BerkiGEM2007Present3

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===The Controller===
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The Controller is an integrated genetic circuit, comprised of two plasmids, that directs the initiation and production of the primary systems proteins through its component operons.
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  <p><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></p>
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  <p> <a href="https://2007.igem.org/BerkiGEM2007Present4">&lt;&lt;Previous Section:  Chassis</a> | <a href="https://2007.igem.org/BerkiGEM2007Present5">Next Section: Genetic Self-Destruct&gt;&gt;</a></p>
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'''Introduction'''
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=The Controller=
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The Bactoblood oranism needs to exist in two different states:  one form that is genetically stable and able to grow under normal laboratory conditions, and a second state that is highly differentiated, unable to grow, and devoid of genetic material.  To bring about this transformation, we needed a controller that could be easily triggered to coordinate the differentiation process.
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The Controller is an integrated genetic circuit, comprised of two plasmids, that directs the copy number and transcription of the primary devices in our system.
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The controller required a large dynamic range between the off and on states, the ability to maintain and overexpress a large number of genes, and ideally employed a low-cost inducer.  Therefore, we designed a controller based on a two plasmid system.  One plasmid stably replicates the variou sbiosynthetic operons of our system at single copy in a transcriptionally-inactive state.  The second plasmid houses the genes necessary for activation of the operon plasmid.  When activated with iron, the copy number of operon plasmid increases to high-copy and the transcription of the operons is activated.
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==Introduction==
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The Bactoblood organism needs to exist in two different states:  one form that is genetically stable and able to grow under normal laboratory conditions, and a second state that is highly differentiated, unable to grow, and devoid of genetic material.  To bring about the transformation to the differentiated state, we needed a controller that could be easily triggered by an external cue.  This controller required a large dynamic range between the off and on states, the ability to maintain and overexpress a large number of genes, and ideally employed a low-cost inducer.  Therefore, we designed a controller based on a two plasmid system.  One plasmid stably replicates the various biosynthetic operons of our system at single copy in a transcriptionally-inactive state.  The second plasmid houses the genes necessary for activation of the operon plasmid.  When activated with iron, the copy number of the operon plasmid increases to high-copy and the transcription of the operons is activated.
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==Design and construction==
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We designed a two-plasmid architecture in which the biosynthetic operons reside on a single-copy bacterial artificial chromosome (BAC).  The operons are under the transcriptional control of T7 promoters of various strengths.  The BAC also contains an R6K origin of replication.  In most strains of ''E. coli'', this origin is silent as it requires the expression of the ''pir'' gene for replication.  The second plasmid in our controller is a low-copy pSC101-derived plasmid that houses the T7 RNA polymerase and ''pir'' genes under the control of an iron-inducible promoter.<br><br>
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Our controller system is composed of two plasmids: a pSC101 Controller Plasmid, and an Amplifiable BAC.
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[[Image:Berk-DT Figure 1.png|575px]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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[[Image:Berk-DT Figure Legend.png|325px]]<br><br>
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===Construction of an iron-responsive PoPS-generating device===
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[[Image:BerkiGEM2007-yfbEcytometry.jpg|thumb|200px|right|'''An iron-inducible promoter.'''  Cells were transformed with an RFP transcriptional reporter device derived from our yfbE promoter part and grown with or without exogenous iron to various densities and then analyzed for fluorescence by cytometry.]]
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[[Image:Berk-DT Figure 1.png]]
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To construct this system, first we needed a promoter that was induced by iron.  Microarray studies suggested that the ''yfbE'' promoter of ''E. coli'' might function as an iron-responsive PoPS-generating device.  We therefore constructed a Biobrick derived from the ''yfbE'' promoter and constructed an RFP reporter composite part derived from this basic part.  We examined the fluorescence of cells harboring this part both as a function of external iron concentration and growth phase.  The ''yfbE'' promoter part had the ideal qualities for our controller:  it is induced 100-fold as the bacteria emerge from the mid-log phase of growth, but only in the presence of exogenous iron.
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[[Image:BerkiGEM2007-Figure-Piron-vector.png|thumb|left|200px|'''Vectorology of the iron promoter characterization construct''']]
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<br><br><br><br><br><br><br><br><br><br><br><br>
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[[Image:Berk-DT Figure Legend.png]]
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===Construction of an iron-dependent transcription device===
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To control gene expression we needed to place the T7 RNA polymerase under the control of the yfbE promoter on a pSC101-derived plasmid.  We therefore made a T7 RNA polymerase basic part and constructed a library of composite parts containing the yfbE part, one of nine ribosome binding site parts of different strengths, and the T7 RNA polymerase gene with a GTG or an ATG start codon.  We constructed these composite parts in the pSC101 Biobrick plasmid I716101 and then examined their activity in an engineered ''E. coli'' strain, GH455G, containing a genome-integrated cassette with GFP under the control of a T7 promoter.  Of the composite parts we constructed, only the composite part with the weakest ribosome binding site and a GTG start codon showed iron-dependent GFP production.  All composite parts with an ATG start were too active and toxic, while the other ribosome binding sites were either constitutively on or off.
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[[Image:BerkiGEM2007-Figure-T7-vector.png|thumb|left|367px|'''Vectorology of the T7 RNA Polymerase characterization construct''']]
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<br><br><br><br><br><br><br><br><br><br><br>
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'''Design and construction'''
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===Construction of an iron-dependent copy number device===
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[[Image:BerkiGEM2007-PirCytometry.jpg|thumb|200px|left|'''Iron-inducible Copy Number.'''  Cells containing an optimized ''yfbE-pir'' controller, a ''pir116'' strain, and a non-pir strain were transformed with an inducible BAC plasmid encoding GFP.  As the copy number increases, so does the amount of GFP produced.  Only the controller cells show iron-dependent copy number.]]
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[[Image:BerkiGEM2007-YfbEPirScreen.jpg|375px]]<br>
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The third component of our controller was an iron-inducible copy number amplifier system.  We therefore constructed a ''pir'' basic part and attempted to make composite parts with the ''yfbE'' promoter part.  We were unable to construct Biobrick-derived cassettes of this composition apparently due to toxicity.  Instead, we constructed a ribosome binding site-free version of the part in the pSC101-derived plasmid.  We subsequently introduced a library of ribosome binding sites by EIPCR in which the start codon was either ATG or GTG and positions 11-13 upstream of start codon were randomized.  We introduced the library plasmids into ''E. coli'' cells harboring plasmid pBACr-AraGFP which is a BAC plasmid with an R6K origin of replication and GFP under an arabinose-inducible promoter.  Due to the lack of a pir gene in these cells, only a low level of GFP production is observed when they are grown in arabinose media.  In contrast, the production of GFP in cells with an induced pir gene is 200-fold greater, similar to the GFP production in the pir116 strain.
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[[Image:BerkiGEM2007-Figure-pir-vector.png|thumb|left|400px|'''Vectorology of the ''pir'' characterization construct''']]
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<br><br><br><br><br><br><br><br><br><br><br>
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We designed a two-plasmid architecture in which the biosynthetic operons reside on a single-copy bacterial artificial chromosome (BAC).  The operons are under the transcriptional control of T7 promoters of various strengths.  The BAC also contains an R6K origin of replication.  In most strains of ''E. coli'', this origin is silent as it requires the expression of the ''pir'' gene for replication.  The second plasmid in our controller is a low-copy pSC101-derived plasmid that houses the T7 RNA polymerase and ''pir'' genes under the control of an iron-inducible promoter.
+
==Conclusion==
-
To construct this system, first we needed a promoter that was induced by iron.  Microarray studies *ref* suggested that the yfbE promoter of ''E. coli'' might function as an iron-responsive PoPS-generating device.  We therefore constructed a Biobrick derived from the ''yfbE'' promoter and constructed an RFP reporter composite part derived from this basic partWe examined the fluorescence of cells harboring this part both as a function of external iron concentration and growth phaseThe ''yfbE'' promoter part had the ideal qualities for our controller:  it is induced 100-fold as the bacteria emerge from the mid-log phase of growth, but only in the presence of exogenous iron.
+
Our controller system allows for a dramatic induction of the operons present on our BAC upon the introduction of free iron into the systemIt accomplishes this by utilizing both the ''pir'' gene to amplify the R6K origin, as well as the T7 RNA polymerase transcription systemThis provides not only excellent dynamic range, but also the ability to tune the relative expression levels of the operons under T7 promoter controlThese capabilities allow the Bactoblood organism to adopt its two distinct phenotypes required for normal growth in the laboratory, and static oxygen carrying in the bloodstream.
-
To control gene expression with the controller, we needed to place the T7 RNA polymerase under the control of the yfbE promoter on a pSC101-derived plasmid.  We therefore made a T7 RNA polymerase basic part and constructed a library of composite parts containing the yfbE part, one of nine ribosome binding site parts of different strengths, and the T7 RNA polymerase gene with a GTG or an ATG start codon.  We constructed these composite parts in the pSC101 Biobrick plasmid I716101 and then examined their activity in an engineered ''E. coli'' strain, GH455G, containing a genome-integrated cassette with GFP under the control of a T7 promoter.  Of the composite parts we constructed, only the composite part with the weakest ribosome binding site and a GTG start codon showed iron-dependent GFP production.
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<p align="justify">&nbsp;</p>
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The third component of our controller was an iron-inducible copy number amplifier system.  We therefore constructed a ''pir'' basic part and attempted to make composite parts with the ''yfbE'' promoter part. We were unable to construct Biobrick-derived cassettes of this composition apparently due to toxicity. Instead, we constructed a ribosome binding site-free version of the part in the pSC101-derived plasmid.  We subsequently introduced a library of ribosome binding sites by EIPCR in which the start codon was either ATG or GTG and the ###-## positions relative to the ribosome binding site were randomized (See table ##).  We introduced the library plasmids into ''E. coli'' cells harboring plasmid pBACr-AraGFP*ref* which is a BAC plasmid with an R6K origin of replication and GFP under an arabinose-inducible promoter.  Due to the lack of a pir gene in these cells, only a low level of GFP production is observed when they are grown in arabinose media.  In contrast, the production of GFP in
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<p align="center"><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></p>
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<p align="center"> <a href="https://2007.igem.org/BerkiGEM2007Present4">&lt;&lt;Previous Section: Chassis</a> | <a href="https://2007.igem.org/BerkiGEM2007Present5">Next Section: Genetic Self-Destruct&gt;&gt;</a></p>
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The pSC101 Controller Plasmid contains both the Self-Destruct Device, as well as the controller operon: The genes pir and the T7 RNA Polymerase under the control of an Iron Promoter. The pir gene activates the R6K origin, which causes the plasmid containing it to jump to high copy - the R6K origin is silent without pirThe T7 RNA Polymerase transcribes genes under the control of T7 Promoters, and will vary with strength depending on the variant of T7 Promoter.  The T7 Promoters are silent without the T7 RNA Polymerase, and have a high level of transcription when active.
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The Amplifiable BAC contains all the primary systems production genes, and two origins of replication.  It contains both the BAC origin and the R6K origin, and so is single-copy when pir is absent, and high-copy when pir is active.  Additionally, the BAC contains all the systems production genes, such as Hemoglobin, Heme (A to D), and the necessary chaperones, reductases, etc.  Each of these operons is under the control of a custom T7 Promoter variant, tuned to produce the relative amount of protein desired for each operon. For example, the heme genes are under a weak T7 Promoter, as the heme genes naturally produce a large amount, where not too much is needed. However, the Hemoglobin operon is under a strong T7 Promoter, as we want a large amount of Hemoglobin present to effectively carry oxygen.
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'''Results and Data'''
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[[Image:072007_yfbEgraph.jpg]]
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'''[[072007_neg | White Cell Negative]]'''
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[ Add Results and More Data Here ]
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<!-- As these incredibly awesome figures and charts clearly demonstrate, our system works ridiculously well, with  42 orders of magnitude of dynamic range (approximately). -->
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Latest revision as of 04:10, 27 October 2007

<<< Return to UC Berkeley iGEM 2007

<<Previous Section: Chassis | Next Section: Genetic Self-Destruct>>

The Controller

The Controller is an integrated genetic circuit, comprised of two plasmids, that directs the copy number and transcription of the primary devices in our system.

Introduction

The Bactoblood organism needs to exist in two different states: one form that is genetically stable and able to grow under normal laboratory conditions, and a second state that is highly differentiated, unable to grow, and devoid of genetic material. To bring about the transformation to the differentiated state, we needed a controller that could be easily triggered by an external cue. This controller required a large dynamic range between the off and on states, the ability to maintain and overexpress a large number of genes, and ideally employed a low-cost inducer. Therefore, we designed a controller based on a two plasmid system. One plasmid stably replicates the various biosynthetic operons of our system at single copy in a transcriptionally-inactive state. The second plasmid houses the genes necessary for activation of the operon plasmid. When activated with iron, the copy number of the operon plasmid increases to high-copy and the transcription of the operons is activated.

Design and construction

We designed a two-plasmid architecture in which the biosynthetic operons reside on a single-copy bacterial artificial chromosome (BAC). The operons are under the transcriptional control of T7 promoters of various strengths. The BAC also contains an R6K origin of replication. In most strains of E. coli, this origin is silent as it requires the expression of the pir gene for replication. The second plasmid in our controller is a low-copy pSC101-derived plasmid that houses the T7 RNA polymerase and pir genes under the control of an iron-inducible promoter.

Berk-DT Figure 1.png       Berk-DT Figure Legend.png

Construction of an iron-responsive PoPS-generating device

An iron-inducible promoter. Cells were transformed with an RFP transcriptional reporter device derived from our yfbE promoter part and grown with or without exogenous iron to various densities and then analyzed for fluorescence by cytometry.

To construct this system, first we needed a promoter that was induced by iron. Microarray studies suggested that the yfbE promoter of E. coli might function as an iron-responsive PoPS-generating device. We therefore constructed a Biobrick derived from the yfbE promoter and constructed an RFP reporter composite part derived from this basic part. We examined the fluorescence of cells harboring this part both as a function of external iron concentration and growth phase. The yfbE promoter part had the ideal qualities for our controller: it is induced 100-fold as the bacteria emerge from the mid-log phase of growth, but only in the presence of exogenous iron.

Vectorology of the iron promoter characterization construct













Construction of an iron-dependent transcription device

To control gene expression we needed to place the T7 RNA polymerase under the control of the yfbE promoter on a pSC101-derived plasmid. We therefore made a T7 RNA polymerase basic part and constructed a library of composite parts containing the yfbE part, one of nine ribosome binding site parts of different strengths, and the T7 RNA polymerase gene with a GTG or an ATG start codon. We constructed these composite parts in the pSC101 Biobrick plasmid I716101 and then examined their activity in an engineered E. coli strain, GH455G, containing a genome-integrated cassette with GFP under the control of a T7 promoter. Of the composite parts we constructed, only the composite part with the weakest ribosome binding site and a GTG start codon showed iron-dependent GFP production. All composite parts with an ATG start were too active and toxic, while the other ribosome binding sites were either constitutively on or off.

Vectorology of the T7 RNA Polymerase characterization construct












Construction of an iron-dependent copy number device

Iron-inducible Copy Number. Cells containing an optimized yfbE-pir controller, a pir116 strain, and a non-pir strain were transformed with an inducible BAC plasmid encoding GFP. As the copy number increases, so does the amount of GFP produced. Only the controller cells show iron-dependent copy number.

BerkiGEM2007-YfbEPirScreen.jpg
The third component of our controller was an iron-inducible copy number amplifier system. We therefore constructed a pir basic part and attempted to make composite parts with the yfbE promoter part. We were unable to construct Biobrick-derived cassettes of this composition apparently due to toxicity. Instead, we constructed a ribosome binding site-free version of the part in the pSC101-derived plasmid. We subsequently introduced a library of ribosome binding sites by EIPCR in which the start codon was either ATG or GTG and positions 11-13 upstream of start codon were randomized. We introduced the library plasmids into E. coli cells harboring plasmid pBACr-AraGFP which is a BAC plasmid with an R6K origin of replication and GFP under an arabinose-inducible promoter. Due to the lack of a pir gene in these cells, only a low level of GFP production is observed when they are grown in arabinose media. In contrast, the production of GFP in cells with an induced pir gene is 200-fold greater, similar to the GFP production in the pir116 strain.

Vectorology of the pir characterization construct












Conclusion

Our controller system allows for a dramatic induction of the operons present on our BAC upon the introduction of free iron into the system. It accomplishes this by utilizing both the pir gene to amplify the R6K origin, as well as the T7 RNA polymerase transcription system. This provides not only excellent dynamic range, but also the ability to tune the relative expression levels of the operons under T7 promoter control. These capabilities allow the Bactoblood organism to adopt its two distinct phenotypes required for normal growth in the laboratory, and static oxygen carrying in the bloodstream.

 

<<< Return to UC Berkeley iGEM 2007

<<Previous Section: Chassis | Next Section: Genetic Self-Destruct>>