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Utah State University

CyanoBricks

Project Description

Synechocystis sp. PCC 6803 (hereafter referred to as PCC 6803) is a cyanobacterium, a photosynthetic bacterium, and one of the major model organisms in microbiology. PCC 6803 has been extensively studied for it photosynthetic pathway and its genome has been completely sequenced. It is capable of acting as both an autotroph, obtaining its energy from light and its carbon from the atmosphere, and as a heterotroph, using glycolysis of sugars as both an energy source and a carbon source. It also possesses the ability to uptake DNA in its environment through natural transformation, making it a useful species for genetic manipulation (Zang, et al., 2007). PCC 6803 possesses many industrial applications including lipid recovery in biofuel production, PHB collection, and as a toxin biosensor (Wu, et al., 2001; Avramescu, et al., 1999).

However, despite the many potential uses and the promises of this bacterium in genetic research, new techniques for manipulating and studying bacterial genetics have not been adapted to this species. This project attempts the first step of the adaptations of the BioBrick biological part system to PCC 6803. This project seeks to create a BioBrick compatible integration vector for use in PCC 6803, since the species is more efficient at integrating genetic material into its chromosomes than maintaining separately replicating plasmids.

This project also seeks to study the behaviors and strengths of 15 promoters from PCC 6803 which potentially are induced in light, darkness, heat stress, or nitrogen stress. Promoter strength and activation will be assessed using GFP, which is already in BioBrick form (BBa_K208000) and has been used to test promoters in PCC 6803 in previous studies. Despite PCC 6803's natural green color, GFP expression is clearly distinguishable from cellular background by spectrofluorometry(Kunert, et al., 2000; Spence, et al., 2003). GFP was also chosen over CFP and RFP due to its excitation and emission wavelengths lying in a region where the photosynthetic system of PCC 6803 has reduced absorbance, which may improve accuracy of quantitative measurements by limiting interference (Figure 1) (Wilde, et al. 1995).

Figure 1. Absorbance spectrum of Synechocystis sp. PCC 6803 and the excitation and emission wavelengths of CFP, GFP, YFP, and RFP. Absorbance spectrum (black line) adaptated from Wilde, et al. (1995). Fluorescent proteins emission and excitation wavelengths shown as a single bar due to small relative distance between wavelengths, bars are color coded to the proteins.

The transcription of genes in PCC 6803 is controlled in part by a system of the nine currently known sigma factors (Table 1) (Imamura and Asayama, 2009). SigA controls the transcription of housekeeping genes, and its promoter will likely be a useful standard, as its concentration varies little over a range of temperatures, cell cycle phases, and stress conditions. SigE is upregulated by light and downregulated in the dark, potentially contributing to circadian rhythm-based gene transcription. The other sigma factors are detailed in Table 1 and are thoroughly reviewed in Imamura and Asayama (2009). These sigma factors act on three classes of promoter sequences. Type I promoters have the typical prokaryotic structure of -10 and -30 regions, and are generally activated by SigA. Type I promoters are potentially functional in other species of bacteria due to their similarity to sigma 70 promoters. Type II promoters have only the -10 region, but may include enhancer sequences for stress response. Type III promoters have -12 and -32 regions, are activated through SigF, and are transcriptionally independent of the other two types (Imamura and Asayama, 2009).

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-Synechocystis sp. PCC 6803 (hereafter referred to as PCC 6803) is a cyanobacterium, a photosynthetic bacterium, and one of the major model organisms in microbiology.  PCC 6803 has been extensively studied for it photosynthetic pathway and its genome has been completely sequenced. It is capable of acting as both an autotroph, obtaining its energy from light and its carbon from the atmosphere, and as a heterotrophy, using glycolysis of sugars as both an energy source and a carbon source. It also possesses the ability to uptake DNA in its environment through natural transformation, making it a useful species for genetic manipulation (Zang, et al., 2007). PCC 6803 possesses many industrial applications including lipid recovery in biofuel production, PHB collection, and as a toxin biosensor (Wu, et al., 2001; Avramescu, et al., 1999).
+Synechocystis sp. PCC 6803 (hereafter referred to as PCC 6803) is a cyanobacterium, a photosynthetic bacterium, and one of the major model organisms in microbiology.  PCC 6803 has been extensively studied for it photosynthetic pathway and its genome has been completely sequenced. It is capable of acting as both an autotroph, obtaining its energy from light and its carbon from the atmosphere, and as a heterotroph, using glycolysis of sugars as both an energy source and a carbon source. It also possesses the ability to uptake DNA in its environment through natural transformation, making it a useful species for genetic manipulation (Zang, et al., 2007). PCC 6803 possesses many industrial applications including lipid recovery in biofuel production, PHB collection, and as a toxin biosensor (Wu, et al., 2001; Avramescu, et al., 1999).
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-This project also seeks to study the behaviors and strengths of 15 promoters from PCC 6803 which potentially are induced in light, darkness, heat stress, or nitrogen stress. Promoter strength and activation will be assessed using GFP (mut3b), which is already in BioBrick form (BBa_E0040) and has been used to test promoters in PCC 6803 in previous studies and is clearly distinguishable by spectrofluorometer despite its natural green color (Kunert, et al., 2000; Spence, et al., 2003). GFP was also chosen over CFP and RFP due to its excitation and emission wavelengths lying in a region where the photosynthetic system of PCC 6803 has reduced absorbance, which may improve accuracy of quantitative measurements by limiting interference (Figure 1) (Wilde, et al. 1995).
+This project also seeks to study the behaviors and strengths of 15 promoters from PCC 6803 which potentially are induced in light, darkness, heat stress, or nitrogen stress. Promoter strength and activation will be assessed using GFP, which is already in BioBrick form (BBa_K208000) and has been used to test promoters in PCC 6803 in previous studies. Despite PCC 6803's natural green color, GFP expression is clearly distinguishable from cellular background by spectrofluorometry(Kunert, et al., 2000; Spence, et al., 2003). GFP was also chosen over CFP and RFP due to its excitation and emission wavelengths lying in a region where the photosynthetic system of PCC 6803 has reduced absorbance, which may improve accuracy of quantitative measurements by limiting interference (Figure 1) (Wilde, et al. 1995).
+<center><a href="https://static.igem.org/mediawiki/2010/c/cb/Construction_pic2.jpg"><img with="900" height="400" src="https://static.igem.org/mediawiki/2010/c/cb/Construction_pic2.jpg"></a></center>
+Figure 1. Absorbance spectrum of Synechocystis sp. PCC 6803 and the excitation and emission wavelengths of CFP, GFP, YFP, and RFP. Absorbance spectrum (black line) adaptated from Wilde, et al. (1995). Fluorescent proteins emission and excitation wavelengths shown as a single bar due to small relative distance between wavelengths, bars are color coded to the proteins.
 <p class="class">
-The transcription of genes in PCC 6803 is controlled in part by a system of nine currently known sigma factors (Table 1) (Imamura and Asayama, 2009). SigA controls the transcription of housekeeping genes, and its promoter will likely be a useful standard, as its concentration varies little over a range of temperatures, cell cycle phases, and stress conditions. SigE is upregulated by light and downregulated in the dark, potentially contributing to circadian rhythm-based gene transcription. The other sigma factors are detailed in Table 1 and are thoroughly reviewed in Imamura and Asayama (2009). These sigma factors act on three classes of promoter sequences. Type I promoters have the typical prokaryotic structure of -10 and -30 regions, and are generally activated by SigA. Type II promoters have only the -10 region, but may include enhancer sequences for stress response. Type III promoters have -12 and -32 regions, are activated through SigF, and are transcriptionally independent of the other two types (Imamura and Asayama, 2009).
+The transcription of genes in PCC 6803 is controlled in part by a system of the nine currently known sigma factors (Table 1) (Imamura and Asayama, 2009). SigA controls the transcription of housekeeping genes, and its promoter will likely be a useful standard, as its concentration varies little over a range of temperatures, cell cycle phases, and stress conditions. SigE is upregulated by light and downregulated in the dark, potentially contributing to circadian rhythm-based gene transcription. The other sigma factors are detailed in Table 1 and are thoroughly reviewed in Imamura and Asayama (2009). These sigma factors act on three classes of promoter sequences. Type I promoters have the typical prokaryotic structure of -10 and -30 regions, and are generally activated by SigA. Type I promoters are potentially functional in other species of bacteria due to their similarity to sigma 70 promoters. Type II promoters have only the -10 region, but may include enhancer sequences for stress response. Type III promoters have -12 and -32 regions, are activated through SigF, and are transcriptionally independent of the other two types (Imamura and Asayama, 2009).
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