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<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> Strain Construction

STRAIN CONSTRUCTION

1. Constructing EX – the bacterial strain that allows selection without use of antibiotics

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.

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.

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.

2. How to select against EX without the vector plasmid? Our alternative selection method

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).

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.

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 "shuffled out" 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.

3. Stepping in the heart of construction - methods of assembly

3.1 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 our EX without conferring it any new antibiotic resistance. For this reason no resistance gene should be found in our dummy plasmid pDummy.

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.

The solution is to develop a mutualistic relation between two plasmids and we planned to exploit positively regulated origin of replications.

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.

Let’s consider the following scenario:
i. G is placed on pDummy with no selection marker but 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

We would obtain three possible outcomes:

1. only pDummy is uptaken
- since pDummy has no selection marker, the host bacteria die under selection pressure and cannot propagate

2. only pToolkit is uptaken
- 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.

3. both pDummy and pToolkit are uptaken
- 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.

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.

3.2 Development of addiction – use of the lambda RED recombination system

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.

The deletion here is mediated through the lambda RED recombination system

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Nat’s part
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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.

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.

When the recombination is kicked started, the essential gene will be swapped out and the reporter gene will be incorporated into the bacteria genome.

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.

3.3 Complementation between reporter genes – manifesting completion of EX engineering

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.

Different methods can achieve the above aim:
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.
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.

3.4 Summary of construction flow:
1. Assembly pDummy and pToolkit
2. Co-transform both plasmid into E Coli and maintain stable strains
3. Introduce linear dsDNAs and induce recombination
4. Isolate recombinants
5. Induce loss of pToolkit

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-<TR>
-<TH ROWSPAN=3 BGCOLOR="#A1C6B2">
-<a name=top></a><p>
-<h3>1. ASM</h3>
-<font color=black>
-</p>
-<p >
-<h4 align=left><a name=theory></a>1.1. Theory – how to select? </h4>
-</p>
-<p align=justify style="margin: 20px 20px 20px 20px">
-The E. trojan is a synthetic E.coli strain that is engineered to lack an essential gene, nadE,  in its genomic DNA. To survive, this strain has to rely on a pre- introduced plasmid (pDummy) bearing the essential gene; thus forcing the bacteria to maintain the plasmid until an alternative source of nadE gene is present.  The pDummy, however, has been designed to have a temperature- sensitive origin of replication which would cease to function if the bacterial cells are incubated under higher incubation temperatures (>42ᵒC???). <br><br>
-For sub-cloning purposes, an E. trojan – compatible vector plasmid is designed. This carrier vector, like the pDummy, contains the nadE essential gene. Once a gene of interest is inserted into this  vector, the plasmid can be transformed to the E. trojan for amplification. Incubating the transformed bacteria at a temperature high enough to inactivate the heat sensitive replication origin  of the pDummy would result in pDummy loss, making it necessary for the cells to retain the insert- bearing pCarrier for survival. Bacterial cells that do not take up the pCarrier and its insert would be deprived of the nadE gene product and die; while those who do would survive and continue dividing.
-<a href=#top>[Top]</a>
-</p>
-<p >
-<h4 align=left><a name=method></a>1.2. Method of assembly</h4>
-</p>
-<p align=justify style="margin: 20px 20px 20px 20px">
-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.
-<br><br>
-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 bacteria strain that can perform plasmid selection without the use of antibiotics, and contains as little antibiotics resistance gene as possible.<br><br>
-<b>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 our EX without conferring it any new antibiotic resistance. For this reason no resistance gene should be found in our dummy plasmid pDummy. <br><br>
-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 extragenomic copies on pDummy), and inconveniently, the addiction can only be achieved after the introduction of the plasmid.<br><br>
-The solution is to develop a mutualistic relation between two plasmids and we planned to exploit positively regulated origin of replications. 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.<br></p>
-<p>
-Let’s consider the following scenario: <br>
-i. G is placed on pDummy with no selection marker but with a normal replication origin<br>
-ii. OR is the sole origin of replication of another plasmid (pToolkit) with a selection marker<br>
-iii. pDummy and pToolkit are co-transformed to a bacterium which is under selection stress</p>
-<br>
-<p>
-We would obtain three possible outcomes:<br>
-<b>1. only pDummy is uptaken</b><br>
-- since pDummy has no selection marker, the host bacteria die under selection pressure and cannot propagate<br><br>
-<b>2. only pToolkit is uptaken</b><br>
-- 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.<br><br>
-<b>3. both pDummy and pToolkit are uptaken</b><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.<br><br></p>
-<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>
-<p align=justify style="margin: 20px 20px 20px 20px">
-<b>Development of addiction – use of the lambda RED recombination system</b><br>
-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-genomic copies after the deletion.<br><br>
-The deletion here is mediated through the lambda RED recombination system.<br><br>
-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. <br><br>
-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. When the recombination is kicked started, the essential gene will be swapped out and the reporter gene will be incorporated into the bacteria genome.<br><br>
-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.<br><br>
-<b>Complementation between reporter genes – manifesting completion of EX engineering</b><br>
-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.<br><br>
-Different methods can achieve the above aim:<br>
-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><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 Förster resonance energy transfer (FRET). This idea is adopted but an alternative set of candidate, split superfolder GFPs (sfGFP), was developed.</p>
-<h5 align=left>Summary of construction flow:</h5>
-<p>1. Assembly pDummy and pToolkit<br>
-. Co-transform both plasmid into E Coli and maintain stable strains<br>
-. Introduce linear dsDNAs and induce recombination<br>
-. Isolate recombinants<br>
-. Induce loss of pToolkit
-<a href=#top>[Top]</a>
-</p>
-<br>
-<p >
-<h4 align=left><a name=component></a>1.3. Component details</h4>
-</p>
-<p align=justify style="margin: 20px 20px 20px 20px">
-Temperature-sensitive origin of replication_oriR101 & repA101-ts (BBa_K524000)
-oriR101 & repA101-ts is a set of low copy origin of replication derived from the pSC101 origin of replication. The repA101-ts gene codes for a heat-labile protein that is required in trans for the initiation of replication at oriR101. In our construct, our characterization has shown that plasmids with this origin of replication can only be maintained below than 300C, and partial maintenance of plasmid was observed within temperature range from 290C to 330C. This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by a nucleotide mutation.<br>
-</p>
-<p align=justify style="margin: 20px 20px 20px 20px">
-split superfolder green fluroscent protein_split sfGFPsplit superfolder green fluroscent <br>
-protein_split sfGFP<br>
-sfGFP1-10 (BBa_K524001)<br>
-sfGFP11 ((BBa_K524002)
-</p>
-<p align=justify style="margin: 20px 20px 20px 20px">
-The sfGFPs are mutated variants of GFPs that has improved folding kinetics and resistance to chemical denaturants. Split sfGFPs at amino acid residues 214 and 215 have been reported to undergo spontaneous complementation to give green fluorescence. The two split constructs were produced from an existing biobrick – pBAD driven sfGFP BBa_I746908. CDS of sfGFP amino acid residues 1-214 were copied out for sfGFP1-10 using PCR and stop codon was added to the end. The sfGFP11 was produced in a similar fashion, with a start codon added to the front of the CDS of amino acid residues 215 to 238.
-</p>
-<p align=justify style="margin: 20px 20px 20px 20px">
-<b>Essential gene nadE (BBa_K524003)</b><br>
-<i>nadE</i> is a vital gene in <i>E. Coli.</i> It codes for NAD+ synthetase. In principle, removal of such gene from the genome would cause addiction of bacteria to a plasmid that has a copy of the gene. CyaR (a sRNA) regulates the expression of nadE post-transcriptionally. This feature is retained in our construct. Transcription of <i>nadE</i> operon requires the sigma-70 factor and is terminated by downstream extragenic sites. The <i>nadE</i> gene was cloned out from the genome of strain BL21(DE3), and was completed the <i>nadE</i> by having B0015 terminator assembled to its end.
-</p>
-<p align=justify style="margin: 20px 20px 20px 20px">
-<b>Replication initiator pi protein and ori-gamma from R6K plasmid</b><br>
-ori-gamma is one of three replication origins (the other two being alpha and beta) of the R6K origin. Initiation of replication at ori-gamma requires the pi protein in trans, which is encoded by the pir gene. Yet doubling the concentration of pi protein would effectively shut down the replication as well. Expression of pi protein is autogenously regulated. The pir construct was cloned out from the genome of strain BW25141 (courtesy of The Coli Genetic Stock Center) and standardized. The ori-gamma was adopted from the R6K origin of replication BBa_J61001.
-</p>
-<p align=justify style="margin: 20px 20px 20px 20px">
-<b>iGEM 2010 Slovenia Split/FRET constructs</b><br>
-The split CFP and YFP from the biobricks of Slovenia team last year were used as alternative reporters. The idea is to put one of the terminal fragments of a split fluorescence protein into the pDummy, and swap out the essential nadE gene from the genome with the other terminal fragment. Driven by pLac R0010, both fragments should express simultaneously when induced by IPTG and fluorescence signal would be observed as an indicator of successful recombination.<a href=#top>[Top]</a>
-</p>
-</font>
-</TH>
-    <TD bgcolor="white" width=160 height=150>
-<p>
-<h2>ASM</h2>
-</font>
-</p>
-<br><br><br>
-<img src="https://static.igem.org/mediawiki/2011/6/62/Ust_21.jpg" width=100 height=100><BR>
-ASM <br>
-<a href=#theory>1.1 Theory – how to select?</a> <br>
-<a href=#method>1.2 Method of assembly</a><br>
-<a href=#component>1.3 Component details</a><br><br>
-</TD>
-    </TR>
-<TR>
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-</TD>
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-<TR>
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-<a href="https://2011.igem.org/Team:HKUST-Hong_Kong" target=_top><font face="Verdana, Arial, Helvetica, sans-serif" size="4" color="#FFE1E1" font color=white><span style="font-weight:700">Home</span></font></a></font></b></p>
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-<p align="center" valign="baseline"><b> <font face="Verdana, Arial, Helvetica, sans-serif" size="3" color="green">
-Our Project</font></b></p>
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-<span style="line-height:1; font-weight:600">Experiments and Results</span><br>
-<a href="asm.html"  target=_top>Strain construction</a> |
-<a href="mic.html"  target=_top>Culture tests</a> |
-<a href="modeling.html"  target=_top>Modeling</a><br>
-<span style="line-height:1; font-weight:600">Miscellaneous</span><br>
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-</table>
+<body>
+<p><strong>STRAIN  CONSTRUCTION</strong></p>
+<p><strong>1. Constructing EX – the bacterial strain that  allows selection without use of antibiotics</strong></p>
+<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>
+<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>
+<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>
+<p><strong>2. How to select against EX without the vector  plasmid? Our alternative selection method</strong></p>
+<p><img src="Eveything you may need 03_10_11/exported diagrams/final product.png" width="695" height="395" border="0" /></p>
+<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>
+<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>
+<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>
+<p><strong>3. Stepping  in the heart of construction - methods of assembly </strong></p>
+<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>
+<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>
+<p><img src="Eveything you may need 03_10_11/exported diagrams/post-swap pt.png" width="722" height="197" /></p>
+<p>The  solution is to develop a mutualistic relation between two plasmids and we  planned to exploit positively regulated origin of replications. </p>
+<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>
+<p><img src="Eveything you may need 03_10_11/exported diagrams/ML of OR.png" width="791" height="445" /></p>
+<p>Let’s consider  the following scenario: <br />
+  i. G is placed  on pDummy with no selection marker but with a normal replication origin</p>
+<p><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</p>
+<p>We would  obtain three possible outcomes:</p>
+<p>1. only  pDummy is uptaken<br />
+  - since pDummy  has no selection marker, the host bacteria die under selection pressure and  cannot propagate</p>
+<p><img src="Eveything you may need 03_10_11/exported diagrams/have pd only.png" width="631" height="200" /><br />
+. only pToolkit  is uptaken<br />
+- 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>
+<p><img src="Eveything you may need 03_10_11/exported diagrams/have pt only.png" width="921" height="200" /></p>
+<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>
+<p><img src="Eveything you may need 03_10_11/exported diagrams/have both.png" width="858" height="240" /></p>
+<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>
+<p><strong>3.2 Development of addiction – use of the lambda  RED recombination system</strong></p>
+<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>
+<p>The  deletion here is mediated through the lambda RED recombination system</p>
+<p>|||||||||||||||||||||||||||||||||||||<br />
+  Nat’s  part<br />
+  ||||||||||||||||||||||||||||||||||||</p>
+<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>
+<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>
+<p><img src="Eveything you may need 03_10_11/exported diagrams/trans dsDNA.png" width="684" height="203" /></p>
+<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>
+<p><img src="Eveything you may need 03_10_11/exported diagrams/recombination.png" width="634" height="784" /></p>
+<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>
+<p><img src="Eveything you may need 03_10_11/exported diagrams/post-swap pd.png" width="696" height="1020" /></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>Different  methods can achieve the above aim:<br />
+  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><strong>3.4 Summary of construction flow:</strong><br />
+. Assembly pDummy  and pToolkit<br />
+. Co-transform  both plasmid into E Coli and maintain stable strains<br />
+. Introduce linear  dsDNAs and induce recombination<br />
+. Isolate recombinants<br />
+. Induce loss of pToolkit</p>
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