Part:BBa_K1045003:Experience

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Applications of BBa_K1045003

The part BBa_K1045003 was used for in vivo and in vitro analysis of protein functionality. The sequence codes for an active adenylate cyclase with a high activity in vivo and a moderate activity in vitro in a reaction solution containing 10 mM MgCl2. The coding sequence was equipped with a 5'-Strep-tag for purification, crystallized and the defraction pattern determined.

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UNIQaa42b1202a6c78b8-partinfo-00000000-QINU UNIQaa42b1202a6c78b8-partinfo-00000001-QINU Part: BBa_K1045003

Diadenylate cyclase domain of Listeria monocytogenes DacA (Lmo2120)

The essential signaling molecule bis-(3‘,5‘)-cyclic dimeric adenosine monophosphate (c-di-AMP) was identified in a crystal structure of the DNA damage checkpoint protein DisA of Bacillus subtilius and Thermotoga maritima (Witte et al., 2008). c-di-AMP is structurally similar to the signaling molecule c-di-GMP but it seem to have a distinct physiological function. c-di-AMP was reported to play a crucial role in cell wall synthesis and spore formation in B. 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 (Yun Luo and Helmann, 2012; Mehne et al., 2013). The presence of proteins containing an adenylate cyclase domain (DAC) was confirmed for Gram-positive bacteria like Bacillus subtilis and important pathogens like Streptococcus pneumoniae, Staphylococcus aureus and Listeria monocytogenes (Corrigan and Gründling, 2013). In striking contrast to the severe effects that alterations in c-di-AMP homeostasis have on Gram-positive bacteria, Escherichia coli as a Gram-negative representative does not produce c-di-AMP, implying that this molecule is not essential in Gram-negatives. Here, we introduce a BioBrick with the coding sequence of the DacA cyclase domain of Listeria monocytogenes. Cloning of the full-length membrane-bound DacA protein failed in E. coli. Therefore, we decided to exclude the trans-membrane domains ending up with a coding sequence of 100 – 273 amino acids of DacA. Unsuitable restriction sites within the gene were removed without changing the amino acid sequence. The protein was expressed from the BioBrick equipped with a Strep-tag on the 5‘-end of the gene. The soluble protein localizes to the cytoplasm and can easily be extracted with standard protein purification methods.

The BioBrick is an active adenylate cyclase in vivo

To analyze the cyclase activity of the truncated DacA protein, we introduced the part BBa_K1045003 into the E. coli strain BL21 with an N-terminal Strep-Tag and under the control of a T7-promoter. To express the protein, an overnight culture was used to inoculate LB-medium containing the appropriate antibiotic. The culture was incubated at 37°C until it reached an OD600 of 0.5 – 0.7. Having induced the expression of the T7-polymerase by adding Isopropyl-β-D-thiogalactopyranosid (IPTG), the culture was then incubated for 3 h. In order to extract c-di-AMP from the cell, a defined volume of the culture was taken and the cells lysed by snap freezing and heating. The overexpression of part BBa_K1045003 was confirmed by SDS gel electrophoresis showing a thick overexpression band with a weight of about 20 kDa (Fig. 1A). The c-di-AMP determination using LC-MS/MS revealed the presence of c-di-AMP in the supernatant of lysed bacterial cells. The measured concentration was determined as ca. 60 µg/mg of the total protein extract (Fig. 1B). The empty vector control did not show a measurable amount of c-di-AMP. The ectopic expression of the L. monocytogenes DAC-domain in E. coli BL21 yielded an active enzyme. Thus, the part BBa_K1045003 codes for an active adenylate cyclase domain which catalyzes the condensation reaction of two molecules ATP to a single molecule c-di-AMP in vivo.