Team:HKUST-Hong Kong/asm.html
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+ | <TH BGCOLOR="#A1C6B2"> | ||
+ | <a name=top></a> | ||
+ | <a href=#method>How to Select</a> · | ||
+ | <a href=#assembly>Methods of Assembly </a> · | ||
+ | <a href=#component>Details of Components</a> · | ||
+ | <a href=#refer>References</a> | ||
+ | <p> | ||
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<font color=black> | <font color=black> | ||
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+ | <a href=#method><img src="https://static.igem.org/mediawiki/2011/a/ac/Ust_select.gif" width=100 height=100 alt="How to select against E. CRAFT cells that fail to take up the vector plasmid - our alternative selection method"></a> | ||
+ | <a href=#assembly><img src="https://static.igem.org/mediawiki/2011/e/e4/Ust_assembly.gif" width=100 alt="Stepping into the heart of construction - methods of assembly" height=100></a> | ||
+ | <a href=#component> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/7/71/Ust_details.gif" width=100 height=100 alt="Details of the components – a closer look to the molecular basis of assembly "></a> | ||
+ | <a href=#refer> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/5/54/Ust_refer.gif" width=100 height=100 alt="References"></a> | ||
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+ | <b><font size=14>Strain Construction</font></b><hr> | ||
+ | <br> | ||
<p> | <p> | ||
- | < | + | <b><a name=method></a>1. How to select against E. CRAFT cells that fail to take up the vector plasmid - our alternative selection method</b> |
</p> | </p> | ||
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+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
+ | <center><img src=https://static.igem.org/mediawiki/2011/4/4a/Ust_Shuffle_pc.jpg width=800></center><br><br> | ||
+ | </p> | ||
<p> | <p> | ||
- | < | + | <b><a name=assembly></a>2. Stepping into the heart of construction - methods of assembly</b> |
</p> | </p> | ||
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- | <center><img src=https://static.igem.org/mediawiki/2011/3/32/Ust_ML_of_OR.png width= | + | <center><img src=https://static.igem.org/mediawiki/2011/3/32/Ust_ML_of_OR.png width=660></center> |
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i. G is placed on the pDummy, which lacks a selection marker but is equipped with a normal replication origin<br> | i. G is placed on the pDummy, which lacks a selection marker but is equipped with a normal replication origin<br> | ||
ii. OR is the sole origin of replication of another plasmid (here we introduce a new plasmid, pToolkit) with a selection marker<br> | ii. OR is the sole origin of replication of another plasmid (here we introduce a new plasmid, pToolkit) with a selection marker<br> | ||
- | iii. pDummy and pToolkit are co-transformed to a bacterium which is under selection stress. | + | iii. pDummy and pToolkit are co-transformed to a bacterium which is under selection stress.<a href=#top>[Top]</a> |
<br><br> | <br><br> | ||
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<b>1. Only pDummy is uptaken</b><br> | <b>1. Only pDummy is uptaken</b><br> | ||
Since pDummy has no selection marker, the host bacterium would die under selection pressure and fail to propagate. | Since pDummy has no selection marker, the host bacterium would die under selection pressure and fail to propagate. | ||
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+ | </p> | ||
+ | |||
+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
+ | <center><img src=https://static.igem.org/mediawiki/2011/c/c4/Ust_Have_pd_only.png width=770></center> | ||
+ | </p> | ||
+ | <br> | ||
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+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
<b>2. Only pToolkit is uptaken</b><br> | <b>2. Only pToolkit is uptaken</b><br> | ||
The host bacterium that uptakes pToolkit survives. During its propagation, however, the pToolkit is not replicated because protein products of G are absent. Therefore, daughter cells of the host bacterium would not receive copies of the pToolkit and die under selection pressure. | The host bacterium that uptakes pToolkit survives. During its propagation, however, the pToolkit is not replicated because protein products of G are absent. Therefore, daughter cells of the host bacterium would not receive copies of the pToolkit and die under selection pressure. | ||
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+ | </p> | ||
+ | |||
+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
+ | <center><img src=https://static.igem.org/mediawiki/2011/1/1a/Ust_Have_pt_only.png width=810></center> | ||
+ | </p> | ||
+ | <br> | ||
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+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
<b>3. Both pDummy and pToolkit are uptaken</b><br> | <b>3. Both pDummy and pToolkit are uptaken</b><br> | ||
In the presence of pDummy, pToolkit would be maintained and confer resistance to selection pressure on the host bacterium. Daughter cells that receive copies of both plasmids would survive and eventually form a colony. | In the presence of pDummy, pToolkit would be maintained and confer resistance to selection pressure on the host bacterium. Daughter cells that receive copies of both plasmids would survive and eventually form a colony. | ||
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</p> | </p> | ||
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+ | <center><img src=https://static.igem.org/mediawiki/2011/6/68/Ust_Have_both.png width=770></center> | ||
+ | </p> | ||
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This in turn will activate <i>E. coli</i>’s alternative DNA repair system, RecFOR Recombination Pathway, whereby RecFOR will recruit RecA on to the ssDNA-dsDNA complex (at the homology sites). As the complementation of the linear Transformation and genomic DNA will result in a Holliday junction, RuvABC will be recruited to “resolve” this junction, cleaving away the target DNA on the bacterial genome and integrating the Transformation DNA into the genome. (It should be noted that RecA and RuvABC are also a shared downstream pathway for the RecBCD Recombination Pathway). | This in turn will activate <i>E. coli</i>’s alternative DNA repair system, RecFOR Recombination Pathway, whereby RecFOR will recruit RecA on to the ssDNA-dsDNA complex (at the homology sites). As the complementation of the linear Transformation and genomic DNA will result in a Holliday junction, RuvABC will be recruited to “resolve” this junction, cleaving away the target DNA on the bacterial genome and integrating the Transformation DNA into the genome. (It should be noted that RecA and RuvABC are also a shared downstream pathway for the RecBCD Recombination Pathway). | ||
- | <br>< | + | <br> |
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+ | </p> | ||
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+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
+ | <center><img src=https://static.igem.org/mediawiki/2011/a/a4/Ust_Recombination.png width=600></center> | ||
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+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
The λ RED recombination cassette is located in our third plasmid “Toolkit”. Upon successful co-transformation of pDummy and pToolkit, loss of genomic essential gene can be stimulated by introducing- into the bacterial cell- linear dsDNA molecules carrying a reporter gene flanked by sequences homologous to those of the essential gene. An expected outcome of this introduction is the swapping out of the <i>nadE</i> gene with the reporter gene. | The λ RED recombination cassette is located in our third plasmid “Toolkit”. Upon successful co-transformation of pDummy and pToolkit, loss of genomic essential gene can be stimulated by introducing- into the bacterial cell- linear dsDNA molecules carrying a reporter gene flanked by sequences homologous to those of the essential gene. An expected outcome of this introduction is the swapping out of the <i>nadE</i> gene with the reporter gene. | ||
- | <br><br> | + | <br> |
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+ | </p> | ||
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+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
+ | <center><img src=https://static.igem.org/mediawiki/2011/c/c7/Ust_Trans_dsDNA.png width=780></center> | ||
+ | </p> | ||
+ | <br> | ||
+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
Since the linear dsDNAs do not have origin of replications, they would not be inherited in daughters unless the swapping has taken place properly. Thus any observable signals from the reporter would allow identification of successful recombination. Once the recombination is completed, the toolkit plasmid and the cell’s antibiotic resistance gene can be eliminated from the host bacterium, giving us the completed strain of E. CRAFT. | Since the linear dsDNAs do not have origin of replications, they would not be inherited in daughters unless the swapping has taken place properly. Thus any observable signals from the reporter would allow identification of successful recombination. Once the recombination is completed, the toolkit plasmid and the cell’s antibiotic resistance gene can be eliminated from the host bacterium, giving us the completed strain of E. CRAFT. | ||
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+ | </p> | ||
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+ | <center><img src=https://static.igem.org/mediawiki/2011/6/6a/Ust_KO_pt.png width=810></center> | ||
+ | </p> | ||
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<b>2.3 Complementation between reporter genes – manifesting completion of E. CRAFT engineering</b> | <b>2.3 Complementation between reporter genes – manifesting completion of E. CRAFT engineering</b> | ||
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<br> | <br> | ||
- | (1) successfully had its essential | + | (1) successfully had its essential nadE gene deleted from the genome; |
<br> | <br> | ||
- | (2) maintained the pDummy, a complementation reporter system between the pDummy and swapped gene is preferred over a single reporter at the swap site. | + | (2) maintained the pDummy, |
+ | <br> | ||
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+ | a complementation reporter system between the pDummy and the swapped gene is preferred over a single reporter at the swap site. | ||
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</p> | </p> | ||
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+ | <center><img src=https://static.igem.org/mediawiki/2011/6/6c/Ust_Reporter.jpg width=700></center> | ||
+ | </p> | ||
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<p align=justify style="margin: 20px 20px 20px 20px"> | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
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<p > | <p > | ||
- | < | + | <b><a name=component></a>3. Details of the components – a closer look to the molecular basis of assembly</b> |
</p> | </p> | ||
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This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by nucleotide mutation. | This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by nucleotide mutation. | ||
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+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
+ | <center><img src=https://static.igem.org/mediawiki/2011/b/b2/Ust_BBa_K524000.png width=640></center> | ||
</p> | </p> | ||
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</p> | </p> | ||
+ | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
+ | <center><img src=https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png width=530> | ||
+ | <br> | ||
+ | <img src=https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png width=510></center> | ||
+ | </p> | ||
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</p> | </p> | ||
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+ | <center><img src=https://static.igem.org/mediawiki/2011/2/2a/Ust_BBa_K524003.png width=600></center> | ||
+ | </p> | ||
<p align=justify style="margin: 20px 20px 20px 20px"> | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
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<b>3.4 π replication initiator protein encoded by pir gene (BBa_K524004) and γ-origin of replication (ori-γ) from R6K plasmid</b><br> | <b>3.4 π replication initiator protein encoded by pir gene (BBa_K524004) and γ-origin of replication (ori-γ) from R6K plasmid</b><br> | ||
- | Ori-γ is one of the three replication origins (the other two being α and | + | Ori-γ is one of the three replication origins (the other two being α and β) of the R6K origin. Initiation of replication at ori-γ is regulated in trans by the π protein encoded by pir gene. [4, 5] While the presence of the appropriate amount of π protein is required for replication initiation, doubling the concentration of the same protein would effectively shut down the process. [8] Expression of π protein is autogenously regulated. [7] |
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</p> | </p> | ||
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+ | <center><img src=https://static.igem.org/mediawiki/2011/e/e3/Ust_BBa_K524004.png width=450></center> | ||
+ | </p> | ||
<p align=justify style="margin: 20px 20px 20px 20px"> | <p align=justify style="margin: 20px 20px 20px 20px"> | ||
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<p > | <p > | ||
- | < | + | <b><a name=refer></a>4. References</b> |
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+ | <table border="0" cellspacing="0" cellpadding="10"> | ||
+ | <tr> | ||
+ | <td width="100px" height="150px"; bgcolor="#980000" > | ||
+ | <p align="center"> | ||
- | </ | + | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong" target=_top> |
- | + | <b><font color="#FFE1E1" size=3>Home</font></b> | |
+ | </p> | ||
+ | </td> | ||
+ | <td width="382px" bgcolor="#CCFF99" valign="baseline"> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <b><font color="green">Our Project</font></b></p> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/overview.html" target=_top>Overview</a><font color="green"> | </font> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/data.html" target=_top>Data Page</a><br></p> | ||
- | </ | + | <p align="center" valign="baseline"> |
+ | <b><font color="green">Experiments and Results</font></b></p> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/asm.html" target=_top>Strain Construction</a><font color="green"> | </font> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/mic.html" target=_top>Culture Tests</a><font color="green"> | </font> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/modeling.html" target=_top>Modeling</a><br></p> | ||
- | < | + | <p align="center" valign="baseline"> |
+ | <b><font color="green">Miscellaneous</font></b></p> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/notebook.html" target=_top>Notebook</a></p> | ||
+ | </td> | ||
+ | <td width="302px" bgcolor="#D09C00" valign="baseline"> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <b><font color="#FFF4D0">iGEM Resources</font></b></p> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/acknowledgement.html" target=_top>Acknowledgements</a></p> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <b><font color="#FFF4D0">The Team</font></b></p> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/team.html" target=_top>iGEM Member List</a><font color="#FFF4D0"> | </font> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/contribution.html" target=_top>Contributions</a><br></p> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <b><font color="#FFF4D0">Achievements</font></b></p> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/medal.html" target=_top>Medal Requirements</a><font color="#FFF4D0"> | </font> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/biosafety.html" target=_top>BioSafety</a><br></p> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <b><font color="#FFF4D0">BioBricks</font></b></p> | ||
+ | <p align="center" valign="baseline"> | ||
+ | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/characterization.html" target=_top>Master List & Characterization Data</a><br></p> | ||
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- | < | + | <td width="180px"bgcolor="#980000"valign="baseline"> |
- | < | + | <p align="center" valign="baseline"> |
- | < | + | <b><font color="#FFE0E0">Human Practice</font></b></p> |
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- | < | + | <p align="center" valign="baseline"> |
- | < | + | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/workshop.html" target=_top>Workshop</a><font color="white"> | </font> |
- | + | <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/survey.html" target=_top>Survey</a><br></p> | |
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Latest revision as of 18:57, 28 October 2011
How to Select ·
Methods of Assembly ·
Details of Components ·
References
1. How to select against E. CRAFT cells that fail to take up the vector plasmid - our alternative selection method
Our E. CRAFT (Escherichia coli Re-engineered for Antibiotics-Free Transformation) is designed to have one of its essential genes (genes that are required for viability) removed from its genome, and relocated into an engineered plasmid “Dummy”. This would result in E. CRAFT’s dependency on this extra- chromosomal copy of the essential gene for survival, and hence the addiction to the pDummy. By having direct control over the replication of pDummy, we dictate the life and death of E. CRAFT (and hence the name pDummy).
This analog plasmid, named “pCarrier”, is essentially our E. CRAFT- compatible vector in cloning. Under an unfavorably high incubation temperature, only E. CRAFT cells that are transformed with the insert-bearing pCarrier would be able to propagate and survive. The remaining E. CRAFT cells would not be able to undergo division and would eventually be eliminated from the population. In this sense, the pDummy can be considered to be "shuffled out" by pCarrier. Our designed selection system, in short, bases itself on plasmid shuffling, with no involvement of antibiotic resistance genes in any cloning step.[Top]
2. Stepping into the heart of construction - methods of assembly
2.1 Construction and maintenance of an antibiotic-resistance-gene-free plasmid through antibiotic selection – the unavoidable evil two plasmid system
The solution to this problem is to develop mutuality between pDummy and another plasmid by exploiting the nature of positively- regulated origins of replication. Well studied examples of such origins include those of pSC101 [2] and R6K plasmids [4, 5, 7, 8], where the origins of replication (OR) appear together with a constitutive gene (G). Initiation of replication happens if and only if the trans- element of the gene is provided.
Let’s consider the following scenario:
Three possible outcomes could be expected:
2. Only pToolkit is uptaken
3. Both pDummy and pToolkit are uptaken
Owing to this mutualistic relation, retention of the desired pDummy would be possible once the host bacterium develops an addiction it, while pToolkit can be lost in bacterial propagation if the expression of G can be shut off manually. Eventually, the bacteria would not obtain any new antibiotic resistance genes but keep pDummy.
2.2 Development of addiction – use of the λ RED recombination system [1]
The λ RED recombination cassette is located in our third plasmid “Toolkit”. Upon successful co-transformation of pDummy and pToolkit, loss of genomic essential gene can be stimulated by introducing- into the bacterial cell- linear dsDNA molecules carrying a reporter gene flanked by sequences homologous to those of the essential gene. An expected outcome of this introduction is the swapping out of the nadE gene with the reporter gene.
Since the linear dsDNAs do not have origin of replications, they would not be inherited in daughters unless the swapping has taken place properly. Thus any observable signals from the reporter would allow identification of successful recombination. Once the recombination is completed, the toolkit plasmid and the cell’s antibiotic resistance gene can be eliminated from the host bacterium, giving us the completed strain of E. CRAFT.
2.3 Complementation between reporter genes – manifesting completion of E. CRAFT engineering
2.4 Summary of construction flow: 3. Details of the components – a closer look to the molecular basis of assembly
3.1 Temperature-sensitive origin of replication_oriR101 & repA101-ts (BBa_K524000)
3.2 split superfolder green fluroscent protein_split sfGFP
3.3 Essential gene nadE (BBa_K524003)
3.4 π replication initiator protein encoded by pir gene (BBa_K524004) and γ-origin of replication (ori-γ) from R6K plasmid
3.5 iGEM 2010 Slovenia Split/FRET constructs
1. Datsenko KA, Wanner BL.(2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5.
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Our Project Experiments and Results
Strain Construction |
Culture Tests |
Modeling Miscellaneous |
iGEM Resources The Team
iGEM Member List |
Contributions Achievements
Medal Requirements |
BioSafety BioBricks |
Human Practice |