcription of the Barnase and is therefore the most active one.

Genetic Approach

Rhamnose promoter P_Rha

The promoter P_Rha (<bbpart>BBa_K914003</bbpart>) naturally regulates the catabolism of the hexose L-rhamnose. The advantage of the operon for further usage is its solely positive regulation. The regulon consists of the promoter P_RhaT, which regulates the expression of the protein RhaT for the uptake of L-rhamnose and the two operons rhaSR and rhaBAD.
The operon rhaSR regulates the genes rhaS and rhaR, whose translated proteins are responsible for the positive activation of the L-rhamnose catabolism, while the operon rhaBAD regulates the genes for the direct catabolism of L-rhamnose.
When L-rhamnose is present, it acts as an inducer by binding to the regulatory protein RhaR. RhaR regulates his own expression and the expression of the regulatory gene rhaS by inhibiting or, in the presence of L-rhamnose, activating the operon RhaSR. Normally the expression level is modest, but it can be enhanced by a higher level of intracellular cAMP, which increases in the absence of glucose. So, in the presence of L-rhamnose and a high concentration of intracellular cAMP, the activator protein RhaR is expressed on higher level, resulting in an activation of the promoter P_RhaT for an efficient L-rhamnose uptake and an activation of the operon rhaBAD. The L-rhamnose is than broken down into dihydroxyacetone phosphate and lactate aldehyde by the enzymes of the operon rhaBAD (Wickstrum et al., 2005).
A brief schematic summary of the regulation is shown in Figure 2.

Figure 2: The catabolism of L-rhamnose in E. coli is turned off in general but inducible by L-rhamnose. The induction activates the transcription of the genes rhaS and rhaR, which regulate the L-rhamnose catablosim by positive activation of the rhamnose uptake (RhaT) and its metabolization (rhaBAD).

Dihydroxyacetone phosphate can be metabolized in the glycolysis pathway, while lactate aldehyde is oxidated to lactate under aerobic conditions and reduced to L-1,2,-propandiol under anaerobic conditions.
This degradation of L-rhamnose can be separated in three steps. In the first step the L-rhamnose is turned into L-rhamnulose by an isomerase (gene rhaA). The catabolism continues by the a kinase (gene rhaB), which phosphorylates the L-rhamnulose to L-rhamnulose-1-phosphate. This is finally hydrolyzed by Aldolase (gene rhaD) to dihydroxyacetone phosphate and lactate aldehyde (Baldoma et al., 1988).
Four our Safety-System the rhamnose promoter P_Rha is used to control the expression of the repressor AraC and the essential alanine racemase, because this promoter has an even lower basal transcription then the arabinose promoter P_BAD. This is needed to tightly repress the expression of the alanine racemase (alr) and therefore take advantage of the double-kill switch. Although the rhamnose promoter P_Rha is characterized by a very low basal transcription it can not be used for the control of the toxic RNase Ba (Barnase), which ist needed for the alanine racemase (alr), the other part of the double-kill switch. In conclusion this promoter is the best choice for the first part of the Biosafety-System, as it is tightly repressed in the absence of L-Rhamnose, but activated in its presence.

Repressor AraC

The araC gene is naturally found in E. coli and is coding for the 32 kDa AraC protein, which regulates the expression of the genes required for the uptake and catabolism of the pentose L-arabinose. The genes for the catabolism of arabinose are under the control of the arabinose promoter P_BAD, which is both positively and negatively regulated by the repressor AraC. Naturally in the presence of L-arabinose the expression of those genes is activated, while it is repressed in its absence. Besides the AraC protein regulates its own transcription under the control of the so called P_C promoter. Compared to the P_BAD promoter the P_C promoter as well as the araC gene are therefore transcribted in opposite direction (Schleif, 2010).

Figure 3: Positive regulation of the arabinose promoter P_BAD. When L-arabinose is present the dimer-structure of AraC is relaxed and the AraC protein functions as activator. The activation can additionally be enhanced by the CRP-cAMP-complex.

Figure 4: Negative regulation of the arabinose promoter P_BAD. In the absence of L-arabinose AraC functions as repressor by forming a dimer, which inhibits the binding of the RNA-polymerase.

Naturally in the absence of L-arabinose the AraC protein is found in its repressor conformation, binding to the O₂-region and I₁-region. Binding two AraC proteins to this domains is leading to a protein-protein-interaction of AraC forming a dimer. This AraC is responsible for the tight repression of the P_C and the P_BAD promoter by forming a DNA-loop to inhibit the initiation of the RNA-polymerase (see Figure 4 above).
When L-arabinose is present in the media the operon changes to the active form. Following this conditions the araC protein functions as activator by binding to the I₁- and I₂-region of the operator region. This causes transcription of the genes behind the P_BAD and the P_C promoter. Depending on the intracellular level of cAMP the transcription of both the genes behind the promoters P_BAD and P_C can even be enhanced. In the absence of glucose the cell reaches a high level of cAMP as a signal molecule. CAMP binds as the typically CRP-cAMP complex binds in front of the AraC activator and increases the binding affinity of the RNA-polymerase, so that the genes behind the P_BAD promoter are expressed on higher levels. Beside the transcription affinity of the P_BAD promoter the AraC protein also regulates the genes araE and the operon consisting the genes araFGH, which are necessary for an efficient uptake of L-arabinose (Cass, 1988).
The auto-regulation of the araC gene itself works both in the presence and absence of L-arabinose. In presence of L-arabinose the DNA-loop which prevents also the transcription of the P_BAD promoter also inhibits the transcription of araC. When L-arabinose is present and the AraC concentration is high enough, its auto-regulates its own transcription by dimerization between the araO₁- and the araO₂-region. This leads to an other DNA-loop, inhibiting solely the transcription of the genes behind the P_C promoter (Hamilton, 1988).
For our Biosafety-System we decided to use the arabinose P_BAD promoter, because this promoter is really tight regulated by AraC, and shows no basal transcription in the complete absence of AraC. Additional this promoter is repressed by glucose and basal transcription is activated in the presence of cAMP.

Alanine racemase Alr

The alanine racemase Alr (EC 5.1.1.1) from the gram-negative enteric bacteria Escherichia coli is a racemase, which catalyses the reversible conversion of L-alanine into the enantiomer D-alanine. For this reaction the cofactor pyridoxal-5'-phosphate (PLP) is typically needed. The constitutive expressed alanine racemase (alr) is naturally responsible for the accumulation of D-alanine, which is an essential component of the bacterial cell wall, because it is used for the crosslinkage of peptidoglykan (Walsh, 1989).
The usage of D-alanine instead of a typically L-amino acids prevents cleavage by peptidases. However a lack of D-alanine causes to a bacteriolytic characteristics. In the absence of D‑alanine dividing cells will lyse rapidly. This perception is used for our Biosafety-Strain, a D-alanine auxotrophic mutant (K-12 ∆alr ∆dadX). The Biosafety-Strain grows only with a plasmid containing the alanine racemase (<bbpart>BBa_K1172901</bbpart>) with a complementation of the D-alanine auxotrophy. Consequently the alanine racemase is essential for bacterial cell division. This approach guarantees a high plasmid stability, which is extremely important when the plasmid contains a toxic gene like the Barnase. In addition this construction provides the possibility for the implementation of a double kill-switch system. Because if the expression of the alanine racemase is repressed and there is no D-alanine-Supplementation in the media, cells will not grow.

Figure 5: The alanine racemase (<bbpart>BBa_K1172901</bbpart>) from E. coli catalyses the reversible conversion from L-alanine to D-alanine. For this isomerisation the cofactor pyridoxal-5'-phosphate is necessary.

Terminator

Terminators are essential for the end of an operon. In procaryots two types of terminators exist. The rho-depending and the rho-independing terminator. Rho-independing terminators are characterized by their stem-loop forming sequence. In general the terminator-region can be divided into four regions. Starting with a GC-rich region, which performs the stem and is followed by the loop-region. The third region is made up from the opposite part of the stem, so that this region concerns also a high GC-content. The terminator region closes with a poly uracil region, which destabilizes the binding of the RNA-polymerase. The stem-loop of the terminator causes a distinction of the DNA and the translated RNA. Consequently the binding of the RNA-polymerase is cancelled and the transcription ends after the stem-loop (Carafa et al., 1990).
For our Bioafety-System araCtive the terminator is necessary to avoid that the expression of the genes under control of the Rhamnose promoter P_Rha, like the Repressor AraC and the Alanine-Racemase (alr) results in the transcription of the genes behind the Arabinose promoter P_BAD, which contains the toxic Barnase <bbpart>BBa_K1172904</bbpart> and would lead to cell death.

Figure 6: Stem-loop structure of the terminator <bbpart>BBa_B0015</bbpart>, which is used for the Biosafety-System araCtive. The terminator is used to make sure that solely both the repressor AraC and the alanine racemase Alr are expressed but the transcription of the toxic RNase Ba (Barnase) is avoided.

Arabinose promoter P_BAD

The arabinose promoter P_BAD controls the expression of the genes, which are necessary for the catabolism of the pentose arabinose. The expression is regulated by the activator and repressor AraC. The genes of this operon convert the pentose to xylose, which can be catalyzed into fructose-1,6-diphosphate and subsequently becomes so part of the glycolysis. The conversion of L-arabinose is realized in three steps. First the isomerase (gene A) catalyses the reaction from L-arabinose into the isomer L-ribulose. In the second step the Ribulose-kinase (gene B) phosphorylates the L-ribulose to L-ribulose-monophosphat. For this reaction ATP is needed. At least the L-ribulose-monophosphate is converted to D-Xylose-5-phosphat by the L-ribulose-5-phosphate-epimerase (gene D, Schleif, 2010).

Figure 7: Overview of the arabinose regulon from E. coli. The transcription of the genes araBAD, which are coding for the enzymes of the L-arabinose catabolism are positivly upregulated and negativly inhibited by the AraC protein, which additionally autoregulates his own transcription.

For our Safety-System we used the arabinose promoter P_BAD for the regulation of the toxic gene product Barnase. As this promoter has very low basal transcription and as the expression of the genes behind this promoter is strict depending on the presence of AraC for activating the transcription, it is possible to use the arabinose promoter P_BAD to control the expression of a toxic gene product without causing cell death.

RNase Ba (Barnase)

The Barnase (EC 3.1.27) is a 12 kDa extracellular microbial ribonuclease, which is naturally found in the gram-positive soil bacteria Bacillus amyloliquefaciens and consists of a single chain of 110 amino acids. The Barnase (RNase Ba) catalyses the cleavage of single stranded RNA, whereat the hydrolysis of the dinucleotides has the highest affinity to the structure GpN. In the first step of the RNA-degradation a cyclic intermediate is formed by transesterification and afterwards this intermediate is hydrolysed yielding in a 3'-nucleotide (Mossakowska et al., 1989).

Figure 8: Enzymatic reaction of the RNA-cleavage by the RNase Ba. First the transesterifiaction by the Glu-73 residue is performed and then this cyclic intermediat is hydrolized by the His-102 of the Barnase.

In Bacillus amyloliquefaciens the activity ot the Barnase (RNase Ba) is inhibited intracellular by the Inhibitor called barstar. Barstar consists of only 89 amino acids and binds with a high affinity to the toxic Barnase. This prevents the cleavage of the intracellular RNA in the host organism (Paddon et al., 1989). Therefore the Barnase naturally acts only outside the cell and is translocated under natural conditions. For the Biosafety-System araCtive we modified this aspect by cloning only the sequence responsible for the cleavage of the RNA, but not the signal sequence of the native Barnase, which is essential for the extracellular transport.
As shown in the graphic below, the transcription of the DNA, which encodes the Barnase produces a 474 nt RNA. The translation of the RNA starts about 25 nucleotides downstream from the transcription start and can be divided into two parts. The first part (colored in orange) is translated into a signal peptide at the amino-terminus of the Barnase coding RNA. This part is responsible for the extracellular translocation of the RNase Ba, while the peptide sequence for the active Barnase starts 142 nucleotides downstream from the transcription start (colored in red).
For the Biosafety-System araCtive we only used the coding sequence (<bbpart>BBa_K1172904</bbpart>) of the Barnase to prevent the extracellular translocation of the toxic gene product. Translation of the barnase gene leads to a rapid cell death if the expression of the Barnase isn't repressed by the repressor AraC of our Biosafety-System.

Figure 9: Sequence of the signal peptide amino terminal of the RNase Ba (Barnase). The Biobrick <bbpart>BBa_K1172904</bbpart> does not include the signal sequence for the extracellular translocation, but only the coding sequence for the mature enzyme.

Biosafety-System araCtive

Combining the gene 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 P_BAD promoter with L-arabinose 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 continously adding L-rhamnose to the medium. As shown in the figure below, this leads to an expression of the essential Alanine-Racemase (alr) and the araC repressor, so that the expression of the RNase Ba is repressed.

Figure 10: Biosafety-System araCtive in the presence of L-rhamnose. The essential alanine racemase (Alr) and the repressor AraC are expressed, resulting in a repression of the expression of the RNAse Ba. Consequently the bacteria show normal growth behaviour.

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 AraC repressor decrease, so that the expression of the toxic RNase Ba (Barnase) is not inhibited as strongly as before. 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 and makes sure that the bacteria can only grow in the defined area.

Figure 11: Active Biosafety-System araCtive outside of a defined enviroment and decreased concentrations of L-rhamnose. Both the expression of the alanine racemase (Alr) and AraC repressor are reduced and ideally completely shutdown. In contrast the expression of the RNase ba (Barnase) is sligthly turned on, leading to cell death by RNA cleavage.

Results

Characterization of the arabinose promoter pBAD

First of all the bacterial growth under the pressure of the unrepressed pBAD promoter was investigated on different carbon source. Therefore the cultivation on M9 minimal media with Glucose or Glycerol was characterized. To identify the transcription rate of the unrepressed arabionse promoter pBAD the expression of the green fluorescence protein GFP <bbpart>BBa_E0040</bbpart> behind the pBAD promoter was used.
As shown in figure 12 below, the bacteria adapted better on glucose then on Glycerol. As glucose is the more powerful energy source, because it posses more carbon atoms than glycerol these result was expected before. So more interesting are the fluorescence measurement shown in the figure 13. As it can be seen both the wild type K-12 as the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC show about the same fluorescence on glucose (blue and black curve) but differ on glycerin. This can be explained by the fact that glucose itself also represses the arabinose promoter, while glycerol does not. In the presence of glucose the intracellular concentration of cAMP is low to repress the inefficient catabolism of arabinose, so that the glucose is catabolized first by the bacteria resulting in an optimal growth. In the absence of glucose increases, which enhances the transcription of the most operons, who regulate the enzymes for the catabolism of an alternative carbon source. Therefore the expression of GFP under the control of the pBAD promoter decreases on glycerol. Another different can be seen between the wild type (orange curve) and the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC (red curve). The Biosafety-Strain shows lower expression then the wild type, but has according to figure 12 about the same growth rate. This is caused by the fact that the araC gene in the Biosafety-Strain was deleted an as the araC gene functions not only as are repressor but also as an activator the transcription rate decreases.

Figure 12: Characterization of the bacterial growth of the pBAD promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC. The M9 media was supplemented with 5 mM D-alanine. It can be seen, that the bacteria grow faster on M9 minimal media glucose than on M9 minimal media glycerol.

Figure 13: Characterization of the fluorescence of pBAD promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC. The bacteria were cultivated on M9 minimal media with 5 mM D-alanine supplemented.

The effect that glucose represses the transcription of the pBAD promoter becomes more clear by the specific production rate, as shown in figure 14. The specific production rate was thereby calculated via equation (1) :

Figure 14: Specific growth rate of GFP behind the pBAD promoter by the use of different carbon sources.

Characterization of the Biosafety-System araCtive

The Biosafety-System araCtive was characterized on M9 minimal medium with glycerol as carbon source. As for the characterization of the pure arabinose promoter pBAD above, the bacterial growth and the fluorescence of GFP <bbpart>BBa-E0040</bbpart> was measured. Therefore the wild type and the Biosafety-Strain E. coli K-12 ∆alr ∆dadX were cultivated once with the induction of 1% L-Rhamnose and once only on glycerol. First of all it is obviously shown in figure 15 that the growth of the bacteria, who are induced with 1 % L-Rhamnose (blue and black curve) is significant slower than on pure glycerol (orange and red curve). This is attributed to the high metabolic pressure of the induced bacteria. The expression of the repressor araC and the Alanine-Racemase (alr) simultaneously causes a high outlay of the cells so that they grow slower then the uninduced cells, who expresses only GFP. Additional the arabinose promoter pBAD is tightly regulated, so that the expression even with a small amount of the repressor AraC is not that high and therefore not as stressful.
Comparing the bacterial growth with the fluorescence in figure 16 it can be seen that the fluorescence seems to follow the same figure than the bacterial growth. The uninduced cells shows approximately an exponantial rise of fluorescence, while the fluorescence of the induced bacteria increases only slow.

Figure 15: Characterization of the bacterial growth of the Biosafety-System on M9 minimal media glycerol. The figure compares the wild tpye k-12 and the Biosafety-Strain K-12 ∆alr ∆dadX and the induction by L-Rhamnose to pure glycerol.

Figure 16: Characterization of the fluorescence of the Biosafety-System araCtive. The figure compares the wild tpye k-12 and the Biosafety-Strain K-12 ∆alr ∆dadX and the induction by L-Rhamnose to pure glycerol.

From the figure above it can not be seen if the expression of the repressor araC does effect the transcription of GFP or not. The slower growth of the bacteria is a first indication that the repressor araC and the Alanine-Racemase are highly expressed, but as the growth of the bacteria shows nearly the same figure than the fluorescence it could be possible that the repressor does not effect the expression level of GFP under the control of the arabinose promoter pBAD. That the Biosafety-System works as aspected by repressing the expression of GFP in the presence of L-Rhamnose can be seen from figure 18 below. The calculated specific production rate (equation 1) differs clearly, so that the production of GFP in the presence of L-Rhamnose is always lower than in its absence.
As the specific production rate was calculated between every single measurement point the curve is not smoothed and so the fluctuations have to be ignored, as they do not stand for are real fluctuations in the transcription in the expression of GFP. They are caused by the growth curve and the fluorescence curve. And this measured curves are not ideal the calculation of the specific production rate causes the fluctuations. But it can be seen very clear that the production of GFP differ an is much lower, when the bacteria are induced with 1% L-Rhamnose. So the Biosafety-System araCtive works.

Figure 17: Specific production rate, calculated by equation (1). The production rate of GFP of the uninduced bacteria is significant higher compared to the bacteria induced with 1% L-Rhamnose. The Biosafety-System AraCtive works.

Conclusion of the Results

As the expression level of GFP is increased in the absence of L-Rhamnose and decreased in its presence, the Biosafety-System araCtive works as aspected. In figure 18 the specific production rates after 7,5 hours are compared. It can be seen that the expression level of the pBAD promoter decreases in the uninduced Safety-Strain compared to the uninduced second part of the Biosafety-System and that the induction with L-Rhamnose leads to a tight repression of the transcription and therefore the expression of GFP.

Figure 18: Comparision of the specific production rate of GFP in the with L-Rhamnose induced Biosafety-System araCtive, the uninduced Biosafety-System araCtive and the second part of the Biosafety-System (pBAD-GFP only).

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