Team:GeorgiaTech/Project

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Overview

Microorganisms respond to environmental stress through several means for survival. This research project aims to engineer Escherichia coli cells to aggregate with one another forming a biofilm given a cold shock and to link this “huddle” response with heat generation. Altering a cell’s reaction to external variations prolongs its ability to survive such a shock, thereby ensuring an evolutionary advantage over other species. Studying and manipulating a prokaryote’s stress response can broaden the prospective on evolutionary mechanisms in general. This research venture will advance biological engineering by furthering current attempts at constructing and understanding complex gene networks. Applications of synthetic, thermogenerating bacteria range from the investigation of cold resistance evolution to the development of a new generation of inexpensive biosensors for environmental monitoring and contaminant detection. The experiment will focus on producing two distinct responses to a decrease in ambient temperature: cell aggregation leading to biofilm formation and over-expression of alternative oxidase (AOX), an enzyme associated with thermogenesis in plants.

Background

The synthesis of ATP is coupled with the flow of electrons through the electron transport chain to a terminal electron acceptor, terminal oxidase via ubiquinone and cytochromes . In addition to this pathway, all higher plants contain a cyanide-resistant pathway involving an enzyme, alternative oxidase. In this route, electrons branch from the cytochrome pathway at ubiquinone (before cytochrome) and terminate with an alternative oxidase.

INSERT IMAGE*

Unlike the free energy from the cytochrome pathway, the free energy generated by the flow of electrons from ubiquinone to alternative oxidase does not result in the generation of ATP but instead is lost as heat. Due to an increase in the alternative oxidase activity in certain plants, heat is generated (up to 20ºC above ambient) to volatilize foul-smelling attractants for insects engaged in the pollination process [1][2]. The occurrence of AOX is found in plants, as well as in many fungi and protists.

Arabidopsis thaliana is widely researched partly due to the breadth of knowledge on the plant including the completion of its genome sequence. Additionally, its AOX pathway has been extensively studied. The two major classes of AOX genes are expressed in mRNA, AOX1 and AOX2. The AOX1 gene family is stress induced and found in both monocotyledons and eudicotyledons while the AOX2 gene family is prominent only in eudicotyledons and expression depends on the tissue and developmental stage [3]. It has been demonstrated that over-expressing AOX1 leads to large increases in AOX protein and alternative pathway capacity in tobacco cells [4]. The presence of thermogenic monocots (such as Arum maculatum) also indicates that in simple prokaryotic systems, AOX1 expression would be sufficient for thermogenesis.

Objectives and Techniques

Cloning AOX genes into E.coli Kumar and Soll have demonstrated thatthe gene for alternative oxidase from Arabidopsis thaliana can be cloned into E. Coli [5]. A series of overlapping DNA fragments will be generated by the exonuclease III and nuclease S1 deletion method. Strains and Growth Conditions The E. coli strains used will be DH5a and the 5-aminolevulinic acid auxotroph SASX41B (HfrPO2A hemA41 metBI relAI). The strains will be grown on M9 minimal medium with glycerol (0.2%) as a carbon source and supplemented with 5-aminolevulinate (50 Ag/ml) and methionine (40 j.g/ml) when required. Ampicillin (100 tug/ml) will be added to the growth medium when indicated. Complementation Plasmid DNA (100 ng) from an Arabidopsis cDNA library in plasmid pcDNAII will be used to transform competent SASX41B cells by electroporation. Aliquots will be plated on M9/ampicillin medium and incubated at 37°C to select colonies that contain putative hemA-complementing plasmids. Overexpressing AOX The expression level of AOX corresponds to the amount of heat produced. AOX protein content in the thermogenic stages of flowering was at least 10-fold higher than that of pre and post-flowering stages, when heat is not produced [6]. To produce high levels of a protein, it is often useful to clone the gene downstream of a well characterized promoter. Inducing transcription from the regulated promoter thus results in elevated expression of the downstream gene product. By choosing a temperature-sensitive promoter, AOX production can be regulated and stopped once a cell has reached an optimal temperature. E.coli’s major cold shock protein CS7.4, is cold shock-induced via the temperature sensitive cspA promoter [7]. Other promoters that are more active around 15-20°C than at 37°C with a 100-fold or greater increase in gene expression have been identified. They include nusA, infA, and infB of the nus-inf operon, pnp of the S15 operon, recA, and aceE,F, and hypB [8]. Qoronfleh et. al. have demonstrated that naturally occurring cold-inducible promoters in E. coli could be exploited to develop more effective systems for low-temperature expression of heterologous proteins in plasmid-based vectors. This was done by isolation of TnS-lac insertions that resulted in low-temperature 3-galactosidase (1-Gal) expression. Two of the promoters were mapped, cloned, and sequenced into WQ11 strain E.coli. Biofilm formation In a large number of microbial species, non-optimal growth conditions and cellular stress stimulate biofilm formation [9]. In E. coli, lower temperatures promote several adhesive factors [10]. These factors are largely dependent on a stress-response gene, RpoS, initiated in the σs subunit of RNA polymerase. Translation of σs increases at 20°C suggesting that RpoS-dependent gene products play a role in cellular adaptation to a cold shock [11]. As a defense mechanism against an environmental stress, cells can produce various extracellular polysaccharides (EPS) that can be manipulated for adhesion and biofilm formation. One EPS signal molecule regulated by RpoS is cyclic-di-guanosine monophoshpate (c-di-GMP). c-di-GMP is synthesized by diguanylate cyclases (DGCs) which is associated with the GGDEF domain [12]. The GGDEF protein YddV can stimulate cell aggregation and EPS production via its diguanylate cyclase activity. Overexpression of the YddV protein results in a major rearrangement of the bacterial cell’s global gene expression pattern causing the culture to transition from planktonic to biofilm growth through upregulation of EPS-related genes and RpoS [13]. YddV increases EPS production through stimulation of specific gene transcriptions leading to expression of the protein CsgD. CsgD activates curli fiber production, which is an adhesion factor in E. coli. Cold shock induced biofilm formation can be heightened by a mutation in the ompR gene, ompR234. This mutation initiates transcription of the CsgD protein thereby resulting in an increase in biofilm formation [14]. The resulting cold activated biofilm formation will be paired with expression of the alternative oxidase (AOX1) gene. Pairing cellular group formation with heat production will provide evolutionary insight and allow for the engineering of microorganisms holding utility for institutions such as the Department of Energy and Department of Defense. </p></font></td> </table>

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-<p><center><font color="#FFFFFF" size=5><b>ABSTRACT</b></font></center></p>
+<p><center><font color="#FFFFFF" size=5><b>Overview</b></font></center></p>
 <center><table width="900" border="0" cellpadding="2" cellspacing="10">
    <tr>
-     <td bgcolor="#964141" width="800"><font color="#FFFFFF"><p>The purpose of the proposed research is to genetically engineer bacteria to generate large amounts of heat in response to a cold shock. This will advance biological engineering by furthering current attempts at constructing and understanding complex gene networks. Applications of synthetic, thermogenerating bacteria range from the investigation of cold resistance evolution to the development of a new generation of inexpensive biosensors for environmental monitoring and contaminant detection.  The experiment will focus on producing two distinct responses to a decrease in ambient temperature: cell aggregation leading to biofilm formation and overexpression of alternative oxidase (AOX), an enzyme associated with thermogenesis in plants. </p>
+     <td bgcolor="#964141" width="800"><font color="#FFFFFF"><p>Microorganisms respond to environmental stress through several means for survival.  This research project aims to engineer Escherichia coli cells to aggregate with one another forming a biofilm given a cold shock and to link this “huddle” response with heat generation. Altering a cell’s reaction to external variations prolongs its ability to survive such a shock, thereby ensuring an evolutionary advantage over other species. Studying and manipulating a prokaryote’s stress response can broaden the prospective on evolutionary mechanisms in general. This research venture will advance biological engineering by furthering current attempts at constructing and understanding complex gene networks. Applications of synthetic, thermogenerating bacteria range from the investigation of cold resistance evolution to the development of a new generation of inexpensive biosensors for environmental monitoring and contaminant detection.  The experiment will focus on producing two distinct responses to a decrease in ambient temperature: cell aggregation leading to biofilm formation and over-expression of alternative oxidase (AOX), an enzyme associated with thermogenesis in plants.
-<p>Microorganisms respond to environmental stress through several means for survival.  This research project also aims to engineer Escherichia coli cells to aggregate with one another forming a biofilm given a cold shock and link this “huddle” response with heat generation. Altering a cell’s reaction to external variations prolongs its ability to survive such a shock, thereby ensuring an evolutionary advantage over other species. Studying and manipulating a prokaryote’s stress response can broaden the prospective on evolutionary mechanisms in general.
 </p></font></td>
 </table>
 <table width="965" border="0" cellpadding="0" cellspacing="0">
+<p><center><font color="#FFFFFF" size=5><b>Background</b></font></center></p>
+<center><table width="900" border="0" cellpadding="2" cellspacing="10">
+  <tr>
+    <td bgcolor="#964141" width="800"><font color="#FFFFFF"><p>The synthesis of ATP is coupled with the flow of electrons through the electron transport chain to a terminal electron acceptor, terminal oxidase via ubiquinone and cytochromes . In addition to this pathway, all higher plants contain a cyanide-resistant pathway involving an enzyme, alternative oxidase. In this route, electrons branch from the cytochrome pathway at ubiquinone (before cytochrome) and terminate with an alternative oxidase. </p>
+*INSERT IMAGE*
+<p>Unlike the free energy from the cytochrome pathway, the free energy generated by the flow of electrons from ubiquinone to alternative oxidase does not result in the generation of ATP but instead is lost as heat. Due to an increase in the alternative oxidase activity in certain plants, heat is generated (up to 20ºC above ambient) to volatilize foul-smelling attractants for insects engaged in the pollination process [1][2]. The occurrence of AOX is found in plants, as well as in many fungi and protists.</p>
+<p>Arabidopsis thaliana is widely researched partly due to the breadth of knowledge on the plant including the completion of its genome sequence. Additionally, its AOX pathway has been extensively studied. The two major classes of AOX genes are expressed in mRNA, AOX1 and AOX2. The AOX1 gene family is stress induced and found in both monocotyledons and eudicotyledons while the AOX2 gene family is prominent only in eudicotyledons and expression depends on the tissue and developmental stage [3]. It has been demonstrated that over-expressing AOX1 leads to large increases in AOX protein and alternative pathway capacity in tobacco cells [4]. The presence of thermogenic monocots (such as Arum maculatum) also indicates that in simple prokaryotic systems, AOX1 expression would be sufficient for thermogenesis.
+</p></font></td>
+</table>
+<table width="965" border="0" cellpadding="0" cellspacing="0">
+<p><center><font color="#FFFFFF" size=5><b>Objectives and Techniques</b></font></center></p>
+<center><table width="900" border="0" cellpadding="2" cellspacing="10">
+  <tr>Cloning AOX genes into E.coli
+Kumar and Soll have demonstrated thatthe gene for alternative oxidase from Arabidopsis thaliana can be cloned into E. Coli [5]. A series of overlapping DNA fragments will be generated by the exonuclease III and nuclease S1 deletion method.
+Strains and Growth Conditions The E. coli strains used will be DH5a and the 5-aminolevulinic acid auxotroph SASX41B (HfrPO2A hemA41 metBI relAI). The strains will be grown on M9 minimal medium with glycerol (0.2%) as a carbon source and supplemented with 5-aminolevulinate (50 Ag/ml) and methionine (40 j.g/ml) when required. Ampicillin (100 tug/ml) will be added to the growth medium when indicated.
+Complementation Plasmid DNA (100 ng) from an Arabidopsis cDNA library in plasmid pcDNAII will be used to transform competent SASX41B cells by electroporation. Aliquots will be plated on M9/ampicillin medium and incubated at 37°C to select colonies that contain putative hemA-complementing plasmids.
+Overexpressing AOX
+The expression level of AOX corresponds to the amount of heat produced. AOX protein content in the thermogenic stages of flowering was at least 10-fold higher than that of pre and post-flowering stages, when heat is not produced [6]. To produce high levels of a protein, it is often useful to clone the gene downstream of a well characterized promoter. Inducing transcription from the regulated promoter thus results in elevated expression of the downstream gene product. By choosing a temperature-sensitive promoter, AOX production can be regulated and stopped once a cell has reached an optimal temperature. E.coli’s major cold shock protein CS7.4, is cold shock-induced via the temperature sensitive cspA promoter [7].  Other promoters that are more active around 15-20°C than at 37°C with a 100-fold or greater increase in gene expression have been identified. They include nusA, infA, and infB of the nus-inf operon, pnp of the S15 operon, recA, and aceE,F, and hypB [8].  Qoronfleh et. al. have demonstrated that naturally occurring cold-inducible promoters in E. coli could be exploited to develop more effective systems for low-temperature expression of heterologous proteins in plasmid-based vectors. This was done by isolation of TnS-lac insertions that resulted in low-temperature 3-galactosidase (1-Gal) expression. Two of the promoters were mapped, cloned, and sequenced into WQ11 strain E.coli.
+Biofilm formation
+In a large number of microbial species, non-optimal growth conditions and cellular stress stimulate biofilm formation [9]. In E. coli, lower temperatures promote several adhesive factors [10]. These factors are largely dependent on a stress-response gene, RpoS, initiated in the σs subunit of RNA polymerase. Translation of σs increases at 20°C suggesting that RpoS-dependent gene products play a role in cellular adaptation to a cold shock [11].
+As a defense mechanism against an environmental stress, cells can produce various extracellular polysaccharides (EPS) that can be manipulated for adhesion and biofilm formation. One EPS signal molecule regulated by RpoS is cyclic-di-guanosine monophoshpate (c-di-GMP). c-di-GMP is synthesized by diguanylate cyclases (DGCs) which is associated with the GGDEF domain [12]. The GGDEF protein YddV can stimulate cell aggregation and EPS production via its diguanylate cyclase activity. Overexpression of the YddV protein results in a major rearrangement of the bacterial cell’s global gene expression pattern causing the culture to transition from planktonic to biofilm growth through upregulation of EPS-related genes and RpoS [13].
+YddV increases EPS production through stimulation of specific gene transcriptions leading to expression of the protein CsgD. CsgD activates curli fiber production, which is an adhesion factor in E. coli. Cold shock induced biofilm formation can be heightened by a mutation in the ompR gene, ompR234. This mutation initiates transcription of the CsgD protein thereby resulting in an increase in biofilm formation [14].
+The resulting cold activated biofilm formation will be paired with expression of the alternative oxidase (AOX1) gene. Pairing cellular group formation with heat production will provide evolutionary insight and allow for the engineering of microorganisms holding utility for institutions such as the Department of Energy and Department of Defense.
+</p></font></td>
+</table>
+<table width="965" border="0" cellpadding="0" cellspacing="0">
    <tr>
      <td bgcolor="#7c1212" width="965"><font color="#FFFFFF"><p></p></font></td>
    </tr>
 </table>