Synthetic Biology Overview

EVERYTHING YOU NEED TO KNOW TO GET STARTED IN SYNTHETIC BIOLOGY

Janie Brennan

Purdue University

5/16/12

Outline

Introduction (definitions and history)Fundamentals of molecular biologyAssembly methodsPart characterizationModelingDesign theoryMore about iGEMOther stuff you might want to know

Disclaimer

This is NOT a comprehensive look at Synthetic Biology.just an introduction

It may not be *everything* you need to know, but Ill try to get closeFor everything else, theres Wikipedia

For more detailed topics, other people will be a much better source of information:Transcriptional control strategies, the Registry, and knowledge about what types of parts are possible: Sean KearneyLab Equipment: Jenna Rickus and Rickus lab grad studentsCloning particulars/Reagents: Dow AgroSciences

What is synthetic biology (synbio)?

Synthetic Biology is:The design and construction of new biological parts, devices, and systems, andThe re-design of existing, natural biological systems for useful purposes.

(from SyntheticBiology.org)

Basically, its genetic engineering (aka molecular biology) for practical purposesTry to look at genetics from engineering paradigmGenes and different genetic components = parts Groups of components = devicesModel devices as circuits

Whos in charge?

The Biobricks Foundation (BBF) is one of the biggest initiatives worldwide driving the development of synbio

Goals of the BBF:Educate the public about synbio and biotechnologyMake biotech simple to do and available to the public Define ethical concerns and practices in synbio

The BBF developed and maintains the Parts Registry

How does iGEM fit into this?

Both the BBF and iGEM were started by Drew Endy and Tom Knight of MIT in the early 2000s

iGEM has increased the public interest in synbio and helped drive the development of standard parts and methods

BBF and iGEM are also related to the BioBuilder Foundation, which hosts the educational BioBuilder website This is where Dr. Clase got a lot of the IT 227 course materialBased a lot on the material developed for the MIT course 20.020, developed by Natalie Kuldell (who is President of BioBuilder)

Outline


Fundamentals of molecular biology

.or What makes up a device?

For this section, I drew a lot of basic information from the Registry: http://partsregistry.org

You can also find most of this (minus the synbio-specific terminology) in any genetics or biochemistry textbook (or Wikipedia)

Some of this is probably review, but I figured it would be better to include too much rather than too little

Synbio jargon

A part is a DNA sequence encoding some part of the genetic machinery, including but not limited to:PromotersRibosome binding sites (RBS)Protein coding regionsTerminators

A device is a group of parts that work together for specific functions, such as:Protein productionSensing/reportingMeasurementSignal inversionCell signalingCell motility

More jargon

A chassis is the organism (host) containing the devicesUsually E. coli or S. cerevisiae (yeast)Also can be bacteriophage (virus), plants, mammals, etcanything, really

Cloning does not refer to Dolly the sheep!Refers to manipulation of recombinant DNA

Promoters

Control transcription of downstream coding region by recruiting RNA polymerasesCan be constitutive (always on) or controlled by external signalsOften constitute the main logical part of your device.The promoter is basically an if statement (to use a programming analogy)Sensitivity to external signals compose the AND/OR/NOT logicPromoters can be sensitive to things like repressor proteins, activator proteins/transcription factors, metals, or other factors in cell signalingPromoters have a wide range of strength and activitiesThey can even be designed to suit your own purposes! http://partsregistry.org/Promoters/Design

Ribosome binding sites (RBS)

Control translation level by recruiting ribosomes to the mRNA transcriptTherefore, an RBS is necessary if you want to make a proteinMost are constitutiveMany strengths are availableLike promoters, RBSs can be designed to suit your project: http://partsregistry.org/Ribosome_Binding_Sites/Design

Protein coding sequences (CDS)

DNA sequences that encode mRNA transcripts that later get translated into proteins by ribosomesBegin with a start codon (ATG) encoding methionineEnd with stop codons (often TAA TAA)Can also include special features at each endAt N-terminus (front end): export tags, attachment tags, or protein cleavage sitesAt C-terminus (tail end): degradation tagsCommon protein products:Reporters (produce a measurable signal like GFP, etc)Transcriptional regulators (activate/repress transcription)Selection markers (antibiotic resistance, etc)Enzymes for biosynthesis, DNA modification (ligases/polymerases/etc.), degradation/cleavage (proteases), etc.Membrane proteins (surface display, transporters, channels, pumps)Receptors/ligandsAs you might have guessed, CDSs can also be designed: http://partsregistry.org/Help:Protein_coding_sequences/Design

Terminators

Causes transcription to stop (causes polymerase to fall off mRNA)Can terminate transcription on the forward strand, reverse strand, or bothDont stop transcription with 100% efficiencyStrength can be tuned/designed, tooCan be rho-dependent or rho-independentRho-independent terminators work based on stem-loop structures formed by GC-rich DNA sequences (pictured below)The Registry only includes rho-independent terminatorsYou need one (or two) of these in all your devices!

Outline


How do I make my device?

Lots of ways to do it, but there are two I would specifically recommend:3A Assembly (similar to Standard Assembly)Based on traditional cloning methodsIf a part is designed to work with these methods, it is said to be a BiobrickGibson AssemblyDeveloped recentlyRapidly becoming one of the methods of choice

The next few slides will give you an idea of both methods, but first lets talk about some of the tools you will be usingEnzymesPlasmids

Restriction Enzymes

Also called restriction endonucleasesDoes the cutting in cut & paste:Recognize specific DNA sequences (recognition sites) and cleave DNA at specific locationsCan cut straight across both DNA strands (blunt ends) or make a staggered cut with overhangs (sticky ends)Named for organism where foundEcoRI is from E. coli, R = strain reference, and I = first enzyme isolated from that strain)

Other Enzymes

LigaseResponsible for the pasting in cut & pasteBonds 2 complementary pieces of DNA

DNase, RNase: break down DNA/RNACIP (Calf Intestinal Phosphatase): Prevents self-ligation by removing phosphate groups from sticky endsPolymerase: constructs DNA/RNA

Plasmids

Most molecular biology work is done with plasmids!Occur naturally in bacteriaWe use specially designed plasmids

Plasmids are circular DNA containing the following:Origin of replication (ori)Antibiotic resistance (or other selection marker)Kills off nearly all bacterial colonies that dont contain your plasmidAlternatively, can use a positive selection marker (GFP, blue/white screening with X-gal) to label the colonies that do have your plasmidYour parts and devices!!Usually contain a region known as a polylinker (MCS) (lots of restriction sites in a row where you can cut with restriction enzymes and insert your parts)

Can be high- or low-copy number (# copies maintained in the cell at any one time)High-copy used for cloning (construction) (ex: pUC)Low-copy used for expression (transcription/translation) (ex: pET vectors and T7 expression vectors)

Traditional cloning methods

Quite literally, involves cutting and pasting DNA fragmentsLots more jargon:Miniprep = small-scale DNA purification 1-20 mL of bacterial cultureLarger scales called midiprep, maxiprep, and megaprepDigestion = cutting DNA with restriction enzymesLigation = pasting DNA pieces togetherTransformation = insertion of a plasmid into a hostCompetent = ready to be transformedCulturing = growing organism in the labRun a gel = separate DNA fragments by size, usually in agarose gelProperly, agarose gel electrophoresisExpression = when the bacteria are actively making the protein from the gene of interest

Lab work flowchart for traditional cloning

Obtain component fragments

Isolate Plasmid

DNA

(Miniprep)

Copy or Cut Out

Fragment

(PCR or Digestion)

Separate

Fragments

(Electrophoresis)

Purify Fragment

(Gel

Purification)

Combine fragments (ligate)

+ Insert into E. coli (transform)

Check Plasmid Accuracy

Isolate Plasmid

DNA

(Miniprep)

Cut into

Fragments

(Digestion)

Separate

Fragments

(Electrophoresis)

Express protein, purify, test properties

Unfinished or Incorrect Plasmids

Finished Plasmids

Transform into expression host

DNA

Sequencing

iGEM/Biobrick Assembly Methods

Use specially-designed polylinker sequences that make it easy to piece parts together in whatever order you wanthttp://partsregistry.org/Help:Standards/Assembly#Registry_Supported_Assembly_StandardsThey are idempotent: newly composed parts will automatically adhere to the standard without any further manipulationSpecific prefixes and suffixes are added to each part so they work with each other and with standard plasmid backbonesThere are several different polylinker sequences availableKnown as RFCsRFC10 = the BioBrick standard.not optimal!RFC23 = the Silver standard (like the BioBrick standard but better)I recommend using RFC23 or RFC25Many of them are compatible with the Standard and 3A Assembly Methods..but not all! Check each part you want to use to make sure its compatible!!To learn more: http://partsregistry.org/Help:Standards

Standard Assembly

The original BioBrick Assembly MethodPuts parts together one-by-oneVery easy, but can be time-consumingAlso, unreliable therefore, this method is no longer recommended by iGEM or the RegistryUses 4 enzymes: EcoRI, XbaI, SpeI, and PstIOverhanging ends for X and S are compatible, but when they are ligated together, they form a new site that cant be cut by either enzyme (ablated site)This site is known as a scar, since it is a region of DNA that doesnt code for anything and cant be gotten rid of

3A Assembly

3A = 3 antibioticAdvantages:97% colonies correctNo gel purificationNo PCREasyBest method if only combining 2 partsUses same restriction sites as Standard Assemblyjust slightly different strategyCompatible with all the same RFCs as Standard Assembly.just requires more antibiotics and lots of different plasmid backbones (available in the Registry)Also produces a scar siteThis is probably the best method to use if you dont want to do any PCR or if you want to put together a long string of partsProtocol: http://partsregistry.org/Help:Protocol/3A_Assembly

Gibson Assembly

Developed recently, but becoming quite popular due to these advantages:Scarless (good for fusion proteins and RBS placement)DNA can come from any source (blunt-end fragments, genomes, biobricks, etc)Great for putting multiple (>2) parts together at the same timeSequences dont have to follow any particular standard (RFC)Disadvantages:Need custom primers for PCR must order in advance (and can be expensive)Will not work for repetitive sequences!This system has been commercialized as gBlocks by IDT/NEB

Gibson Assembly

How it works:PCR out genes with specialized primersAdjoining DNA fragments should have a

20 bp overlap

Obtain a series of fragments with overlapping sequence homologyPurified amplified DNA fragments are then mixed with a Gibson Master Mix consisting of T5 exonuclease, Phusion polymerase, and Taq ligaseT5 exonuclease chews back on the 5 endThe overlapping ends are then annealed together (the newly-added homologous regions line up)Finally, the fragments are repaired and fused with Phusion polymerase and Taq ligasePuts all parts together simultaneously without any scars!!!

Great website: http://synbio.org.uk/dna-assembly/guidetogibsonassembly.html

Synthesized Parts

Another way to get scarless fusion of all your parts (and without much work) is to get them synthesized by a company!Can be rather expensive (~$400 per kb)Need to make sure that you can clone it into standard iGEM plasmids afterwardsOtherwise, will not qualify as a part for the iGEM competition (and it wont be much use to anyone else who wants to use it)Add restriction sites to either side of your part/device so that it works with standard RFCs/assembly methods

Where do I get my parts? The Registry!

http://partsregistry.org/Main_PageMany of them dont actually work.Be careful to use only parts with a Registry star and/or a green WGood source for basic stuff including: RBS, terminators, GFP, characterization devices, logic devices, some promotersEven if the part is listed in the Registry, it may not have been included in the iGEM RepositoryTo check, go to partsregistry.org, click on DNA Repositories, and then type in your part number into the top blue box (Part list) this will search the repositories and tell you where you might find itOther option: on parts main page, click Get This Part in the upper right hand corner will take you to a page with the availability and location in current and past repositories, as well as quality control informationSometimes, the Registry can send you a part, even if its not in the repository: http://partsregistry.org/DNA_Requests

If its not in the Registry, what do I do?

You have 2 options:

1. Get it straight from the source: PCR out of a natural genome

Organism may not be readily available.Organism genome may not be compatible with your host organism genome (i.e., a part from a plant may not work if placed into E. coli because of different codon biases)Need to add correct prefix and suffix so will work with Registry standards (RFCs)

2. Order it (synthesize it) from IDT (iGEM partner) or GenScript

You will need the DNA sequenceNatural DNA sequences can be found from many sources:Academic papers (search through Web of Science Database available through Purdue Libraries)GenBank: http://www.ncbi.nlm.nih.gov/genbank/BRENDA (enzyme database): http://www.brenda-enzymes.info/

If using a natural or synthesized sequence.

.be sure to optimize the codon sequences to work in your chassis!! (although dont change the prefix and suffix they must still work with the assembly methods!!!)

Codon Optimization protocol:

Go to http://genomes.urv.cat/OPTIMIZER/

Insert the DNA sequence you would like to optimize.

Select a) to use the OPTIMIZER data for codon frequency.

Select the organism in which you will be expressing (most likely E. Coli K12)

Choose Codon Usage (HEG) for highly expressed genes.

Choose the genetic code you would like to use (Eubacterial)

Use guided random for the method.

Click next and a report will be created with the optimized sequence.

Once you have your optimized sequence, be sure to check that you didnt accidentally add an extraneous restriction sites that might interfere with your assembly method!!!!

In silico cloning

BEFORE you ever work in the lab, it is imperative to make sure that everything will work!!Do this with computer programs that will simulate assembly proceduresDNA2.0 (iGEM is a partner)https://www.dna20.com/genedesigner2/Geneious (this is what my lab uses.I really really like it)pDRAW32 (never used thisI found it onlineI think its free)VectorNTI (this is the industrial-level software.what Dow Agro uses its really expensive)

Another thing to check: Rare codons

Besides checking that there arent any restriction sites within your sequence (none that will be used in assembly, anyway), you also need to check for rare codonsRare codons are codons that arent used very often by your chassis, so there arent very many tRNAs corresponding to themTherefore, a rare codon can either slow translation or even cause it to stop (especially if there are multiple rare codons)Even though you optimized your coding sequence, the assembly process still has the potential to introduce rare codons into the overall sequenceTherefore, you need to use in silico cloning to check for them in the fully assembled deviceTo check for rare codons (E. coli only), use this tool: http://nihserver.mbi.ucla.edu/RACC/

For more information

Browse the Registry and iGEM websitesAll this information and more is therebut it can be hard to find

Read the following paper (in the Dropbox under Background Info):

Engineering BioBrick vectors from BioBrick parts by Reshma P Shetty, Drew Endy, and Thomas F Knight Jr, Journal of Biological Engineering, 2:5, 2008.

Outline


Ive made my device.now what?

You need to characterize it!!http://partsregistry.org/Characterization_of_PartsUsually, characterization methods use fluorescent tags (GFP = green fluorescent protein, RFP = red fluorescent protein, etc)As part is made/used, the cells fluoresceMeasure the fluorescence levels and quantify!A standardized way to measure protein expression is PoPS, or Polymerase Per Second http://partsregistry.org/PoPSDefined as the number of times that RNA polymerase passes a specific site on DNA per unit of timeUsed by inserting test gene into same regulatory region with a fluorescent protein coding region measure fluorescence

More on characterization

To learn more about characterization methods, read the following paper (in the Dropbox under Background Info):Refinement and standardization of synthetic biological parts and devices by Barry Canton, Anna Labno, and Drew Endy, Nature Biotechnology, 26:7, 2008.

The paper also contains a sample datasheet showing what kind of data you need to collect for proper characterization

Outline


Modeling

We at Purdue think its important to not just make the parts and devices, but have a good understanding of how they workTherefore, it is important that you model your system!There are 2 main ways you can do this:Graphical software: TinkerCell, CellDesignerMathematical method: MATLAB (Mathworks is a partner with iGEM)No matter which way you pick, youll still need to have a basic understanding of the math!!!Its not as bad as it sounds, I promise!Examples of iGEM projects that use good models (theres not very many):Purdue 2009 (I did this one! So yes, Im a little biased)British Columbia 2011 (won Best Model at iGEM Americas)Good documentation at: http://2011.igem.org/Team:British_Columbia/Model1

Intro to the maths

It all boils down to solving a set of differential equationsThe differential equations are derived from knowledge of how the system worksFirst, draw a pathway of what occurs physically in your systemThen fill in the defining equationsMost enzymes work via well-defined kinetics, i.e. Michaelis-MentenOther phenomena can usually be estimated by zero, 1st, or 2nd order reactions (just like in freshman chemistry!)If necessary, account for cell growth with the logistic model of cell growthOnce you have your set of differential equations, you will have lots of unknown parameters you have to supplyYou can sometimes find these in literatureFor enzymes, the BRENDA database has a LOT of kinetic constantsOtherwise, you can estimate them based on physiologically relevant values (find these in academic papers, etc)Good animation for introduction to enzyme kinetics: http://www.wiley.com/college/pratt/0471393878/student/animations/enzyme_kinetics/Also, the Wikipedia article is quite good: http://en.wikipedia.org/wiki/Enzyme_kinetics

Outline


Design theory and strategies

As we talked about in IT227, there are some things you want to keep in mind when designing your system:

You want to make it have sections which can be tested individually

This is not unlike computer programming, where you want to divide your code up so you can test individual functions

Later, you can put all the pieces together once you know they all work alone

There are many ways in which a genetic circuit can function you might want to think of alternatives when you start designing your device (for example, http://openwetware.org/wiki/BioBuilding:_Synthetic_Biology_for_Students:_Lab_1)

Once you have your device put together, you might want to optimize it to work better

Before you put everything together, think about how you might do this

Optimization can be done in tandem with modeling: once you have your basic system, you can model it. Then change some parameters in your model to see how you can make the output better! Translate that into your device and repeat.

Outline


iGEM!

So, youre on an iGEM team. What exactly does that mean?The best way to find out is to look at lots of older projectsTry to look mostly at the winning projects.(although the rest can often give you good insight as to what to avoid in your own project)Goals of iGEM:Learn about synbio!Spread the word to the publicEnforce safety and ethical concerns regarding recombinant DNAStimulate progress in synbio Several iGEM teams have actually published their results!Provide a venue for undergraduates to conduct their own creative research projects

How iGEM works

The competition (Jamboree) is in the fall (Oct/Nov)The rest of the year (primarily in summer), teams from around the world develop a new and creative projectIn fall, teams attend regional competitions (new in 2011)Each team is graded on certain criteria (more later)If a team satisfies all of the criteria, a gold medal is awardedYou can also achieve a silver or a bronze medal Other awards are also given, including Best Model, Best New Biobrick, etc.The best teams from each regional move on to the World competition at MIT where they compete for the Golden BioBrick!

Getting a gold medal

More info: http://2012.igem.org/JudgingIn summary, you will need to do the following:Register your team (already done!)Complete the judging form (when you get closer to the Jamboree)Develop a Team WikiIncludes a project description, modeling information, a lab notebook, information about team members, safety, and anything else you want people to see before the JamboreeThis is your first impression to the judges, so make it thorough!Present a poster and a talk at the JamboreeSubmit at least one new BioBrick part or device before the deadlineYou can also *extensively* document an older part used for a new application insteadThe new part *must* work with existing RFCs (i.e., have the correct prefix and suffix and be in an iGEM plasmid)Show that your new part/device works as expectedCharacterize operation of at least one new BioBrick part or device and enter information into the RegistryPlus one of the following:Improve an existing part and document it in the RegistrySignificantly help another iGEM team (debug their part, model their system, etc)Outline a **new** approach to an issue in Human Practice in synbio (dont just do a survey!! The judges have seen zillions of them!)

Things to note

It is VERY difficult to successfully complete an iGEM projectmostly due to time and cost constraintsTherefore, do NOT waste time!

The first few weeks (when you nail down your design) are CRUCIAL, and they can easily get you behind scheduleIt takes a lot more reading and searching than you probably think

Also, lots of other teams have lots more funding, dedicated grad students/post docs, and/or courses that focus on developing the summers iGEM project (i.e. MIT)You have to compete against them, even though you may not have the same resourcesEuropean teams especially have started growing in strength schools there are putting a lot of focus and time into developing a good iGEM team

Outline


Other useful resources

When doing a literature review, the best place to find academic articles is the Web of Science databaseYes, its better than Google ScholarAccess through Purdue Libraries:Go to http://lib.purdue.edu and click on the orange Databases tabFind Web of Science in the drop-down menuUse the search tools to find what you wantEach article will have a link that looks like this: Click it, and it will take you to a page with links to the pdf

If its not available on the internet, Purdue Libraries can do an Interlibrary Loan they find it at another school, scan it, and send it to you

For the basics behind all the lab protocols, a good resource is Wiley Current Protocols in Molecular BiologyAccess via same Database list (Current Protocols series)Note: these detail the traditional protocols.more often than not, people nowadays use kits to do many of these techniquesHowever, these will give you a good idea of how the kits workMolecular Cloning: A Laboratory Manual by T. ManiatisThis is basically the bible of molecular biologyIntroduction to Genetic Engineering by W. SoferGood intro and history section for how molecular biology developed (and the basic terminology)

Thats all, folks!

Or rather, thats all I can think of right now.If you have any other questions, dont hesitate to ask. If I cant answer them, we can find someone who can.I really want to see Purdue succeed this yearI think we have a good shot at a Gold medal, as long as we stay on track and dont get lost in the details. I will do my best to make sure this happens!

Documents

Synthetic Biology Overview