Difference between revisions of "Part:BBa K299806"
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==Experimental results== | ==Experimental results== | ||
| − | MinC was measured using following | + | MinC was measured using following construct: <partinfo>BBa_K299807</partinfo>. Part <partinfo>BBa_K299808</partinfo> was created as a control to compare stronger B0034 <partinfo>BBa_B0034</partinfo> RBS present in this construct with the weaker <partinfo>BBa_B0032</partinfo> B0032 present in the <partinfo>BBa_K299807</partinfo> construct. During the experiment, part <partinfo>BBa_K299807</partinfo> inhibited <i>E. coli</i> growth at IPTG concentration of 2,5 μM after approx. 90 minutes. Complete measurement results are [http://2010.igem.org/Team:Warsaw/Stage2/Results here]. The part <partinfo>BBa_K299808</partinfo> showed overexpression during the experiment - no colonies were obtained, even without IPTG induction. |
<p>The efficiency of the <partinfo>BBa_K299807</partinfo> and <partinfo>BBa_K299808</partinfo> constructs was measured by following means:</p> | <p>The efficiency of the <partinfo>BBa_K299807</partinfo> and <partinfo>BBa_K299808</partinfo> constructs was measured by following means:</p> | ||
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<p>* stationary measurement of OD</p> | <p>* stationary measurement of OD</p> | ||
<p>* stationary measurement of CFU (colony-forming units)</p> | <p>* stationary measurement of CFU (colony-forming units)</p> | ||
| + | <br> | ||
| + | <div>For the measurement BL21 RIL strain of <i>E. coli</i> (<i>F− ompT hsdS(rB −mB −) dcm +Ter<sup>r</sup> gal λ (DE3) endA Hte (argU ileY leuW Cam<sup>r</sup></i>)) was transformed with the following plasmids: | ||
| + | <ul> | ||
| + | <li>pSB1A2 containing MinC under T7 promoter and B0032 RBS (<partinfo>BBa_K299807</partinfo> part) | ||
| + | <li>pSB1A2 containing MinC without a promoter or RBS</li> | ||
| + | </ul> | ||
| + | <br> | ||
| − | |||
| − | |||
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| − | |||
| − | |||
| − | |||
<h3>Dynamic performance of MinC</h3> | <h3>Dynamic performance of MinC</h3> | ||
| + | <p align="justify">Approximately 100 μl of over-night culture were used to inoculate 50 ml of LB medium with ampicilin and chloramphenicol (volume of inoculate was adjusted to ensure equal initial OD values). One flask was inoculated with pSB-MinC and two more with pSB-pT7-B0032-MinC. Cultures were incubated in 37 <sup>o</sup>C (with shaking) for 30 min. Following this initial incubation, OD was measured and a small volume of culture was plated (on LA medium with amp and cm) for CFU measurement. pSB-MinC and one of the pSB-pT7-B0032-MinC cultures were induced by addition of IPTG to the final concentration of 10 μM. Cultures were again placed on a shaker at 37 <sup>o</sup>C. Samples for OD and CFU measurements were then taken every 30 min until the OD began to exceed 1,5. | ||
| + | Results of the measurements are shown on the plots below:</p> | ||
| + | |||
https://static.igem.org/mediawiki/2010/a/a7/Od1.jpg | https://static.igem.org/mediawiki/2010/a/a7/Od1.jpg | ||
<p> Optical density measurement (OD) of <i>E. coli</i> BL21 RIL strain bearing <partinfo>BBa_K299807</partinfo> part – describing the function of <partinfo>BBa_K299807</partinfo> represented by bacterial culture concentraction in the inoculate due to IPTG induction time. | <p> Optical density measurement (OD) of <i>E. coli</i> BL21 RIL strain bearing <partinfo>BBa_K299807</partinfo> part – describing the function of <partinfo>BBa_K299807</partinfo> represented by bacterial culture concentraction in the inoculate due to IPTG induction time. | ||
| − | The blue curve represents negative control <i>E. coli</i> BL21 RIL strain bearing pSB plasmid with MinC | + | The blue curve represents negative control <i>E. coli</i> BL21 RIL strain bearing pSB plasmid with MinC, without RBS and promoter, IPTG induced. The yellow curve represents <i>E. coli</i> BL21 RIL strain bearing pSB with <partinfo>BBa_K299807</partinfo> without IPTG induction, our control for leaky expression. The pink curve curve represents <i>E. coli</i> BL21 RIL strain bearing pSB plasmid with <partinfo>BBa_K299807</partinfo> induced by IPTG. IPTG induction started on the 30 minute of the experiment. Each data point represent individual measurement (30 minutes intervals between measurements). </p> |
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<p> Calculating the number of colony forming units on agar plate per mililiter (cfu/ml) – describing the function of <partinfo>BBa_K299807</partinfo> represented by <i>E. coli</i> BL21 RIL strain cell growth on agar plate due to IPTG induction time. | <p> Calculating the number of colony forming units on agar plate per mililiter (cfu/ml) – describing the function of <partinfo>BBa_K299807</partinfo> represented by <i>E. coli</i> 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 <partinfo>BBa_K299807</partinfo> without IPTG induction, our control for leaky expression. The pink curve curve represents pSB plasmid carrying <partinfo>BBa_K299807</partinfo> induced by IPTG. IPTG induction started on the 30 minute of the experiment. The data points represent individual measurements (30 minutes intervals between measurements). </p> | 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 <partinfo>BBa_K299807</partinfo> without IPTG induction, our control for leaky expression. The pink curve curve represents pSB plasmid carrying <partinfo>BBa_K299807</partinfo> induced by IPTG. IPTG induction started on the 30 minute of the experiment. The data points represent individual measurements (30 minutes intervals between measurements). </p> | ||
| + | |||
| + | <h3>Static performance of MinC</h3> | ||
| + | |||
| + | https://static.igem.org/mediawiki/2010/5/5f/OD2.jpg | ||
| + | |||
| + | <p>Optical density measurement (OD) of <i>E. coli</i> BL21 RIL strain bearing <partinfo>BBa_K299807</partinfo> part – describing the function of <partinfo>BBa_K299807</partinfo> represented by bacterial culture concentration in the inoculate due to IPTG concentration. Each data point on the blue curve represents single OD measurement of <i>E. coli</i> culture under certain IPTG concentration.</p> | ||
| + | |||
| + | https://static.igem.org/mediawiki/2010/4/45/CFU2.jpg | ||
| + | <p>Colony forming units per militer (cfu/ml) calculation of <i>E. coli</i> BL21 RIL strain bearing <partinfo>BBa_K299807</partinfo> part - functional description of <partinfo>BBa_K299807</partinfo> represented by bacterial cell growth on agar plates due to IPTG concentration. Each data point on the blue curve represents single measurement of cfu/ml under certain IPTG concentration.</p> | ||
<h3>References:</h3> | <h3>References:</h3> | ||
Revision as of 19:26, 27 October 2010
This part is licensed under
Creative BioCommons
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 construct: BBa_K299807. Part BBa_K299808 was created as a control to compare stronger B0034 BBa_B0034 RBS present in this construct with the weaker BBa_B0032 B0032 present in the BBa_K299807 construct. During the experiment, part BBa_K299807 inhibited E. coli growth at IPTG concentration of 2,5 μM after approx. 90 minutes. Complete measurement results are [http://2010.igem.org/Team:Warsaw/Stage2/Results here]. The part BBa_K299808 showed overexpression during the experiment - no colonies were obtained, even without IPTG induction.
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)
- pSB1A2 containing MinC under T7 promoter and B0032 RBS (BBa_K299807 part)
- pSB1A2 containing MinC without a promoter or RBS
Dynamic performance of MinC
Approximately 100 μl of over-night culture were used to inoculate 50 ml of LB medium with ampicilin and chloramphenicol (volume of inoculate was adjusted to ensure equal initial OD values). One flask was inoculated with pSB-MinC and two more with pSB-pT7-B0032-MinC. Cultures were incubated in 37 oC (with shaking) for 30 min. Following this initial incubation, OD was measured and a small volume of culture was plated (on LA medium with amp and cm) for CFU measurement. pSB-MinC and one of the pSB-pT7-B0032-MinC cultures were induced by addition of IPTG to the final concentration of 10 μM. Cultures were again placed on a shaker at 37 oC. Samples for OD and CFU measurements were then taken every 30 min until the OD began to exceed 1,5. Results of the measurements are shown on the plots below:
Optical density measurement (OD) of E. coli BL21 RIL strain bearing BBa_K299807 part – describing the function of BBa_K299807 represented by bacterial culture concentraction in the inoculate due to IPTG induction time. The blue curve represents negative control E. coli BL21 RIL strain bearing pSB plasmid with MinC, without RBS and promoter, IPTG induced. The yellow curve represents E. coli BL21 RIL strain bearing pSB with BBa_K299807 without IPTG induction, our control for leaky expression. The pink curve curve represents E. coli BL21 RIL strain bearing pSB plasmid with BBa_K299807 induced by IPTG. IPTG induction started on the 30 minute of the experiment. Each data point represent individual measurement (30 minutes intervals between measurements).
Calculating the number of colony forming units on agar plate per mililiter (cfu/ml) – describing the function of BBa_K299807 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).
Static performance of MinC
Optical density measurement (OD) of E. coli BL21 RIL strain bearing BBa_K299807 part – describing the function of BBa_K299807 represented by bacterial culture concentration in the inoculate due to IPTG concentration. Each data point on the blue curve represents single OD measurement of E. coli culture under certain IPTG concentration.
Colony forming units per militer (cfu/ml) calculation of E. coli BL21 RIL strain bearing BBa_K299807 part - functional description of BBa_K299807 represented by bacterial cell growth on agar plates due to IPTG concentration. Each data point on the blue curve represents single measurement of cfu/ml under certain IPTG concentration.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 652
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 652
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 652
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 652
Illegal AgeI site found at 211 - 1000COMPATIBLE WITH RFC[1000]