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[[Category:CH391L_S13]]
= Introduction to Synthetic Biology =
 
  
This quotation about the Molecular Biology revolution of the first part of the twentieth century sets the stage for synthetic biology.
+
Ashley Kessel's first assignment: Cloned wiki (Feedback in discussion)
  
<blockquote>
+
==Introduction==
It was a quarter-century ago that Watson and Crick, playing with cardboard cutouts and wire-and-sheet-metal models and sorting out the few controlling facts from a hotchpotch of data, elucidated the molecular architecture of the genetic material itself, the double-railed circular staircase of deoxyribonucleic acid. What has been learned in the years since is full of surprises, full of wit and beauty, full of the most gratifying illumination. The culmination is now approaching of the great endeavor of biology that has swept on for a century and a quarter—an achievement of imagination that rivals the parallel, junior enterprise in physics that began with relativity and quantum mechanics. Biologists' pursuit of complete and explicit understanding has begun to list the exact molecular sequences that encode the hereditary message, instruction by instruction; it has tweezed apart the springs and gears by which the message is expressed in the building of the cell, and the ratchets and pawls by which that expression is regulated; it has accustomed men to speak apparently without wonder of the structural transformations by which a single protein molecule, an enzyme, will break or build other proteins, or by which, for example, a molecule of hemoglobin will flex its broad shoulders and bend its knees to pick up oxygen.
+
The ability to introduce exogenous DNA into an organism to alter its genetic program is one of the most crucial tools in modern biology. Early work showed that certain bacteria could acquire the traits of a related strain through the addition of heat-killed cells.  Although it was not well understood at the time, the transfer of gene-encoding DNA from one strain to another facilitated this.  This concept was turned into a useful tool upon the advent of bacterial plasmid transformations in the early 1970's, which allowed genes of interest to be easily inserted into ''E. coli''.  Over the years, methods have been developed to introduce exogenous genes into a wide range of useful organisms, including bacteria, yeasts, plants, and animal tissues.  These methods vary enormously in efficiency however, necessitating a way to identify and isolate cells which contain the DNA of interest. This can be accomplished either by screening for successfully modified cells, or through selection.<cite>Cohen72</cite>
</blockquote>
+
<blockquote>
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To be sure, the discoveries have not produced the great practical payout that has repeatedly been anticipated for them. Biologists have no atomic power stations and no bombs to point to, or at least not yet. No baby has been cured of a congenital deficiency by insertion of a missing gene into its cells. There is no vaccine against human leukemia, not even a cure for hay fever. Though some of the rewards are at last imminent, most scientists have learned that they must speak guardedly and emphasize to laymen the gaps to be filled in.
+
</blockquote>
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<blockquote>
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''The Eighth Day of Creation'', Horace Freeland Judson, 1979 <cite>Judson1979</cite>
+
</blockquote>
+
  
== The Culture of Synthetic Biology ==
+
===Screening vs. Selection===
 +
The process of screening requires checking every cell (or colony in the case of bacteria) for the gene(s) of interest.  This is often done with the aid of a screenable marker, like a fluorescent protein expressing gene, that allows rapid identification of transformed cells.  Alternatively, exogenous genes in bacterial colonies can be detected by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] or a variety of other methods without the use of a marker.  Screening is often time-consuming and expensive, particularly when the fraction of modified cells is low.  A much more efficient strategy, selection, involves growing cells in conditions which only allow for survival of those with the desired genes.  Selectable marker genes can be combined with any other genes of interest and used to select for their insertion without additional screening steps.  In bacteria, the most commonly used selectable markers provide resistance to antibiotics, allowing for positive selection of plasmids following transformation.
  
On the one hand, synthetic biology has its roots in conventional fields of science, including molecular biology, metabolic engineering, protein design, and bioengineering. One could say that it is simply a clever re-branding of advances in those fields. On the other hand, much of the "newness" of synthetic biology has been due to the impact of a do-it-yourself (DIY) mindset associated with the open-source movement and even hacker culture that is not traditional to the field of molecular biology. The idea is that putting the tools in the hands of young and creative scientists will result in rapid and creative progress. This is a philosophy of the [http://igem.org iGEM competition], for example. Critics of this approach would contend that such rapid progress and wide dissemination of these technologies without oversight risks having unintended adverse ecological and societal impacts. See the [[CH391L/S13/Ethics|synthetic biology ethics topic]].
+
==Antibiotic Resistance Markers: ''ampR''==
 +
Perhaps the most commonly used selectable marker in bacteria is the ''ampR'' gene, which provides resistance to certain beta-lactam antibiotics such as [http://en.wikipedia.org/wiki/Ampicillin ampicillin] (amp) and its more stable relative [http://en.wikipedia.org/wiki/Carbenicillin carbenicillin] (carb).  [http://en.wikipedia.org/wiki/Beta-lactam_antibiotic Beta-lactam antibiotics] are [http://en.wikipedia.org/wiki/Penicillin penicillin] derivatives that inhibit synthesis of bacterial [http://en.wikipedia.org/wiki/Peptidoglycan peptidoglycan] cell walls, arresting cell division and ultimately leading to cell death. Although beta-lactams are primarily functional against gram-positive bacteria due to their larger cell walls, some examples, such as amp and carb, are capable of killing gram-negative E. coli.  All antibiotics in the family share a central four atom ring structure known as the beta-lactam ring, which serves as a cleavage target for enzymes known as beta-lactamases.  Due to the shared structural features of beta-lactam antibiotics, these enzymes often have promiscuous activities that target multiple drugs.   Upon cleavage of the lactam ring, antibiotics such as amp lose their toxicity, allowing cell growth and division to resume.<cite>Sutcliffe78</cite> 
  
Links related to the flavor and hype of synthetic biology:
+
[[Image:amp.png|thumb|center|500px| The structure of ampicillin, including the central beta-lactam ring cleaved by the ''ampR'' gene product [http://en.wikipedia.org/wiki/Ampicillin Ampicillin on Wikipedia]]]
* [[Adventures|Adventures in Synthetic Biology Comic Book]]
+
* [[Media:16cribsheet.pdf|SEED Magazine's Synthetic Biology Cribsheet]]
+
* [http://www.fieldtest.us/bio.shtml Field Test Film Core Synthetic Biology documentary]
+
* [http://www.ted.com/talks/craig_venter_is_on_the_verge_of_creating_synthetic_life.html 2008 TED talk given by J.Craig Venter about the 1st Synthetic Genome Assembled ]
+
* [http://www.youtube.com/watch?v=ushmgPM7HT8 Imperial College iGEM team Wellcome Trust documentary (50 min)]
+
* [http://vimeo.com/19759432 E. chromi video]
+
* [https://www.facebook.com/IgemMemes iGEM memes]
+
  
== Timeline of Synthetic Biology ==
+
The common ''ampR'' gene, as used in the ''E. coli'' pBR322 plasmid, was naturally derived from [http://en.wikipedia.org/wiki/Salmonella ''Salmonella''] bacteria through [http://en.wikipedia.org/w/index.php?title=Transposition_(horizontal_gene_transfer)&redirect=no transposition].  Because it efficiently cleaves amp and carb, it's one of the most useful markers in ''E. coli''.  Ampicillin does not kill the bacteria quickly, so cells transformed with the ''ampR'' marker can be immediately plated on selective media, before they've produced a significant amount of beta-lactamase.  One downside to the use of amp is that it's rapid removal from selective media by beta-lactamase can allow for growth of cells that lack resistance.  This is often witnessed in the form of satellite colonies on amp plates.  The use of carbenicillin for selection reduces the problem, as carb is less rapidly degraded by the ''ampR'' beta-lactamase, but carb is significantly more expensive than amp. <cite>Sutcliffe78</cite> <br>
  
{| cellpadding="4"
 
|- bgcolor="lightgray"
 
! Year !! Event
 
|-
 
| 1970 || Discovery of first site-specific restriction enzyme <cite>Smith70</cite>
 
|-
 
| 1972 || First publication on recombinant DNA <cite>Jackson1972</cite>
 
|-
 
| 1976 || First genome sequenced- Bacteriophage MS2 <cite>Fiers1976</cite>
 
|-
 
| 1981 || Introduction of phosphoramidite method of DNA oligonucleotide synthesis. <cite>Matteucci1981</cite>
 
|-
 
|1983 || First genetically modified plant is produced <cite>Fraley</cite>
 
|-
 
|2000 || The "Repressilator" <cite>Elowitz2000</cite>
 
|-
 
|2002 || Creation of the first synthetic virus <cite>Cello2002</cite>
 
|-
 
| 2003 || First use of the term "BioBrick" <cite>Knight2003</cite> First chemically synthesized bacteriophage  genome assembled <cite>Smith2003</cite>
 
|-
 
| 2004 || First iGEM Jamboree
 
|-
 
| 2005 || "Adventures in Synthetic Biology" ''Nature'' Cover
 
|-
 
| 2006 || Human Genome Project completed <cite>Gregory2006</cite>
 
|-
 
| 2007 || First application of context-free grammars in CAD software for Synthetic Biology ([http://www.genocad.org GenoCAD]) <cite>Cai2007</cite>
 
|-
 
| 2008 || First synthetic genome assembled <cite>Gibson2008</cite>
 
|-
 
| 2010 || Creation of ''Mycoplasma mycoides'' JCVI-syn1.0, the first microbe with a self-replicating synthetic genome. <cite>Gibson2010</cite>
 
|-
 
| 2012 || Storage of digital information using DNA popularized in the media, ''Economist'' ([http://www.economist.com/news/science-and-technology/21570671-archives-could-last-thousands-years-when-stored-dna-instead-magnetic Archives Could Last Thousands of Years when Stored on DNA])
 
|}
 
  
== What is synthetic biology? ==
+
Marker genes that provide resistance to [http://en.wikipedia.org/wiki/Kanamycin kanamycin] (kan) and [http://en.wikipedia.org/wiki/Chloramphenicol chloramphenicol] (cap) are also popular for use in ''E. coli'', as kan and cap are more stable than ampicillin.  Bacterial cells transformed with kan and cap resistance markers must be allowed to recover before plating however.  Additionally, multiple antibiotics may also be necessary to simultaneously select for markers on separate plasmids.  For more information about how they work, see the links below: <br>
 +
[http://en.wikipedia.org/wiki/Kanamycin Kanamycin on Wikipedia] <br>
 +
[http://en.wikipedia.org/wiki/Chloramphenicol Chloramphenicol on Wikipedia] <br>
  
Types of studies referred to as synthetic biology (and other fields that might also claim them).
+
==Antibiotic/Non-Antibiotic Dual Selection: ''tetA(C)''==
 +
===Tetracycline Positive Selection===
 +
The ''tetA(C)'' gene is primarily used for positive selection in bacteria, similar to ''ampR''.  ''tetA(C)'' encodes a membrane-bound transporter that rapidly pumps the antibiotic [http://en.wikipedia.org/wiki/Tetracycline tetracycline] out of bacterial cells.  This process is energy dependent, as the protein uses the influx of H+ ions from the surrounding environment to drive the process.  Tetracycline is a broad-spectrum, polyketide antibiotic derived from ''Streptomyces'' that inhibits bacterial translation.  The antibiotic binds the 30s subunit of bacterial ribosomes, blocking entry of aminoacyl-tRNAs to the A site of the ribosome.<cite>McNicholas92</cite> 
  
* "The goal of synthetic biology is to extend or modify the behavior of organisms and engineer them to perform new tasks."<cite>Andrianantoandro2006</cite>
+
[[ The structure of tetracycline. [http://en.wikipedia.org/wiki/Tetracycline Tetracycline on Wikipedia]]]
* "Synthetic biologists come in two broad classes. One uses unnatural molecules to reproduce emergent behaviours from natural biology, with the goal of creating artificial life. The other seeks interchangeable parts from natural biology to assemble into systems that function unnaturally." <cite>Benner2005</cite>
+
* Bottom-up assembly of genes, organelles and organisms.
+
** In contrast to traditional "top-down" genetic approaches that look for mutated versions of existing organisms.
+
** Ex:Re-factoring and re-writing genomes from scratch.
+
** Create chemical systems with biological behaviors (e.g., self-replication).
+
* Application of engineering principles to biology.
+
** Standardized parts that give predictable outcomes when put together in different combinations.
+
** Instantiating algorithms and problems from physics and math into biology. (e.g., oscillators)
+
** Ex: circuits, DNA computing, metabolic engineering
+
** Rewriting biological sequences in ways that could not be achieved (quickly) by natural evolution
+
  
== Synthetic Biology Workshops ==
+
Because tetracycline resistance is gained through expression of a transporter and not a modifying enzyme, selective media maintains its antibiotic levels during growth of resistant cells.  High levels of ''tetA'' expression can have a detrimental effect on the cells however, due the energy cost of pumping tetracycline and increased vulnerability to certain extracellular conditions.  This often results in fewer viable cells when using tetracycline selection compared to selection with antibiotics like ampicillin and kanamycin.<cite>Podolsky96</cite>
  
* [http://meetings.cshl.edu/courses/2013/c-synbio13.shtml 2003 Cold Spring Harbor Laboratory Synthetic Biology Course]
+
===Nickel Salt Negative Selection===
 +
Expression of ''tetA'' in bacteria has the side effect of making cells more vulnerable to poisoning by either lipophilic chelating agents or metal salts. Although ''tetA'' expressing ''Salmonella'' strains were shown to be extremely sensitive to chelating agents such as fusaric or quinaldic acids, these compounds are only marginally effective on ''E. coli''.  Based on earlier observations that metals like cadmium inhibited growth of tetracycline-resistant bacteria, a technique was developed that uses nickel salts to select against ''tetA'' expressing E. coli with much greater efficiency.  Cheap, non-toxic Nickel Chloride is the most commonly used selection agent.  Although originally used to select for cells that had lost a ''tetA'' marker, this method has proven useful in dual selection schemes for evolving regulators of gene expression. <cite>Podolsky96,Nomura07</cite>
  
== Synthetic Biology Conferences/Sessions ==
+
===Engineering Riboswitches using ''tetA'' Dual Selection===
 +
The evolution of functional [http://www.openwetware.org/wiki/CH391L/S13/Riboswitches Riboswitches] requires selection of a library of mutants in both an ON state and an OFF state.  This removes variants that either activate or repress gene expression regardless of the small molecule used to regulate them.  Previous attempts at dual selection required the use of both positive and negative selection markers.  This necessitated intermediate steps to purify plasmids from the pool and re-transform them along with either the positive or negative marker.  Aside from being labor intensive, this method also increased the rate of false positives in the pool <cite>Collins06,Muranaka09</cite>.
  
Looking over the schedules of recent conferences is an excellent way to find new topics of interest or finds labs that are involved in synthetic biology.
+
The fact that a single ''tetA'' gene can be used for both positive and negative selection greatly simplifies the process of selecting for riboswitches, as a culture of cells expressing a library of riboswitches can be alternately swapped between media that selects for or against expression of the tetracycline transporter.  This strategy was used by the [http://yokobayashilab.net/ Yokobayashi Lab] at [http://www.ucdavis.edu UC Davis] to select for mutants of the ''E. coli'' TPP riboswitch (from ''thiM'') that activated downstream gene expression in the presence of thiamine instead of repressing it.  A library of TPP riboswitch variants was cloned upstream of a ''tetA'' marker and transformed into ''E. coli''.  The first round of selection involved plating transformants on media with thiamine and tetracycline to select for mutants that were turned ON.  The survivors were then transferred to media with nickel chloride and no thiamine to remove any variants that did not turn off in the absence of thiamine.  A similar approach can be carried out in liquid culture, as shown below.<cite>Nomura07</cite>.
  
[http://sb6.biobricks.org/about/ SBx.0 Conference Series]
+
[[Image:DualSelection.png|thumb|center|550px| A ''tetA'' dual selection scheme as used by the [http://2011.igem.org/Team:Peking_R/Project/RNAToolkit4 2011 Peking R iGEM team] to select for ribozymes based on a TPP aptamer fused to the hammerhead ribozyme. A similar scheme was used to switch the ON/OFF behavior of the TPP riboswitch. [http://2011.igem.org/Team:Peking_R/Project/RNAToolkit4 Team Website]]]
* Synthetic Biology 6.0 (2013) [http://sb6.biobricks.org/ main page]
+
* Synthetic Biology 5.0 (2011) [http://sb5.biobricks.org/schedule/ schedule]
+
  
ASM
+
===Using a ''tetA'' Fusion Protein for Monitoring Selection===
* American Society for Microbiology General Meeting (2013) [http://gm.asm.org/ schedule] [[:Image:CH391L_S13_ASM_Program_1.png | session1]] [[:Image:CH391L_S13_ASM_Program_1.png | session2]]
+
More recent work by the [http://yokobayashilab.net/ Yokobayashi Lab] involved combining the ''tetA'' marker with GFP to allow screening of ''thiM'' riboswitch function following each round of dual selection. The ''tetA'' transporter remains functional when GFP is fused to its c-terminal end, facilitating selection and screening steps with the same gene product. To measure function of different riboswitch variants in the selection pool, samples were plated on LB and individual colonies were picked and used to seed culture in non-selective media.  GFP fluorescence of each culture was measured to determine the amount of gene expression generated by that particular riboswitch, and functional variants were sequenced<cite>Muranaka09</cite>.  
* American Society for Microbiology General Meeting (2012) [[:Image:CH391L_S12_ASM2012Program.png | session]]
+
  
Keystone
+
==Auxotrophic Marker Selection: GFAT==
* Keystone Conference: Precision Genome Engineering and Synthetic Biology [http://www.keystonesymposia.org/index.cfm?e=web.Meeting.Program&meetingid=1238 schedule]
+
An alternative method of positive selection involves the use of auxotrophs, or cells that lack the ability to synthesize a compound necessary for their own growth.  In bacteria and yeast, useful auxotrophic strains are easily generated by knocking out a single gene involved in a chemical synthesis pathway. The resulting strains can be grown in media containing the essential product but require complementation with an exogenous copy of the missing gene (the auxotrophic marker) to remain viable in media lacking it. Auxotrophic markers are extremely useful in yeast, where the range of useful antibiotic markers is limited, but they are also used in bacterial applications where introduction of antibiotic-resistance genes could be problematic. Recently, a single gene involved in glucosamine synthesis was shown to be a useful auxotrophic marker in ''E. coli'' and fission yeast (''S. pombe'').
  
ICBE—International Conference on Biomolecular Engineering
+
Glutamine:fructose-6-phosphate aminotransferase (GFAT) is an enzyme required for the biosynthesis of hexosamines in both bacteria and eukaryotes.  GFAT catalyzes the rate-limiting first step in the pathway, converting fructose-6-phosphate into glucosamine-6-phosphate.  In ''E. coli'', GFAT is expressed from the ''glmS'' gene, which is necessary for growth in minimal media.  Recent work has shown that ''E. coli'' lacking ''glmS'' are capable of growing at near wild type levels when supplemented with glucosamine however, and complementation with a plasmid-encoded ''glmS'' gene restores hexosamine synthesis and growth<cite>Wu11</cite>.
*4th ICBE (2013) [http://www.aiche.org/sbe/conferences/international-conference-biomolecular-engineering-icbe/2013/icbe-conference-schedule schedule]
+
 
 +
[[Image:GFAT.png|thumb|center|400px| '''The growth condition of E. coli K12 ΔglmS in different medium.''' (A) The growth condition of E. coli K12 ΔglmS in M9 minimal medium supplemented with glucosamine and different concentration of yeast extract respectively. (B) The growth condition of E. coli K12 ΔglmS in MmGTV, MT, MG, MGTV, MGT medium (M: M9 medium, Mm: M9 medium subtracting Mg2+ and glucose, G: glucosamine, T: tryptone, V: vitamin mixture)<cite>Wu11</cite>. doi:10.1371/journal.pone.0017082.g004]]
 +
 
 +
Selection of ''glmS'' expressing cells is complicated by the fact that rich media such as LB contains sufficient glucosamine to support limited growth of ''glmS'' knockout cells.  Growth tests using M9 minimal media with individual LB components added to it showed that yeast extract is the source of glucosamine that allows growth of the knockout strain.  Thus, selections with GFAT markers are limited to minimal media, but the addition of tryptone increases growth rates while still allowing selection.  Under these conditions, selection for ''glmS'' was shown to be an effective replacement for ''ampR'' selection<cite>Wu11</cite>.
 +
 
 +
==Future Directions==
 +
Although the currently available set of selectable markers has helped us to overcome many of the challenges involved in synthetic biology, the continued discovery of new markers may greatly simplify the process of designing organisms with useful functions.  The variety of useful markers available for engineering of higher eukaryotes, including animals and human tissue culture lines pales in comparison to those available for use in bacteria or even yeast.  Thus, the discovery of new selection schemes may allow experiments that are not currently feasible.
 +
 
 +
Another area of ongoing progress is the discovery of non-antibiotic markers to potentially replace commonly used genes like ''ampR'' in bacterial engineering.  Although antibiotic resistance is an increasingly common trait in wild bacteria, the possibility of engineered organisms transferring their resistance to otherwise susceptible bacteria is a frequent cause for alarm.  If engineered bacteria are to prove useful for tasks like bio-remediation, we will have to engineer them without the benefit of antibiotic selection or markers that require growth in minimal media.
 +
 
 +
See Also: <br>
 +
[http://openwetware.org/wiki/CH391L/S12/Selectablegeneticmarkers Selectable Genetic Markers] <br>
 +
[http://openwetware.org/wiki/CH391L/S12/ToxinAntitoxins Toxin/Antitoxin Systems] <br>
 +
[http://openwetware.org/wiki/CH391L/S12/CounterSelection Counterselection] <br>
 +
 
 +
==iGEM Connections==
 +
The [http://2011.igem.org/Team:Peking_R 2011 Peking_R iGEM team] from Peking University used ''tetA'' dual selection to engineer a ribozyme-based "genetic rheostat."  Their approach involved fusing the TPP aptamer domain from the ''thiM'' riboswitch to a section of the hammerhead ribozyme.  Portions of the construct were randomized and selections were carried out in a similar fashion to those performed by the [http://yokobayashilab.net/ Yokobayashi Lab]<cite>Muranaka09</cite>.  This approach did increase the ratio of ON/OFF ribozymes in their pool, and similar selections using an adenine aptamer domain also demonstrated the potential of the [http://2011.igem.org/Team:Peking_R/Project/RNAToolkit4 approach].
  
==Read More==
 
Special Synthetic Biology Edition in the [http://www.sciencedirect.com/science/journal/13675931/16/3 August 2012 Current Opinion in Chemical Biology ]
 
  
 
== References ==  
 
== References ==  
  
 
<biblio>
 
<biblio>
#Smith70 Smith H and Welcox KW. A Restriction enzyme from Hemophilus influenzae ☆: I. Purification and general properties. 1970. pmid:5312500 http://www.sciencedirect.com/science/article/pii/002228367090149X
+
#Cohen72 pmid=4559594
#Jackson1972 pmid=4342968 http://www.sciencedirect.com/science/article/pii/002228367090149X
+
//CaCl<sub>2</sub> bacterial transformations
//Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of ''Escherichia coli''.
+
#Collins06 pmid=16715074
#Elowitz2000 pmid=10659856
+
//Dual selection with separate markers
#Fraley  Fraley, RT et al. (1983) Expression of bacterial genes in plant cells. Proc. NatL. Acad. Sci. USA 80: 4803–4807  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC384133/
+
#McNicholas92 pmid=1459940
#Knight2003 Knight, T. Idempotent Vector Design for Standard Assembly of Biobricks. 2003.
+
//The tetA(C) gene from pBR322
http://hdl.handle.net/1721.1/21168
+
#Muranaka09 pmid=19190095
#Smith2003 pmid=14657399
+
//Riboswitch selection/screening using a tetA-GFP fusion marker
//Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides.
+
#Nomura07 pmid=17944473
#Judson1979 isbn=0879694785
+
//Reengineering the TPP riboswitch using ''tetA'' dual selection
#Gregory2006 Gregory et al. The DNA sequence and biological annotation of human chromosome 1. 2006.
+
#Podolsky96 pmid=8954882
http://www.nature.com/nature/journal/v441/n7091/full/nature04727.html
+
//Nickel selection with ''tetA''
#Andrianantoandro2006 pmid=16738572
+
#Sutcliffe78 pmid=358200
// Synthetic biology: new engineering rules for an emerging discipline
+
//Background on the ''ampR'' gene from pBR322
#Benner2005 pmid=15995697
+
#Wu11 pmid=21340036
// Synthetic biology
+
//GFAT as a selectable marker
#Gibson2010 pmid=20488990
+
<!--below:First publication of a CAD program for synthetic bio-->
+
#Cai2007 pmid=17804435
+
#Gibson2008 pmid=18218864
+
#Cello2002 pmid=12114528
+
#Fiers1976 Fiers, W et al. 1976. Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene.
+
http://www.regensci.org/twiki/pub/IGEM2009/BrainStorming/ms2article.pdf 
+
#Matteucci1981 Matteucci MD and Caruthers MH. 1981. Synthesis of Deoxyoligonucleotides on a Polymer Support. http://pubs.acs.org/doi/abs/10.1021/ja00401a041?prevSearch=matteucci%252C%2Bcaruthers%252C%2B1981&searchHistoryKey=
+
 
</biblio>
 
</biblio>

Latest revision as of 20:26, 28 January 2014


Ashley Kessel's first assignment: Cloned wiki (Feedback in discussion)

Contents

Introduction

The ability to introduce exogenous DNA into an organism to alter its genetic program is one of the most crucial tools in modern biology. Early work showed that certain bacteria could acquire the traits of a related strain through the addition of heat-killed cells. Although it was not well understood at the time, the transfer of gene-encoding DNA from one strain to another facilitated this. This concept was turned into a useful tool upon the advent of bacterial plasmid transformations in the early 1970's, which allowed genes of interest to be easily inserted into E. coli. Over the years, methods have been developed to introduce exogenous genes into a wide range of useful organisms, including bacteria, yeasts, plants, and animal tissues. These methods vary enormously in efficiency however, necessitating a way to identify and isolate cells which contain the DNA of interest. This can be accomplished either by screening for successfully modified cells, or through selection.[1]

Screening vs. Selection

The process of screening requires checking every cell (or colony in the case of bacteria) for the gene(s) of interest. This is often done with the aid of a screenable marker, like a fluorescent protein expressing gene, that allows rapid identification of transformed cells. Alternatively, exogenous genes in bacterial colonies can be detected by PCR or a variety of other methods without the use of a marker. Screening is often time-consuming and expensive, particularly when the fraction of modified cells is low. A much more efficient strategy, selection, involves growing cells in conditions which only allow for survival of those with the desired genes. Selectable marker genes can be combined with any other genes of interest and used to select for their insertion without additional screening steps. In bacteria, the most commonly used selectable markers provide resistance to antibiotics, allowing for positive selection of plasmids following transformation.

Antibiotic Resistance Markers: ampR

Perhaps the most commonly used selectable marker in bacteria is the ampR gene, which provides resistance to certain beta-lactam antibiotics such as ampicillin (amp) and its more stable relative carbenicillin (carb). Beta-lactam antibiotics are penicillin derivatives that inhibit synthesis of bacterial peptidoglycan cell walls, arresting cell division and ultimately leading to cell death. Although beta-lactams are primarily functional against gram-positive bacteria due to their larger cell walls, some examples, such as amp and carb, are capable of killing gram-negative E. coli. All antibiotics in the family share a central four atom ring structure known as the beta-lactam ring, which serves as a cleavage target for enzymes known as beta-lactamases. Due to the shared structural features of beta-lactam antibiotics, these enzymes often have promiscuous activities that target multiple drugs. Upon cleavage of the lactam ring, antibiotics such as amp lose their toxicity, allowing cell growth and division to resume.[2]

File:Amp.png
The structure of ampicillin, including the central beta-lactam ring cleaved by the ampR gene product Ampicillin on Wikipedia

The common ampR gene, as used in the E. coli pBR322 plasmid, was naturally derived from Salmonella bacteria through transposition. Because it efficiently cleaves amp and carb, it's one of the most useful markers in E. coli. Ampicillin does not kill the bacteria quickly, so cells transformed with the ampR marker can be immediately plated on selective media, before they've produced a significant amount of beta-lactamase. One downside to the use of amp is that it's rapid removal from selective media by beta-lactamase can allow for growth of cells that lack resistance. This is often witnessed in the form of satellite colonies on amp plates. The use of carbenicillin for selection reduces the problem, as carb is less rapidly degraded by the ampR beta-lactamase, but carb is significantly more expensive than amp. [2]


Marker genes that provide resistance to kanamycin (kan) and chloramphenicol (cap) are also popular for use in E. coli, as kan and cap are more stable than ampicillin. Bacterial cells transformed with kan and cap resistance markers must be allowed to recover before plating however. Additionally, multiple antibiotics may also be necessary to simultaneously select for markers on separate plasmids. For more information about how they work, see the links below:
Kanamycin on Wikipedia
Chloramphenicol on Wikipedia

Antibiotic/Non-Antibiotic Dual Selection: tetA(C)

Tetracycline Positive Selection

The tetA(C) gene is primarily used for positive selection in bacteria, similar to ampR. tetA(C) encodes a membrane-bound transporter that rapidly pumps the antibiotic tetracycline out of bacterial cells. This process is energy dependent, as the protein uses the influx of H+ ions from the surrounding environment to drive the process. Tetracycline is a broad-spectrum, polyketide antibiotic derived from Streptomyces that inhibits bacterial translation. The antibiotic binds the 30s subunit of bacterial ribosomes, blocking entry of aminoacyl-tRNAs to the A site of the ribosome.[3]

[[ The structure of tetracycline. Tetracycline on Wikipedia]]

Because tetracycline resistance is gained through expression of a transporter and not a modifying enzyme, selective media maintains its antibiotic levels during growth of resistant cells. High levels of tetA expression can have a detrimental effect on the cells however, due the energy cost of pumping tetracycline and increased vulnerability to certain extracellular conditions. This often results in fewer viable cells when using tetracycline selection compared to selection with antibiotics like ampicillin and kanamycin.[4]

Nickel Salt Negative Selection

Expression of tetA in bacteria has the side effect of making cells more vulnerable to poisoning by either lipophilic chelating agents or metal salts. Although tetA expressing Salmonella strains were shown to be extremely sensitive to chelating agents such as fusaric or quinaldic acids, these compounds are only marginally effective on E. coli. Based on earlier observations that metals like cadmium inhibited growth of tetracycline-resistant bacteria, a technique was developed that uses nickel salts to select against tetA expressing E. coli with much greater efficiency. Cheap, non-toxic Nickel Chloride is the most commonly used selection agent. Although originally used to select for cells that had lost a tetA marker, this method has proven useful in dual selection schemes for evolving regulators of gene expression. [4, 5]

Engineering Riboswitches using tetA Dual Selection

The evolution of functional Riboswitches requires selection of a library of mutants in both an ON state and an OFF state. This removes variants that either activate or repress gene expression regardless of the small molecule used to regulate them. Previous attempts at dual selection required the use of both positive and negative selection markers. This necessitated intermediate steps to purify plasmids from the pool and re-transform them along with either the positive or negative marker. Aside from being labor intensive, this method also increased the rate of false positives in the pool [6, 7].

The fact that a single tetA gene can be used for both positive and negative selection greatly simplifies the process of selecting for riboswitches, as a culture of cells expressing a library of riboswitches can be alternately swapped between media that selects for or against expression of the tetracycline transporter. This strategy was used by the Yokobayashi Lab at UC Davis to select for mutants of the E. coli TPP riboswitch (from thiM) that activated downstream gene expression in the presence of thiamine instead of repressing it. A library of TPP riboswitch variants was cloned upstream of a tetA marker and transformed into E. coli. The first round of selection involved plating transformants on media with thiamine and tetracycline to select for mutants that were turned ON. The survivors were then transferred to media with nickel chloride and no thiamine to remove any variants that did not turn off in the absence of thiamine. A similar approach can be carried out in liquid culture, as shown below.[5].

File:DualSelection.png
A tetA dual selection scheme as used by the 2011 Peking R iGEM team to select for ribozymes based on a TPP aptamer fused to the hammerhead ribozyme. A similar scheme was used to switch the ON/OFF behavior of the TPP riboswitch. Team Website

Using a tetA Fusion Protein for Monitoring Selection

More recent work by the Yokobayashi Lab involved combining the tetA marker with GFP to allow screening of thiM riboswitch function following each round of dual selection. The tetA transporter remains functional when GFP is fused to its c-terminal end, facilitating selection and screening steps with the same gene product. To measure function of different riboswitch variants in the selection pool, samples were plated on LB and individual colonies were picked and used to seed culture in non-selective media. GFP fluorescence of each culture was measured to determine the amount of gene expression generated by that particular riboswitch, and functional variants were sequenced[7].

Auxotrophic Marker Selection: GFAT

An alternative method of positive selection involves the use of auxotrophs, or cells that lack the ability to synthesize a compound necessary for their own growth. In bacteria and yeast, useful auxotrophic strains are easily generated by knocking out a single gene involved in a chemical synthesis pathway. The resulting strains can be grown in media containing the essential product but require complementation with an exogenous copy of the missing gene (the auxotrophic marker) to remain viable in media lacking it. Auxotrophic markers are extremely useful in yeast, where the range of useful antibiotic markers is limited, but they are also used in bacterial applications where introduction of antibiotic-resistance genes could be problematic. Recently, a single gene involved in glucosamine synthesis was shown to be a useful auxotrophic marker in E. coli and fission yeast (S. pombe).

Glutamine:fructose-6-phosphate aminotransferase (GFAT) is an enzyme required for the biosynthesis of hexosamines in both bacteria and eukaryotes. GFAT catalyzes the rate-limiting first step in the pathway, converting fructose-6-phosphate into glucosamine-6-phosphate. In E. coli, GFAT is expressed from the glmS gene, which is necessary for growth in minimal media. Recent work has shown that E. coli lacking glmS are capable of growing at near wild type levels when supplemented with glucosamine however, and complementation with a plasmid-encoded glmS gene restores hexosamine synthesis and growth[8].

File:GFAT.png
The growth condition of E. coli K12 ΔglmS in different medium. (A) The growth condition of E. coli K12 ΔglmS in M9 minimal medium supplemented with glucosamine and different concentration of yeast extract respectively. (B) The growth condition of E. coli K12 ΔglmS in MmGTV, MT, MG, MGTV, MGT medium (M: M9 medium, Mm: M9 medium subtracting Mg2+ and glucose, G: glucosamine, T: tryptone, V: vitamin mixture)[8]. doi:10.1371/journal.pone.0017082.g004

Selection of glmS expressing cells is complicated by the fact that rich media such as LB contains sufficient glucosamine to support limited growth of glmS knockout cells. Growth tests using M9 minimal media with individual LB components added to it showed that yeast extract is the source of glucosamine that allows growth of the knockout strain. Thus, selections with GFAT markers are limited to minimal media, but the addition of tryptone increases growth rates while still allowing selection. Under these conditions, selection for glmS was shown to be an effective replacement for ampR selection[8].

Future Directions

Although the currently available set of selectable markers has helped us to overcome many of the challenges involved in synthetic biology, the continued discovery of new markers may greatly simplify the process of designing organisms with useful functions. The variety of useful markers available for engineering of higher eukaryotes, including animals and human tissue culture lines pales in comparison to those available for use in bacteria or even yeast. Thus, the discovery of new selection schemes may allow experiments that are not currently feasible.

Another area of ongoing progress is the discovery of non-antibiotic markers to potentially replace commonly used genes like ampR in bacterial engineering. Although antibiotic resistance is an increasingly common trait in wild bacteria, the possibility of engineered organisms transferring their resistance to otherwise susceptible bacteria is a frequent cause for alarm. If engineered bacteria are to prove useful for tasks like bio-remediation, we will have to engineer them without the benefit of antibiotic selection or markers that require growth in minimal media.

See Also:
Selectable Genetic Markers
Toxin/Antitoxin Systems
Counterselection

iGEM Connections

The 2011 Peking_R iGEM team from Peking University used tetA dual selection to engineer a ribozyme-based "genetic rheostat." Their approach involved fusing the TPP aptamer domain from the thiM riboswitch to a section of the hammerhead ribozyme. Portions of the construct were randomized and selections were carried out in a similar fashion to those performed by the Yokobayashi Lab[7]. This approach did increase the ratio of ON/OFF ribozymes in their pool, and similar selections using an adenine aptamer domain also demonstrated the potential of the approach.


References

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All Medline abstracts: PubMed | HubMed