Team:HKUST-Hong Kong/asm.html

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<title>Strain Construction</title>
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<p><strong>STRAIN  CONSTRUCTION</strong></p>
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<p><strong>1. Constructing EX – the bacterial strain that  allows selection without use of antibiotics</strong></p>
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<table align=top style="border-collapse: collapse">
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<p>To study the population dynamics and behavior of a certain antibiotics sensitive strain  of E Coli in a medium of antibiotic, our E. Trojan that is introduced into the culture medium must not process a wide spectrum of antibiotic resistance that impose  a selective advantage. At the same time, E. Trojan needs to be transformed with  the T4MO gene to carry out its job of signal disruption. </p>
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<TR>
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<p>Summarizing  the above criteria, a solution where the bacteria can be transform with the gene  of interest while remaining sensitive to antibiotics is needed. Therefore the requisite  is to construct a new bacterial strain that can perform plasmid selection without  the use of antibiotics, and contains as little antibiotics resistance gene as  possible.</p>
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<TH BGCOLOR="#A1C6B2">
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<p>With  advancements in molecular cloning, nowadays different selection methods are  available for different purposes. Application of antibiotics as the external  selection pressure remains the most common and routine method, but it might interfere  with the results in our study of relationship between MICs and indole  degradation. Selection systems that generate internal stresses, such as those  that base on auxotrophy and toxin-antitoxins interactions, are also available. Yet,  nutrient statues and toxicity suppression might introduce more variables. Thus,  we propose the idea of constructing a new strain of bacteria EX, that perform  selection from internal pressure on holding an essential gene. Our EX might  eliminate use of antibiotics in cloning, simplifies transformation and hopefully  proof itself useful to the versatile field of selection methods.</p>
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<a name=top></a>
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<p><strong>2. How to select against EX without the vector plasmid? Our alternative selection method</strong></p>
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<a href=#method>How to Select</a> ·
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<p><img src="Eveything you may need 03_10_11/exported diagrams/final product.png" width="695" height="395" border="0" /></p>
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<a href=#assembly>Methods of Assembly </a> ·
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<p>Our EX  will have one of its essential genes (genes that are required for viability) removed from its genome, and relocated onto an engineered plasmid pDummy. As  illustrated, in order to survive, EX must rely on those extra-chromosomal copies  of the essential gene; therefore, EX is addicted to pDummy. By having direct control over the replication of pDummy, we dictate the life and death of EX (and hence the name pDummy).</p>
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<a href=#component>Details of Components</a> ·
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<p>Here, we introduce a heat-sensitive origin of replication as the only origin of pDummy. When we intend to switch off the replication of pDummy, we can incubate EX at above 30 degree Celsius. This origin would then cease to function, and pDummy cannot be maintained. Deprived of the essential gene and the corresponding vital product, EX cannot propagate, unless, it receives an alternative but heat insensitive analog of pDummy. </p>
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<a href=#refer>References</a>
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<p>This analog, named pCarrier, is the essentially our vector in cloning. Under an unfavorably high temperature, only those EX that are transformed with the insert-bearing pCarrier will be able to propagate and survive, while the others cannot undergo division and are virtually eliminated from the population. Eventually, the pDummy can be considered to be &quot;shuffled out&quot; by pCarrier. Our designed selection system, in short, bases itself on plasmid shuffling, and thus eliminates involvement of antibiotic resistance genes in any of the cloning steps.</p>
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<p>
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<p><strong>3. Stepping in the heart of construction - methods of assembly </strong></p>
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<br>
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<p><strong>3.1 Construction and maintenance of an antibiotic-resistance-gene-free plasmid through antibiotic selection – the unavoidable evil two plasmid system</strong></p>
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-
<p>Our ultimate goal is to construct our EX without conferring it any new antibiotic resistance. For this reason no resistance gene should be found in our dummy plasmid pDummy. </p>
+
 
-
<p>Yet, such a plasmid would not be maintained by itself unless the host bacterium develops an  addiction to it (i.e. losses the essential gene in its genome and depends on extra-chromosomal copies on pDummy), and inconveniently, the addiction can only be achieved after the introduction of the plasmid.</p>
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<font color=black>
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<p><img src="Eveything you may need 03_10_11/exported diagrams/post-swap pt.png" width="722" height="197" /></p>
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<p>The solution is to develop a mutualistic relation between two plasmids and we  planned to exploit positively regulated origin of replications. </p>
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-
<p>Well studied examples are those in pSC101 and R6K origins of replication, 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.</p>
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-
<p><img src="Eveything you may need 03_10_11/exported diagrams/ML of OR.png" width="791" height="445" /></p>
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<p>Let’s consider the following scenario: <br />
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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>
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   i. G is placed on pDummy with no selection marker but with a normal replication origin</p>
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<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>
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<p><br />
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<a href=#component>
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  ii. OR is the sole origin of replication of another plasmid (here we introduce a new plasmid pToolkit) with a selection marker<br />
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<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>
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  iii. pDummy and pToolkit are co-transformed to a bacterium which is under selection stress</p>
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<a href=#refer>
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<p>We would  obtain three possible outcomes:</p>
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<img src="https://static.igem.org/mediawiki/2011/5/54/Ust_refer.gif" width=100 height=100 alt="References"></a>
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<p>1. only  pDummy is uptaken<br />
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-
  - since pDummy has no selection marker, the host bacteria die under selection pressure and cannot propagate</p>
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<b><font size=14>Strain Construction</font></b><hr>
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<p><img src="Eveything you may need 03_10_11/exported diagrams/have pd only.png" width="631" height="200" /><br />
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<br>
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  2. only pToolkit is uptaken<br />
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<p>
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- the  host bacterium that uptakes pToolkit survives. Yet during propagation, pToolkit is not replicated because proteins of G are absent. Therefore daughter cells of the host bacterium will not receive copies of the pToolkit and die under selection pressure.</p>
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<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>
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<p><img src="Eveything you may need 03_10_11/exported diagrams/have pt only.png" width="921" height="200" /></p>
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</p>
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<p>3. both pDummy and pToolkit are uptaken<br />
+
 
-
  - in  presence of pDummy, pToolkit is maintained and confers the host bacterium with stress  resistance. Daughters that receive copies of both plasmids will survive and eventually develop into a colony.</p>
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<p><img src="Eveything you may need 03_10_11/exported diagrams/have both.png" width="858" height="240" /></p>
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<p align=justify style="margin: 20px 20px 20px 20px">
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<p>Using this mutualistic relation, the desired pDummy can be maintained once the host bacterium develops an addiction it, and pToolkit can be lost in bacteria propagation if the expression of G can be shut off manually. Eventually, the bacteria not obtain any new antibiotic resistance genes but keep pDummy.</p>
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<center><img src=https://static.igem.org/mediawiki/2011/d/d0/Ust_Final_product.png width=540></center>
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<p><strong>3.2 Development of addiction – use of the lambda  RED recombination system</strong></p>
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</p>
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<p>To  develop the addiction in the host bacterium to pDummy, an essential gene for survival  is to be deleted from the bacteria genome, provided that the bacteria can  survive on extra-chromosomal copies after the deletion.</p>
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<p>The  deletion here is mediated through the lambda RED recombination system</p>
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<p>|||||||||||||||||||||||||||||||||||||<br />
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  Nat’s  part<br />
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  ||||||||||||||||||||||||||||||||||||</p>
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Our E. CRAFT (<i>Escherichia coli</i> 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).
-
<p>The lambda  RED recombination cassette is located on the pToolkit (and hence the name of the plasmid). Once the recombination is successful, it can be eliminated from  the host bacterium together with the antibiotic resistance gene. </p>
+
<br><br>
-
<p>Therefore, once the co-transformation of pDummy and pToolkit is successful, linear dsDNAs having  a reporter gene flanked by homologous sequences to the essential gene can be  introduced into the bacteria. </p>
+
 
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<p><img src="Eveything you may need 03_10_11/exported diagrams/trans dsDNA.png" width="684" height="203" /></p>
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Here, we introduce a heat-sensitive origin of replication as the sole origin of pDummy. When we intend to switch off the pDummy’s replication, we can incubate E. CRAFT at a temperature above its optimum 30ᵒC . This origin would then cease to function, and pDummy cannot be maintained.[11] Deprived of the essential gene and its corresponding vital product, E. CRAFT will not be able to propagate unless it receives a heat insensitive analog of pDummy.
-
<p>When the recombination is kicked started, the essential gene will be swapped out and the reporter gene will be incorporated into the bacteria genome.</p>
+
<br>
-
<p><img src="Eveything you may need 03_10_11/exported diagrams/recombination.png" width="634" height="784" /></p>
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<p>Since the linear dsDNAs do not have origin of replications, they are not inherited in daughters unless they are swapped into the genomes. Thus, any observable signals from the reporter would allow identification of successful recombination. Identified colonies can then be further treated to induce loss of pToolkit, which afterwards would be the completed strain of EX.</p>
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-
<p><img src="Eveything you may need 03_10_11/exported diagrams/post-swap pd.png" width="696" height="1020" /></p>
+
</p>
-
<p><strong>3.3 Complementation between reporter genes – manifesting completion of EX engineering</strong></p>
+
 
-
<p>To ensure that the final strain of EX has: 1. successfully had its essential gene deleted from genome, 2. maintained the pDummy, a complementation reporter system between the pDummy and swapped gene is preferred over a single reporter at the swapped site.</p>
+
<p align=justify style="margin: 20px 20px 20px 20px">
-
<p>Different methods can achieve the above aim:<br />
+
<center><img src=https://static.igem.org/mediawiki/2011/1/18/Keep_or_kill.jpg width=750></center>
-
  i. Alpha  complementation can be used in E. Coli strains where the lacZ gene is  completely removed. The larger fragment ω can be swapped for the essential gene while the smaller α fragment can stay on pDummy. In a X-gal rich medium, blue colonies suggest the desired engineered strains.<br />ii. Complementation between split fluorescent proteins (sFP). 2010 iGEM Slovenia team has demonstrated the principle that N-terminal and C-terminal fragments of sFPS are able to complement in vivo and two sets of sfFPS are able to undergo Forster resonance energy transfer (FRET). This idea is adopted but an alternative set  of candidate, split superfolder GFPs (sfGFP), was developed.</p>
+
</p>
-
<p><strong>3.4 Summary of construction flow:</strong><br />
+
 
-
  1. Assembly pDummy and pToolkit<br />
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<p align=justify style="margin: 20px 20px 20px 20px">
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  2. Co-transform  both plasmid into E Coli and maintain stable strains<br />
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-
  3. Introduce linear dsDNAs and induce recombination<br />
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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.<a href=#top>[Top]</a><br>
-
  4. Isolate recombinants<br />
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-
  5. Induce loss of pToolkit</p>
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</p>
 +
 
 +
<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>
 +
<b><a name=assembly></a>2. Stepping into the heart of construction - methods of assembly</b>
 +
</p>
 +
 
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
<b>2.1 Construction and maintenance of an antibiotic-resistance-gene-free plasmid through antibiotic selection – the unavoidable evil two plasmid system</b><br>
 +
 
 +
Our ultimate goal is to construct the E. CRAFT without conferring any new antibiotic resistance on it. For this reason, no resistance gene should be found in our dummy plasmid: the pDummy.
 +
<br><br>
 +
 
 +
Yet, ensuring the maintenance of such a plasmid in its host bacterium would be a challenge, unless the cell needs the plasmid for survival (essential- gene- loss induced addiction: loss of the essential gene in bacterial genome causes dependence on the extra-chromosomal copy in pDummy). Inconveniently, however, this addiction can only be achieved after the introduction of the plasmid.
 +
<br>
 +
 
 +
</p>
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 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
<center><img src=https://static.igem.org/mediawiki/2011/7/7e/Ust_Post-swap_pd.png width=640><br>
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<img src=https://static.igem.org/mediawiki/2011/5/59/Ust_Post-swap_pt.png width=750></center>
 +
</p>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
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.
 +
<br>
 +
 
 +
</p>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
<center><img src=https://static.igem.org/mediawiki/2011/3/32/Ust_ML_of_OR.png width=660></center>
 +
</p>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
<p>
 +
 
 +
Let’s consider the following scenario: <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>
 +
iii. pDummy and pToolkit are co-transformed to a bacterium which is under selection stress.<a href=#top>[Top]</a>
 +
<br><br>
 +
 
 +
</p>
 +
 
 +
 
 +
<p>
 +
 
 +
Three possible outcomes could be expected:<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.
 +
 
 +
</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>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
<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.
 +
 
 +
</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>
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</p>
 +
<br>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
<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.
 +
 
 +
</p>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
<center><img src=https://static.igem.org/mediawiki/2011/6/68/Ust_Have_both.png width=770></center>
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</p>
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<br>
 +
 
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
<p>
 +
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.
 +
</p>
 +
 
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
<b>2.2 Development of addiction – use of the λ RED recombination system [1]</b><br>
 +
 
 +
Performed with a purpose to induce host bacterium’s addiction to the pDummy, removal of the essential gene <i>nadE</i> from the bacterial genomic DNA is mediated by the phage λ RED recombination system.
 +
<br><br>
 +
 
 +
DNA Recombination is in fact inherent in <i>E. coli</i>, and this can be done via the RecBCD Recombination Pathway. Even so, it is not the most ideal method for engineering new strains of <i>E. coli</i> containing genomic modification, in part because this pathway requires a recognition site known as “χ” on the linear “to-be inserted” DNA sequence (called “Transformation DNA” henceforth). While this makes work for a molecular biologist more challenging, naturally, this is quite logical as the RecBCD pathway is meant to be a DNA Repair pathway when the <i>E. coli</i>’s genome is damaged (rather than a means to introduce new DNA material into the genome).
 +
<br><br>
 +
 
 +
Even so, <i>E. coli</i> has its own natural “enemies” and one known enemy is the phage λ. As phage λ is a lysogenic phage (phage able to enter lysogenic phase), it has mechanisms to ensure that its DNA can be integrated into the host’s genome. One mechanism involves the proteins γ. <i>Exonuclease</i> (<i>Exo</i>) and β. Collectively, these three proteins are called “λ-RED”. Essentially, λ-RED hijacks the DNA repair machinery of the <i>E. coli</i> to promote site-specific homologous recombination. While the exact mechanism is still being contested and investigated, some insights have been gained:
 +
<br><br>
 +
 
 +
γ will first bind to <i>E. coli</i>’s RecBCD, inhibiting RecB’s nuclease activity, thus shutting down this recombination pathway. <i>Exo</i> will then begin degrading the linear Transformation DNA (dsDNA) in the 5’&rarr;3’ direction, resulting in either 3’ ssDNA overhangs on both sides of the dsDNA or a sole ssDNA (proposed mechanistic differences). β will then bind to the 3’ ssDNA and encourage the ssDNA to anneal to the target dsDNA.
 +
<br><br>
 +
 
 +
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>
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 +
 
 +
</p>
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<p align=justify style="margin: 20px 20px 20px 20px">
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<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">
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 +
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>
 +
 
 +
</p>
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 +
<p align=justify style="margin: 20px 20px 20px 20px">
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<center><img src=https://static.igem.org/mediawiki/2011/c/c7/Ust_Trans_dsDNA.png width=780></center>
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</p>
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<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.
 +
<br>
 +
 
 +
</p>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
<center><img src=https://static.igem.org/mediawiki/2011/6/6a/Ust_KO_pt.png width=810></center>
 +
</p>
 +
 
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
<b>2.3 Complementation between reporter genes – manifesting completion of E. CRAFT engineering</b>
 +
<br>
 +
 
 +
To ensure that the final strain of E. CRAFT has:
 +
<br>
 +
 
 +
(1) successfully had its essential nadE gene deleted from the genome;
 +
<br>
 +
 
 +
(2) maintained the pDummy,
 +
<br>
 +
 
 +
a complementation reporter system between the pDummy and the swapped gene is preferred over a single reporter at the swap site.
 +
<br><br>
 +
 
 +
Different methods can be employed to achieve this aim:
 +
<br>
 +
 
 +
i. α complementation can be used in <i>E. coli</i> strains which have had their lacZ gene completely removed. The larger fragment ω can be swapped in place for the essential gene while the smaller α fragment can stay on pDummy. In an X-gal rich medium, blue colonies would indicate the desired engineered strains.
 +
<br>
 +
 
 +
ii. Complementation between split fluorescent proteins (sFP). 2010 iGEM Slovenia team has demonstrated the principle that N-terminal and C-terminal fragments of sFPS are able to complement in vivo, and two sets of sfFPS are able to undergo Forster Resonance Energy Transfer (FRET). Using this system has been considered in this project, but an alternative reporter candidate, the split superfolder GFPs (sfGFP) [6, 12, 13] invented by other researchers, was tested instead.
 +
 
 +
</p>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
<center><img src=https://static.igem.org/mediawiki/2011/6/6c/Ust_Reporter.jpg width=700></center>
 +
</p>
 +
<br>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
<b>2.4 Summary of construction flow:</b><br>
 +
 
 +
1. Assembly pDummy and pToolkit<br>
 +
2. Co-transformation of both plasmids into <i>E. coli</i> and maintenance of stable strains<br>
 +
3. Introduction of linear ds DNAs and induction of recombination<br>
 +
4. Isolation of recombinants<br>
 +
5. Induction pToolkit loss
 +
 
 +
<a href=#top>[Top]</a>
 +
 
 +
<br>
 +
</p>
 +
 
 +
 
 +
<p >
 +
<b><a name=component></a>3. Details of the components – a closer look to the molecular basis of assembly</b>
 +
</p>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
<b>3.1 Temperature-sensitive origin of replication_oriR101 & repA101-ts (BBa_K524000)</b><br>
 +
 
 +
oriR101 & repA101-ts is a set of low copy origins of replication derived from the pSC101 replication origin.[11] The repA101-ts gene encodes for a heat-labile protein that is required in trans for the initiation of replication at oriR101. Characterization of our construct has shown that plasmids with this origin of replication could only be fully maintained if incubated at lower than 30ᵒC, while partial maintenance of plasmid was observed at 33ᵒC. The origin ceases function at 37ᵒC.
 +
<br><br>
 +
 
 +
This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by nucleotide mutation.
 +
 
 +
 
 +
<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 align=justify style="margin: 20px 20px 20px 20px">
 +
<b>3.2 split superfolder green fluroscent protein_split sfGFP<br>
 +
sfGFP1-10 (BBa_K524001) [Twins: BBa_K524006]<br>
 +
sfGFP11 (BBa_K524002) [Twins: BBa_K524007]</b><br>
 +
 
 +
The sfGFPs are mutant variants of GFPs, with characteristics of improved folding kinetics and resistance to chemical denaturants. [10] Split sfGFPs have been reported to be able to undergo complementation at amino acid residues 214 and 215 to give green fluorescence. [6, 12, 13]
 +
<br>
 +
 
 +
</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>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
<b>3.3 Essential gene <i>nadE</i> (BBa_K524003)</b><br>
 +
 
 +
<i>nadE</i>, which encodes the enzyme NAD+ synthetase, is a vital gene in <i>E. coli</i>. [2] In principle, removal of such a gene from the bacterial genome would cause the cells to be addicted to a plasmid that has a copy of the gene. CyaR (a sRNA) regulates the expression of <i>nadE</i> post-transcriptionally, and this feature is retained in our construct. Transcription of <i>nadE</i> operon requires the sigma-70 initiation factor and is terminated by downstream extragenic sites.
 +
<br><br>
 +
 
 +
The <i>nadE</i> gene was cloned out from the genome of <i>E. coli</i> strain BL21(DE3), and its construction was completed by ligating B0015 double terminator to its end.
 +
<br>
 +
 
 +
</p>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
<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">
 +
 
 +
<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 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]
 +
<br><br>
 +
 
 +
In the fabrication of these two constructs, the pir gene was cloned out from the genome of BW25141 <i>E. coli</i> strain (courtesy of The Coli Genetic Stock Center) and standardized.
 +
<br><br>
 +
 
 +
The ori-γ was adopted from the R6K origin of replication BBa_J61001.
 +
<br>
 +
 
 +
</p>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
<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">
 +
 
 +
<b>3.5 iGEM 2010 Slovenia Split/FRET constructs</b><br>
 +
 
 +
The split Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP) biobricks constructed by the 2010 Slovenia Team are used as alternatives to the superfolder GFP reporter. The idea here is to put one of the terminal fragments of either CFP or YFP into the pDummy, and swap out the essential <i>nadE</i> gene from the bacterial genome with the other terminal fragment of the fluorescent protein. Driven by pLac R0010, both fragments should be expressed simultaneously when induced by IPTG. Fluorescent signal observed could be used as an indicator of successful recombination.
 +
 
 +
<a href=#top>[Top]</a>
 +
<br>
 +
 
 +
</p>
 +
 
 +
 
 +
 
 +
<hr>
 +
 
 +
<br>
 +
 
 +
</p><p>
 +
 
 +
 
 +
 
 +
<p >
 +
<b><a name=refer></a>4. References</b>
 +
</p>
 +
 
 +
<p align=justify style="margin: 20px 20px 20px 20px">
 +
 
 +
1. Datsenko KA, Wanner BL.(2000). One-step inactivation of chromosomal genes in <i>Escherichia coli</i> K-12 using PCR products, Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5.
 +
<br><br>
 +
 
 +
2. D. Manen, L. Caro.(1991). The replication of plasmid pSC101, Mol Microbiol. 1991 Feb;5(2):233-7.
 +
<br><br>
 +
 
 +
3. Ecocyc: Encyclopedia of <i>Escherichia coli</i> K-12 Genes and Metabolism
 +
<br><br>
 +
 
 +
4. F Wu, I Goldberg, and M Filutowicz.(1992). Roles of a 106-bp origin enhancer and <i>Escherichia coli</i> DnaA protein in replication of plasmid R6K, Nucleic Acids Res. 1992 February 25; 20(4): 811–817.
 +
<br><br>
 +
 
 +
5. F Wu, I Goldberg, and M Filutowicz.(1994). Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K γ origin replication mediated by elevated levels of R6K π protein, J Bacteriol. 1994 November; 176(22): 6795–6801.
 +
<br><br>
 +
 
 +
6. Jun Zhou, Jian Lin, Cuihong Zhou, Xiaoyan Deng and Bin Xia.(2011). An improved bimolecular fluorescence complementation tool based on superfolder green fluorescent protein, Acta Biochim Biophys Sin 43 (3): 239-244.
 +
<br><br>
 +
 
 +
7. M Filutowicz, G Davis, A Greener, and D R Helinski.(1985). Autorepressor properties of the π-initiation protein encoded by plasmid R6K, Nucleic Acids Res. 1985 January 11; 13(1): 103–114.
 +
<br><br>
 +
 
 +
8. M Filutowicz, M J McEachern, and D R Helinski.(1986). Positive and negative roles of an initiator protein at an origin of replication, Proc Natl Acad Sci U S A. 1986 December; 83(24): 9645–9649.
 +
<br><br>
 +
 
 +
9. Peubez I, Chaudet N, Mignon C, Hild G, Husson S, Courtois V, De Luca K, Speck D, Sodoyer R.(2010). Antibiotic-free selection in <i>E. coli</i>: new considerations for optimal design and improved production, Microb Cell Fact. 2010 Sep 7;9:65
 +
<br><br>
 +
 
 +
10. Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS.(2006). Engineering and characterization of a superfolder green fluorescent protein, Nat Biotechnol. 2006 Jan;24(1):79-88. Epub 2005 Dec 20.
 +
<br><br>
 +
 
 +
11. Phillips GJ.(1995). New Cloning Vectors with Temperature-Sensitive Replication, Plasmid. 1999 Jan;41(1):78-81.
 +
<br><br>
 +
 
 +
12. Stéphanie Cabantous, Thomas C Terwilliger & Geoffrey S Waldo.(2004). Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein, Nature Biotechnology 23, 102 – 107
 +
<br><br>
 +
 
 +
13. Stéphanie Cabantous & Geoffrey S Waldo.(2006). In vivo and in vitro protein solubility assays using split GFP, Nature Methods - 3, 845 – 854
 +
<br><br>
 +
 
 +
<a href=#top>[Top]</a><br><br>
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</font>
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</TH>
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</TD>
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  </TR>
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</table>
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<table border="0" cellspacing="0" cellpadding="10">
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<tr>
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<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>
 +
 
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<td width="382px" bgcolor="#CCFF99" valign="baseline">
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<p align="center" valign="baseline">
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<b><font color="green">Our Project</font></b></p>
 +
 
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<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>
 +
 
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<p align="center" valign="baseline">
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<b><font color="green">Experiments and Results</font></b></p>
 +
 
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<p align="center" valign="baseline">
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<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">
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<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>
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</td>
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<td width="302px" bgcolor="#D09C00" valign="baseline">
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<p align="center" valign="baseline">
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<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>
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<p align="center" valign="baseline">
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<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>
 +
 
 +
 
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<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>
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<td width="180px"bgcolor="#980000"valign="baseline">
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<p align="center" valign="baseline">
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<b><font color="#FFE0E0">Human Practice</font></b></p>
 +
 
 +
<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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</td>
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Latest revision as of 18:57, 28 October 2011

How to Select · Methods of Assembly · Details of Components · References


How to select against E. CRAFT cells that fail to take up the vector plasmid - our alternative selection method Stepping into the heart of construction - methods of assembly Details of the components – a closer look to the molecular basis of assembly References Strain Construction



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).

Here, we introduce a heat-sensitive origin of replication as the sole origin of pDummy. When we intend to switch off the pDummy’s replication, we can incubate E. CRAFT at a temperature above its optimum 30ᵒC . This origin would then cease to function, and pDummy cannot be maintained.[11] Deprived of the essential gene and its corresponding vital product, E. CRAFT will not be able to propagate unless it receives a heat insensitive analog of 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
Our ultimate goal is to construct the E. CRAFT without conferring any new antibiotic resistance on it. For this reason, no resistance gene should be found in our dummy plasmid: the pDummy.

Yet, ensuring the maintenance of such a plasmid in its host bacterium would be a challenge, unless the cell needs the plasmid for survival (essential- gene- loss induced addiction: loss of the essential gene in bacterial genome causes dependence on the extra-chromosomal copy in pDummy). Inconveniently, however, this addiction can only be achieved after the introduction of the plasmid.


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:
i. G is placed on the pDummy, which lacks a selection marker but is equipped with a normal replication origin
ii. OR is the sole origin of replication of another plasmid (here we introduce a new plasmid, pToolkit) with a selection marker
iii. pDummy and pToolkit are co-transformed to a bacterium which is under selection stress.[Top]

Three possible outcomes could be expected:
1. Only pDummy is uptaken
Since pDummy has no selection marker, the host bacterium would die under selection pressure and fail to propagate.


2. Only pToolkit is uptaken
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.


3. Both pDummy and pToolkit are uptaken
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.


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]
Performed with a purpose to induce host bacterium’s addiction to the pDummy, removal of the essential gene nadE from the bacterial genomic DNA is mediated by the phage λ RED recombination system.

DNA Recombination is in fact inherent in E. coli, and this can be done via the RecBCD Recombination Pathway. Even so, it is not the most ideal method for engineering new strains of E. coli containing genomic modification, in part because this pathway requires a recognition site known as “χ” on the linear “to-be inserted” DNA sequence (called “Transformation DNA” henceforth). While this makes work for a molecular biologist more challenging, naturally, this is quite logical as the RecBCD pathway is meant to be a DNA Repair pathway when the E. coli’s genome is damaged (rather than a means to introduce new DNA material into the genome).

Even so, E. coli has its own natural “enemies” and one known enemy is the phage λ. As phage λ is a lysogenic phage (phage able to enter lysogenic phase), it has mechanisms to ensure that its DNA can be integrated into the host’s genome. One mechanism involves the proteins γ. Exonuclease (Exo) and β. Collectively, these three proteins are called “λ-RED”. Essentially, λ-RED hijacks the DNA repair machinery of the E. coli to promote site-specific homologous recombination. While the exact mechanism is still being contested and investigated, some insights have been gained:

γ will first bind to E. coli’s RecBCD, inhibiting RecB’s nuclease activity, thus shutting down this recombination pathway. Exo will then begin degrading the linear Transformation DNA (dsDNA) in the 5’→3’ direction, resulting in either 3’ ssDNA overhangs on both sides of the dsDNA or a sole ssDNA (proposed mechanistic differences). β will then bind to the 3’ ssDNA and encourage the ssDNA to anneal to the target dsDNA.

This in turn will activate E. coli’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).

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
To ensure that the final strain of E. CRAFT has:
(1) successfully had its essential nadE gene deleted from the genome;
(2) maintained the pDummy,
a complementation reporter system between the pDummy and the swapped gene is preferred over a single reporter at the swap site.

Different methods can be employed to achieve this aim:
i. α complementation can be used in E. coli strains which have had their lacZ gene completely removed. The larger fragment ω can be swapped in place for the essential gene while the smaller α fragment can stay on pDummy. In an X-gal rich medium, blue colonies would indicate the desired engineered strains.
ii. Complementation between split fluorescent proteins (sFP). 2010 iGEM Slovenia team has demonstrated the principle that N-terminal and C-terminal fragments of sFPS are able to complement in vivo, and two sets of sfFPS are able to undergo Forster Resonance Energy Transfer (FRET). Using this system has been considered in this project, but an alternative reporter candidate, the split superfolder GFPs (sfGFP) [6, 12, 13] invented by other researchers, was tested instead.


2.4 Summary of construction flow:
1. Assembly pDummy and pToolkit
2. Co-transformation of both plasmids into E. coli and maintenance of stable strains
3. Introduction of linear ds DNAs and induction of recombination
4. Isolation of recombinants
5. Induction pToolkit loss [Top]

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)
oriR101 & repA101-ts is a set of low copy origins of replication derived from the pSC101 replication origin.[11] The repA101-ts gene encodes for a heat-labile protein that is required in trans for the initiation of replication at oriR101. Characterization of our construct has shown that plasmids with this origin of replication could only be fully maintained if incubated at lower than 30ᵒC, while partial maintenance of plasmid was observed at 33ᵒC. The origin ceases function at 37ᵒC.

This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by nucleotide mutation.

3.2 split superfolder green fluroscent protein_split sfGFP
sfGFP1-10 (BBa_K524001) [Twins: BBa_K524006]
sfGFP11 (BBa_K524002) [Twins: BBa_K524007]

The sfGFPs are mutant variants of GFPs, with characteristics of improved folding kinetics and resistance to chemical denaturants. [10] Split sfGFPs have been reported to be able to undergo complementation at amino acid residues 214 and 215 to give green fluorescence. [6, 12, 13]


3.3 Essential gene nadE (BBa_K524003)
nadE, which encodes the enzyme NAD+ synthetase, is a vital gene in E. coli. [2] In principle, removal of such a gene from the bacterial genome would cause the cells to be addicted to a plasmid that has a copy of the gene. CyaR (a sRNA) regulates the expression of nadE post-transcriptionally, and this feature is retained in our construct. Transcription of nadE operon requires the sigma-70 initiation factor and is terminated by downstream extragenic sites.

The nadE gene was cloned out from the genome of E. coli strain BL21(DE3), and its construction was completed by ligating B0015 double terminator to its end.

3.4 π replication initiator protein encoded by pir gene (BBa_K524004) and γ-origin of replication (ori-γ) from R6K plasmid
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]

In the fabrication of these two constructs, the pir gene was cloned out from the genome of BW25141 E. coli strain (courtesy of The Coli Genetic Stock Center) and standardized.

The ori-γ was adopted from the R6K origin of replication BBa_J61001.

3.5 iGEM 2010 Slovenia Split/FRET constructs
The split Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP) biobricks constructed by the 2010 Slovenia Team are used as alternatives to the superfolder GFP reporter. The idea here is to put one of the terminal fragments of either CFP or YFP into the pDummy, and swap out the essential nadE gene from the bacterial genome with the other terminal fragment of the fluorescent protein. Driven by pLac R0010, both fragments should be expressed simultaneously when induced by IPTG. Fluorescent signal observed could be used as an indicator of successful recombination. [Top]



4. References

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.

2. D. Manen, L. Caro.(1991). The replication of plasmid pSC101, Mol Microbiol. 1991 Feb;5(2):233-7.

3. Ecocyc: Encyclopedia of Escherichia coli K-12 Genes and Metabolism

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