Biosafety System Lac of Growth

Overview

Figure 1: Biosafety-System Lac of growth.

Naturally the lac operon regulates the catabolism of the disaccharide lactose (4-O-(β-D-Galactopyranosyl)-D-glucopyranose) in E. coli. The operon contains the lactose promoter (plac) and the genes for the catabolism of the Lactose to Glucose and Galactose. Upstream of the lac operator exists the coding sequence for the repressor lacI under the control of a weak promoter (Jacob et al., 1961).

Genetic Approach

Rhamnose promoter

The promoter rhaBAD regulates naturally the catabolism of the hexose L-rhamnose. The advantage of the operon is its positively regulation. All in all the regulon consists of the promoter pRhaT, who regulates the expression of the protein RhaT for the uptake of L-Rhamnose, the 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 activating, in the presence of L-Rhamnose, 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 activor protein RhaR is expressed on higher level, resulting in an activation of the promoter pRhaT 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).

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, who regulate the L-ramnose catablosim by positive activation of the rhmanose uptake (rhaT) and metabolization (rhaBAD).

Dihydroxyacetone phosphate can be metabolized in the glycolysis pathway, while the lactate aldehyde is oxized 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), who 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 rhaBAD is used to control the expression of the repressor araC and the essential Alanine-Racemase, because this promoter has even a lower basal transcription then the arabinose promoter pBAD. This is needed to tightly repress the expression of the Alanine-Racemase (alr) and take so advantage of the double-kill switch. So although the rhamnose promoter rhaBAD is characterized by a very low basal transcription it can not be used for the control of the toxic Barnase, because the regulation is mainly organized by an activation and not an active repression, but this is optimal for the first containing the Alanine-Racemase. So 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.

lacI

Naturally the lac operon regulates the catabolism of the disaccharide lactose (4-O-(β-D-Galactopyranosyl)-D-glucopyranose) in E. coli. The operon contains the lactose promoter (plac) and the genes for the catabolism of the Lactose to Glucose and Galactose. Upstream of the lac operator exists the coding sequence for the repressor lacI under the control of a weak promoter (Jacob et al., 1961).
Compared to the catabolism of the sugars L-Rhamnose or L-arabinose Lactose, as a disaccharide has a higher energy content and is therefore used more preferable. This is a reason, why the basal transcription of this promoter is even more higher. The leakiness of the lac promoter is caused by the fact that the lacY need to be expressed for an efficient Lactose uptake, while in the arabinose system the uptake is regulated separate (Görke et al., 2008).
In our Safety-System the lacI (<bbpart>BBa_C0012</bbpart>) is used for the repression of the lactose promoter (plac).

Alanine Racemase

The alanine-racemase alr (EC 5.1.1.1) from the gram-negative enteric bacteria Escherichia coli is a racemase, which catalyses the reversible reaction from L-alanine into the enantiomer D-alanine. For this reaction the cofactor pyridoxal-5'-phosphate (PLP) is typically needed. The constitutive alanine-racemase (alr) is naturally responsible for the accumulation of D-Alanin, which is an essential component of the bacterial cell wall, because it is used for the crosslinkage of the peptidoglykan (Walsh, 1989).
The use of D-Alanine instead of a typically L-amino acids prevents the cleavage by peptdidases, but a lack of D-Alanine leeds to a bacteriostatic characteristic. So in the absence of D‑Alanine dividing cells will lyse rapidly. This approach is used by our Biosafety-Strain, a D-alanine auxotrophic mutant (K-12 ∆alr ∆dadX). The Safety-Strain grows only with a plasmid containing the Alanine-Racemase (<bbpart>BBa_K1172901</bbpart>) for the complementation of the D-alanine auxotrophic. Because the Alanine-Racemase is therefore 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 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, the cells would not increase.

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

Terminator

Terminator are essential for the end of an operon. In procaryot exists rho-depending and independing terminator. Rho-independing terminators are characterized by an stem-loop, which is caused by special sequence. In general the terminator-region can be divided into four regions. Starting with a GC-rich region, which performs the stem and followed by the loop-region. The third region is made up from the opposite part of the stem, so that this region concerns also GC-rich portion. After that the terminator ends by an 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, so that the binding of the RNA-polymerase is canceld and the transcription ends after the stem-loop (Carafa et al., 1990).
For our Safety-System the terminator is necessary to avoid that the expression of the genes under the control of the Rhamnose promoter pRHA, like the Repressor araC and the Alanine-Racemase (alr), are transcripted but not the genes of the Arabinose promoter pBAD, which contains the toxic Barnase 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. The terminator is used to make sure that only the repressor and the Alanine-Racemase is transcripted and avoids a transcription of the toxic Barnase.

Lactose promoter (plac)

Naturally the lac operon regulates the catabolism of the disaccharide lactose (4-O-(β-D-Galactopyranosyl)-D-glucopyranose) in E. coli. The operon consists of a CAP-binding site, the lac promoter, the lac operator and the genes lacZ, lacY and lacA downstream of the promoter. The transcription of the lactose promoter is regulated by the lacI gene, which is found upstream of the operon under the control of a weak promoter. In the absence of lactose the transcription of the genes behind the lactose promoter is blocked caused by the binding of the lacI pressor. While in the presence of Lactose the repressor is released from the operator and the genes can be transcripted. Typically the transcription is enhanced by a high intracellular level of cAMP (Busby, 1999).

Figure 5:Structure of the lactose operon and its regulatory units. In the absence of Lactose the trascription of the genes behind the lactose promoter is blocked. In the presens of Lactose a side reaction of the ß-Galactosidase (gene lacZ) synthesis allolactose, who causes a change in conformation of the lacI. The repressor lacI releases the operator sequnece and the transcription of the lactose operons starts.

The lactose promoter thereby regulates the transcription of the genes lacZ, lacY and lacA. The lacZ gene encodes for the ß-Galactosidase a enzyme, who breaks down the lactose to glucose and galactose. The ß-Galactosidase catalyses additional the degradation from Lactose to Allolactose. By binding on the lacI repressor, it changes his conformation an is not any more able to bind on the operator sequnece and to block the transcription. As only one enzyme is necessary to gain a substrate of the glycolysis is becomes clear, why the degradation of Lactose is more preferable compared to L-arabinose or L-rhamnose.
To realize a preference of lactose, the transcription of the lactose promoter is not repressed as that strong. This is caused by the fact that the lacY gene, coding for the integral membrane protein lactose permease, is necessary for the lactose uptake and has to be transcripted on a low level.
The last gene of the lac operon, lacA, encodes for a Transacetylase, who acetylizes glycosides that can not be metabolized. The acetylated glycosides are transported outside the cell to avoid the accumulation of lactose.
In our Biosafety-System the lac-promoter is used for the regulation of GFP or the Barnase. As the lac promoter shows a high basal transcription, its might not ideal for the regulation of a toxic gene product, but the the Biosafety-System Lac of growth is ideal for comparison with the other Systems to measure the level of basal transcription under repressed and unrepressed conditions. Besides we improved the leakiness of the lactose promoter by adding a second lacI-binding site 12 nt downstream of the excisting bining site. As this distance corresponds to about one whorl of the double helix, this should allow an additional lacI repressor to bind on the other site of the DNA and tighten the repression of the lactose promoter. Unfortunately the improvement of the so called double lac promoter could not be quantified, because lac of time (Lewis, 2005).

Barnase

The Barnase (EC 3.1.27) is an 12 kDa extracellular microbial ribonuclease, which is naturally found in the gram-positive soil bacteria Bacillus amyloliquefaciens and consist a single chain of 110 amino acids. The Barnase (RNase Ba) catalyses the cleavage of single stranded RNA, where 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: Chemical 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 intracelluar by the Inhibitor called barstar. Barstar consists only about 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 acts naturally only outside the cell and is translocated under natural conditions. For the Biosafety-System we tried to modified this aspect by cloning only the sequence responsible for the cleavage of the RNA, but not the part 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 of this ribonuclease 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 N-Terminus of the Barnase. 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 we used only the coding sequence (<bbpart>BBa_K1172904</bbpart>) of the Barnase itself to prevent the extracellular translocation of the toxic gene product. This leads to a rapid cell death if the expression of the Barnase isn't repressed by the repressor of the Biosafety-System.

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

Biosafety-System Lac of growth

<p align="justify"> Together with the Biosafety-Strain K-12 ∆alr ∆dadX the Biosafety-System Lac of Growth takes advantage of this genes by combine them to a powerful device, which allows to control the bacterial cell division. The control of the bacterial growth is thereby active and passive possible. Active by inducing the lactose 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 an defined environment, like the MFC by adding continously L-Rhamnose to the media. As shown in the figure below, this leads to an expression of the essential Alanine-Racemase (alr) and the lacI repressor, so that the expression of the RNase Ba is repressed.

Figure 10: Biosafety-System araCtive in the presens of L-Rhamnose. The essentail Alanine-Racemase (alr) and the repressor araC are expressed, so that the expression of the RNAse Ba is repressed and the Bacteria grow.

When the bacteria exit the defined environment of the MFC or L-Rhamnose is not added any more to the media, the expression of the Alanine-Racemase (alr) and the lacI repressor decreases, so that the expression of the toxic RNase Ba (Barnase) is not inhibted so strong any more. The cleavage of the intracellular RNA by the Barnase and ideally also the lack of synthesized D-alanine, caused by the repressed Alanine-Racemase inhibits the cell division and makes sure that the bacteria can only grow in the defined area.

Figure 11: Biosafety-System Lac of growth outside the defined conditions and a decreased concentration of L-Rhamnose. The expression of the Alanine-Racemase and lacI repressor is reduced and ideally completly 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 lactose promoter plac

First of all the bacterial growth under the pressure of the unrepressed lactose promoter plac 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 lactose promoter plac the expression of the green fluorescence protein GFP <bbpart>BBa_E0040</bbpart> behind the plac 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 also the fluorescence depends on the carbon source, but not as strong as it can be seen by the arabinose promoter pBAD.
This can be explained by the fact that glucose itself also represses the lactose promoter, while glycerol does not and in comparision to the arabinose system the lactose promoter is known to be more leaky. So in the presence of glucose the intracellular concentration of cAMP is low and represses the inefficient catabolism of lactose, so that the glucose is catabolized first by the bacteria resulting in an optimal growth. In the absence of glucose the concentration of cAMP 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 lactose promoter decreases on glycerol.

Figure 12: Characterization of the bacterial growth of the lactose promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX. 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 plac promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC.

Figure 13: Characterization of the fluorescence of plac promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC.

Characterization of the Biosafety-System Lac of growth

Figure 13: Characterization of the fluorescence of plac promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC.

Figure 13: Characterization of the fluorescence of plac promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC.

Figure 13: Characterization of the fluorescence of plac promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC.

Conclusion of the Results

Figure 13: Characterization of the fluorescence of plac promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC.

References

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