Difference between revisions of "Part:BBa K299806"

Revision as of 16:19, 27 October 2010

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Authors

• Orginal idea to use MinC - Jarosław Pankowski
• Cloning and measurement experiments - Kuba Piątkowski and Jarosław Pankowski

MinC - theoretical bakground

MinC is the component of MinCDE system. Together with nucleoid occlusion it ensures that cell division will occur in the middle of a cell. Three proteins – MinC, MinD and MinE play different roles in preventing formation of division complex too close to cell poles [1]. MinC is directly responsible for stopping the early stage of division by inhibiting the polymerization of FtsZ protein monomers into a structure known as the Z-ring [8].

FtsZ is a tubuline homologue capable of creating chains, lariats and rings independently of other cellular factors [3]. The stability and decay of these structures is dependent on GTPase activity of FtsZ proteins [7]. At the beginning of cell division FtsZ monomers form the Z-ring to which other proteins responsible for the division are recruited. The presence of N-terminal domain of MinC prevents additional Z-rings from being established [9].

In bacterial cells MinC is recruited to membrane by the second protein of the system – MinD. This process involves C-terminal domain of MinC, which is also responsible for its oligomerisation. Recruitment to membrane is necessary for the inhibiting effect to occur at the physiological concentration of protein [4]. Finally the third protein - MinE - is responsible for keeping the MinCD complex from acting in the midcell region, thus enabling the formation of Z-ring in the proper location [5]. It was shown that mutants in either minC or minD divide at cell pole much more often than wild type, resulting in creation of nucleoid-free minicells. This mutations, however are not lethal because enough of the cells in population divide properly to sustain growth [6].

Natural occurrence:

Although MinCDE is mostly analysed in E.coli, it’s elements were found in many different bacteria groups including Proteobacteria, Deinococcus-Thermus and Firmicutes phyla [1]. In B. subtilis the MinE protein is replaced by DivIVa which locates itself at the cell poles and with help of MinJ binds MinCD complex, decreasing it’s concentration at midcell [2].

Application in synthetic biology:

When expressed at high level MinC is capable of preventing bacterial cell from dividing. As a result, of this the cell becomes filamentous. This effect requires only high level of MinC, not MinD. It has been proven that overexpression of C-terminal domain of MinC alone is sufficient to inhibit cell division [9].

Experimental results

MinC was measured using following constructs: BBa_K299807 and BBa_K299808. It inhibited E. coli growth at IPTG concentration of 2,5 uM after approx. 90 minutes. Complete measurement results are [http://2010.igem.org/Team:Warsaw/Stage2/Results here]

The efficiency of the BBa_K299807 and BBa_K299808 constructs was measured by following means:

* dynamic measurement of OD

* dynamic measurement of CFU (colony-forming units)

* stationary measurement of OD

* stationary measurement of CFU (colony-forming units)

Static performance of MinC

Colony forming units per militer (cfu/ml) calculation of E. coli BL21 RIL strain bearing BBa_K299807 part on pSB plasmid - functional description of BBa_K299807 represented by bacterial cell growth on agar plates due to different IPTG concentrations. Each data point on the blue curve represents single measurement of cfu/ml under certain IPTG concentration during the experiment.

Dynamic performance of MinC

Calculating the number of colony forming units on agar plate per mililiter (cfu/ml) – describing the function of BBa_K299807 part represented by E. coli BL21 RIL strain cell growth on agar plate due to IPTG induction time. The blue curve represents negative control BL21 carrying pSB plasmid with MinC without RBS and promoter, IPTG induced. The yellow curve represents pSB plasmid carrying BBa_K299807 without IPTG induction, our control for leaky expression. The pink curve curve represents pSB plasmid carrying BBa_K299807 induced by IPTG. IPTG induction started on the 30 minute of the experiment. The data points represent individual measurements (30 minutes intervals between measurements).

Optical density bacteria measurement (OD) – describing the function of BBa_K299807 part represented by bacterial cell growth concentraction due to IPTG induction time. The blue curve represents negative control BL21 carrying pSB plasmid with MinC without RBS and promoter, IPTG induced. The yellow curve represents pSB plasmid carrying BBa_K299807 without IPTG induction, our control for leaky expression. The pink curve curve represents pSB plasmid carrying BBa_K299807 induced by IPTG. IPTG induction started on the 30 minute of the experiment. The data points represent individual measurements (30 minutes intervals between measurements).

References:

1. “Themes and variations in prokaryotic cell division”, William Margolin, FEMS Microbiology Reviews 24 (2000) 531-548

2. “The MinCDJ System in Bacillus subtilis Prevents Minicell Formation by Promoting Divisome Disassembly”, Suey van Baarle and Marc Bramkami, PLoS One. 5 (2010)

3. “The bacterial cell division protein FtsZ assembles into cytoplasmic ring in fission yeast”, Ramanujam Srinivasan et al. Genes Dev. 22 (2008) 1741-1746

4. “The Switch I and II Regions of MinD Are Required for Binding and Activating MinC”, Huaijin Zhou and Joe Lutkenhaus, J Bacteriol. 186 (2004) 1546–1555.

5. “The MinE ring required for proper placement of the division site is a mobile structure that changes its cellular location during the Escherichia coli division cycle.”, Fu X et al. Proc. Natl. Acad. Sci. U S A 98. (2001)

6. “FtsZ ring cluster in min and partition mutants: Role of both the Min system and the nucleoid in regulation FtsZ ring location”, Yu et al. Mol. Microbiol. 32 (1999) 315-326

7. “FtsZ from Divergent Foreign Bacteria Can Function for Cell Division in Escherichia coli”, Masaki Osawa and Harold P. Erickson, J. Bacteriol. 188 (2006) 7132-7140

8. “FtsZ, a tubulin homologue in prokaryote division”, Harold P. Erickson.Trends. Cell Biol. 7 (1997) 362-367

9. “Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ”, Zonglin Hu and Joe Lutkenhaus, J. Bacteriol. 182 (2000) 3965-3971

Sequence and Features