Team:Goettingen/Team/DAC

From 2013.igem.org

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===DAC Team===
===DAC Team===
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In order to find new antibacterial compounds, we focused on the signaling molecule bis-(3’,5’)-cyclic dimeric adenosine monophosphate (c-di-AMP) as it has proven to be an essential second messenger in many pathogenic Gram-positive bacteria (Witte ''et al.'', 2008). It was reported to have a crucial functions in cell wall synthesis and spore formation in Bacillus subtilis (Oppenheimer-Shaanan ''et al.'', 2011; Mehne ''et al.'', 2013). Interestingly, both absence and excess of c-di-AMP have detrimental effects on cell growth and morphology (Luo and Helmann, 2012; Mehne et al, 2013).  
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<p>Another approach of our project is the determination of the 3D structure of diadenylate cyclase (DAC) from the human pathogenic bacterium <i>Listeria monocytogenes</i> by crystallography. Due to the fact that the DAC from the closely related bacterium <i>Bacillus subtilis</i> is difficult to purify and thus to crystallize, we have decided to crystallize the DAC protein from <i>Listeria</i>. In addition to this, we will try to crystallize the DACs from other Gram-positive bacteria like <i>Streptococcus</i> and <i>Staphylococcus</i> .</p>
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<p>Once having a 3D structure of a protein in hand potential inhibitor binding sites can be identified by computational modeling. Moreover, a functional DAC can be used to develop a powerful <i>in vitro</i> screening system in order to identify potent inhibitors of the enzyme. Promising inhibitors that interfere with DAC activity may serve the development of drugs with improved inhibitory efficiency.</p>
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Knowing this information it makes sense that we take a closer look at the enzyme, the diadenylate cyclase, which produces c-di-AMP.
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The cyclase domain is conserved among several Gram-positive bacteria like B. subtilis, Streptococcus pneumoniae, Staphylococcus aureus and Listeria monocytogenes (Corrigan and Gründling, 2013). So far, the diadenylate cyclase DisA from B. subtilis has been purified (Witte ''et al.'', 2008). However, the purification and crystallization of DisA comes along with some difficulties so we decided to concentrate on the diadenylate cyclase in ''L. monocytogenes'', DacA. As the cloning of the full-length (273 aa) membrane-bound DacA (Lmo2120) into Escherichia coli failed, we excluded the trans-membrane domains meaning we chopped off the first 100 amino acids. Nevertheless, the resulting truncated part still included the essential cyclase domain, and therefore represents one of our favorite BioBricks: [http://parts.igem.org/Part:BBa_K1045003 BBa_K1045003]!
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Conducting several experiments we proved that the truncated DacA protein ([http://parts.igem.org/Part:BBa_K1045003 BBa_K1045003]) was not only active in vivo, but also in vitro. Moreover, we were able to purify the diadenylate cyclase in large scale for determining its protein structure! One can now further search for chemical compounds that interfere with the activity of the cyclase by computational modeling.
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Following, the experiments will be explained in more detail. However, if you wish to get even more details, please visit the Parts Registry or our LabBook.
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The truncated DacA protein ([http://parts.igem.org/Part:BBa_K1045003 BBa_K1045003]) was extended with an N-terminal Step-tag allowing easy purification steps. Moreover, this construct was brought under the control of a T7-promoter enabling us to induce the expression by addition of Isopropyl--D-thiogalactopyranosid (IPTG). This was then cloned into the ''E. coli'' strain BL21. (The Gram-negative bacterium ''E. coli'' does not produce c-di-AMP and is not severely affected by the signaling molecule in contrast to Gram-positive bacteria.)
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In order to analyze the cyclase activity in vivo, the ''E. coli'' clones were induced to express the protein by the addition of IPTG. The cells were then lysed to extract c-di-AMP from the cells.
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By performing SDS gel electrophoresis it was nicely shown that the desired protein was highly expressed (Fig. 1). Furthermore, the presence of c-di-AMP in the supernatant of the lysed bacteria was confirmed using LC-MS/MS. Thus, one can conclude that our truncated DacA protein codes for an active adenylate cyclase domain ''in vivo''.
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Revision as of 10:26, 1 October 2013





The beast and its Achilles heel:

 A novel target to fight multi-resistant pathogenic bacteria



DAC Team

In order to find new antibacterial compounds, we focused on the signaling molecule bis-(3’,5’)-cyclic dimeric adenosine monophosphate (c-di-AMP) as it has proven to be an essential second messenger in many pathogenic Gram-positive bacteria (Witte et al., 2008). It was reported to have a crucial functions in cell wall synthesis and spore formation in Bacillus subtilis (Oppenheimer-Shaanan et al., 2011; Mehne et al., 2013). Interestingly, both absence and excess of c-di-AMP have detrimental effects on cell growth and morphology (Luo and Helmann, 2012; Mehne et al, 2013).

Knowing this information it makes sense that we take a closer look at the enzyme, the diadenylate cyclase, which produces c-di-AMP. The cyclase domain is conserved among several Gram-positive bacteria like B. subtilis, Streptococcus pneumoniae, Staphylococcus aureus and Listeria monocytogenes (Corrigan and Gründling, 2013). So far, the diadenylate cyclase DisA from B. subtilis has been purified (Witte et al., 2008). However, the purification and crystallization of DisA comes along with some difficulties so we decided to concentrate on the diadenylate cyclase in L. monocytogenes, DacA. As the cloning of the full-length (273 aa) membrane-bound DacA (Lmo2120) into Escherichia coli failed, we excluded the trans-membrane domains meaning we chopped off the first 100 amino acids. Nevertheless, the resulting truncated part still included the essential cyclase domain, and therefore represents one of our favorite BioBricks: [http://parts.igem.org/Part:BBa_K1045003 BBa_K1045003]!

Conducting several experiments we proved that the truncated DacA protein ([http://parts.igem.org/Part:BBa_K1045003 BBa_K1045003]) was not only active in vivo, but also in vitro. Moreover, we were able to purify the diadenylate cyclase in large scale for determining its protein structure! One can now further search for chemical compounds that interfere with the activity of the cyclase by computational modeling.


Following, the experiments will be explained in more detail. However, if you wish to get even more details, please visit the Parts Registry or our LabBook.

The truncated DacA protein ([http://parts.igem.org/Part:BBa_K1045003 BBa_K1045003]) was extended with an N-terminal Step-tag allowing easy purification steps. Moreover, this construct was brought under the control of a T7-promoter enabling us to induce the expression by addition of Isopropyl--D-thiogalactopyranosid (IPTG). This was then cloned into the E. coli strain BL21. (The Gram-negative bacterium E. coli does not produce c-di-AMP and is not severely affected by the signaling molecule in contrast to Gram-positive bacteria.)

In order to analyze the cyclase activity in vivo, the E. coli clones were induced to express the protein by the addition of IPTG. The cells were then lysed to extract c-di-AMP from the cells.

By performing SDS gel electrophoresis it was nicely shown that the desired protein was highly expressed (Fig. 1). Furthermore, the presence of c-di-AMP in the supernatant of the lysed bacteria was confirmed using LC-MS/MS. Thus, one can conclude that our truncated DacA protein codes for an active adenylate cyclase domain in vivo.

 

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