Difference between revisions of "Part:BBa K299806"
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==Experimental results== | ==Experimental results== | ||
| − | MinC was measured <i>in vivo</i>, in the expression system of <i>E. coli</i> BL21 RIL strain using the construct <partinfo>BBa_K299807</partinfo>. Construct <partinfo>BBa_K299808</partinfo> was created as a control to compare stronger <partinfo>BBa_B0034</partinfo> RBS, present in <partinfo>BBa_K299808</partinfo> with the weaker <partinfo>BBa_B0032</partinfo>, present in <partinfo>BBa_K299807</partinfo> | + | MinC was measured <i>in vivo</i>, in the expression system of <i>E. coli</i> BL21 RIL strain using the construct <partinfo>BBa_K299807</partinfo>. Construct <partinfo>BBa_K299808</partinfo> was created as a control to compare stronger <partinfo>BBa_B0034</partinfo> RBS, present in <partinfo>BBa_K299808</partinfo> with the weaker <partinfo>BBa_B0032</partinfo>, present in <partinfo>BBa_K299807</partinfo>, to see the effects of higher expression level (see our wiki for the [http://2010.igem.org/Team:Warsaw/Stage1/RBSMeas details about the RBSes]). |
<p>After IPTG induction start our static and dynamic measurements indicated that <partinfo>BBa_K299807</partinfo> inhibited <i>E. coli</i> growth at IPTG concentration of 2,5 μM after approx. 90 minutes. The part <partinfo>BBa_K299808</partinfo> was generating no transformants in BL21 strain in no IPTG induction conditions. Such result was probably due to leaky expression of MinC from T7 promoter. Presence of a strong <partinfo>BBa_B0034</partinfo> RBS resulted in constitutive growth inhibition. </p> | <p>After IPTG induction start our static and dynamic measurements indicated that <partinfo>BBa_K299807</partinfo> inhibited <i>E. coli</i> growth at IPTG concentration of 2,5 μM after approx. 90 minutes. The part <partinfo>BBa_K299808</partinfo> was generating no transformants in BL21 strain in no IPTG induction conditions. Such result was probably due to leaky expression of MinC from T7 promoter. Presence of a strong <partinfo>BBa_B0034</partinfo> RBS resulted in constitutive growth inhibition. </p> | ||
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Revision as of 23:02, 27 October 2010
This part is licensed under
Creative BioCommons
Contents
Authors
- Orginal idea to use MinC - Jarosław Pankowski
- Cloning and measurement experiments - Kuba Piątkowski and Jarosław Pankowski
- Text at parts.igem edited by: Milena Bażlekowa
MinC - theoretical bakground
Natural role
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 in vivo, in the expression system of E. coli BL21 RIL strain using the construct BBa_K299807. Construct BBa_K299808 was created as a control to compare stronger BBa_B0034 RBS, present in BBa_K299808 with the weaker BBa_B0032, present in BBa_K299807, to see the effects of higher expression level (see our wiki for the [http://2010.igem.org/Team:Warsaw/Stage1/RBSMeas details about the RBSes]).
After IPTG induction start our static and dynamic measurements indicated that BBa_K299807 inhibited E. coli growth at IPTG concentration of 2,5 μM after approx. 90 minutes. The part BBa_K299808 was generating no transformants in BL21 strain in no IPTG induction conditions. Such result was probably due to leaky expression of MinC from T7 promoter. Presence of a strong BBa_B0034 RBS resulted in constitutive growth inhibition.
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
Complete measurement results and comments are [http://2010.igem.org/Team:Warsaw/Stage2/Results here].
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 growth curve represents negative control E. coli BL21 RIL strain bearing pSB plasmid with MinC, without RBS and promoter, IPTG induced. The yellow growth curve represents E. coli BL21 RIL strain bearing pSB with BBa_K299807 without IPTG induction, our control for leaky expression. The pink growth 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).
Induced BL21 carrying BBa_K299807 stop growing before they reach the OD=0.4. MinC negative control without promoter grows normally. Uninduced BL21 carrying BBa_K299807 has a slighlty slower grow compared to the MinC negative control.
MinC in our induced construct slows the bacteria cell growth, leaky expression from the promoter isn't as significant as IPTG induction.
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
Cultures for stationary measurement were grown in identical conditions as described above, only with increasing concentrations of IPTG. Measurements were taken 3h following induction. Results are displayed 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 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]