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THE HOW-TOS OF CONSTRUCT DESIGN

One of the main aims of genetic engineering is to get bacteria to make proteins that they cannot normally synthesise. For bacteria to be able to do this, we must take the DNA that codes for these new proteins and transfer it into bacteria, so that they can understand it and make proteins from it. One of the ways that DNA can be moved into bacteria is by using a plasmid.

A plasmid is a circular piece of DNA. The DNA that codes for the new proteins can be added into a plasmid, which can then be put into bacteria (through a process called transfection) and, providing the plasmid contains all the necessary extra features for gene expression, the bacteria will be able to make the brand new proteins. For example, human insulin is made using bacteria that have been genetically-engineered in this way. It used to be that the only way to obtain insulin was purifying it from the pancreas of cows and pigs that had been slaughtered for food. These days, we can put the gene for human insulin into a plasmid, put this plasmid into bacteria and have the bacteria make insulin for us.

The DNA sequences that you want to add to a plasmid can come from a variety of sources. In the simplest case, the sequence you are looking for is already a biobrick, such as the protein DNase. For this component of our project, we used the iGEM parts registry to loacte the DNA sequence. Many of the parts we need to use for our project are already biobricks. This includes the promoter regions, which are regions of DNA that initiate the expression of a particular gene (in effect an on/off switch), and the ribosome binding sites that facilitate the initiation of protein synthesis. If the sequence you need is not a biobrick, you can find it on online databases such as GeneBank.

In other cases, you may have the amino acid sequence of a protein you want to express, but not the DNA sequence that you need to insert into the plasmid. For us, this was the case with the protein Art175. The paper from which we sourced the sequence of Art175 only gave the amino acid sequence. We used the IDT codon optimisation to get the DNA sequence. This software gives the DNA sequence when you enter the amino acids. This programme also optimises the DNA sequence for expression in E. coli (or whichever bacteria species you are wanting to express your proteins of interest in). This is an important step, for which I will now describe the biochemistry.

Amino acids are the building blocks of proteins. There are 20 amino acids and each is coded for by three DNA bases, which are called codons. As there are 4 different nucleotides (A, T, C, G), there are 64 different possible codons. With only 20 amino acids, this means that some amino acids are coded for by more than one codon. For example, AGA, AGG, AGT and ACG all code for the amino acid serine. The relative frequency of codon use vaires widely depending on the organism and organelle. For example, AGA is the most commonly used codon for serine in E. coli. When taking the amino acid sequence of the protein you want to express in E. coli, the codon optimisation software gives the DNA sequence using the codons that are most commonly used by E. coli. This is important to avoid very slow or incorrect protein synthesis.

When you have the codon optimised DNA sequences, restriction enzymes are then used to cut and stick the sequences and plasmids. These enzymes cut at specific sequences of DNA called restriction sites and allow us to stick our pieces of DNA together. iGEM specifies certain restriction sites at the start and end of DNA sequences so that any sequences can be cut and stuck together using only a few restriction enzymes. If the restriction enzyme sites were present within any of the DNA sequences (and not only within the prefixes and suffixes) then the restriction enzymes would cut within and mix up all the sequences, rendering them useless. Before using restriction enzymes, it is therefore crucial to check all of the DNA sequences for any disallowed restriction enzyme sites.

For example, the DNA sequence where the EcoRI restriction enzyme cuts is GAATTC. This sequence is present in the DNA sequence for the protein Dispersin B, meaning that EcoRI would cut within the coding sequence, leading to a shortened amino acid sequence and a non-functional protein. With this in mind, it is necessary to change the codon to alter the restriction site whilst at the same time ensuring that the same amino acid is coded for (a different amino acid means an incorrect protein). GAA codes for glutamic acid but so does GAG; thus by changing the third nucleotide from A to G, the final polypeptide sequence remains unchanged (glutamic acid is still coded for) but there is no longer an EcoRI restriction wite within the DspB coding sequence.

Once you have all the DNA sequences for everything you need, it's then time to assemble your constructs in a software like SnapGene. Together, the coding DNA sequences and the control elements make the constructs that need designing. For example, a part of our project looks at different ways of secreting anti-biofilm agents out of E. coli. If we're considering artilysin (one of the proteins we want to secrete), the construct needs to contain the DNA sequence for artilysin as well as a secretion signal (a tag that directs the protein to a secretion system), a histidine tag (enables us to purify the protein at a later stage) and start and stop sequences (enable the bacteria to know where to start reading the DNA and where to stop). We put all of these parts together in the right order so that the bacteria can understand what to make and how to make it. These constructs have to be madr for each individual component of the project. The DNA order can be made after ensuring that the constructs are suitable for DNA synthesis by a company such as IDT.

This is the stage we are at with our project. Most of the Easter vacation was spent finalising our project and the last few weeks have involved sorting our DNA sequences and playin around with SnapGene. Currently, we're making sure that our constructs comply with IDT DNA synthesis rules and double checking that everything is in the right place and right order to put into plasmids when the DNA arrives. After adding the final touches to our constructs, we should be ready to order by the end of next week.

THE POLYMERASE CHAIN REACTION - IMPLICATIONS OF DNA AMPLIFICATION

How does the reaction work?

The reaction works by cycling through a series of different temperatures to allow the different stages of the reaction to occur. Firstly, the reaction mix is heated to 98oC to split the double stranded DNA into single stranded DNA, giving two strands of template.

After this the temperature of the reaction is lowered to allow the primers to anneal to the template. The precise temperature needed is determined by the enzyme being used, and the primer sequences. Our annealing temperatures ranged from about 65-72 degrees Celsius.

The final temperature in the cycle is the optimum temperature for the enzyme you’re using. This allows primer extension to occur at the faster rate possible.

These temperatures are then cycled, so you get 25-30 cycles of denaturing, annealing and extending. During each cycle, you double the amount of DNA that you started with, meaning that this is a very efficient technique for amplifying.

Applications

In the lab, DNA amplification is important for increasing your DNA concentration for future reactions. When we received our DNA stocks, they were at concentrations that were far too low for decent transformations, meaning that we wouldn’t have been able to achieve appreciable results if we used the DNA as it was. By amplifying it, we were able to make our DNA go further, saving money, and get more meaningful data from our bacteria.

Outside the lab, PCR is still important. One interesting application is in forensic science. At crime scenes, there are often very few pieces of evidence which point towards who performed the crime. Finger prints can be very useful, but sometimes there aren’t any. However, often you can find some biological trace of the criminal, such as blood, or skin cells. These contain the DNA of the criminal, which, if it can be sequenced, can point the finger towards the culprit. However, the DNA present at the crime scene will be at very low concentrations. The solution? It needs to be amplified! The scientists doing the PCR need to be very careful in this situation though, because if their DNA gets into the sample, they could be implicated in the investigation.

UTI-INSPIRED, BIOFILM-TARGETED MICROBIAL WEAPONRY

Urinary tract infection (UTI) is one of the most common infectious human diseases in the world, with some 150 million cases occurring globally per year. It is also the most common cause of hospital-acquired infections in the developed world, accounting for approximately 40% of documented cases in the United States in 2009. On average, UTI accounts for more than 1 million hospitalisations and $1.6 billion in medical expenses each year in the USA alone.

Uropathogenic E. coli is the leading cause of UTIs. It is responsible for 80-85% of community-acquired UTIs, whereas for hospital-acquired UTIs, other bacteria such as P. aeruginosa also play a significant role. Among the UTIs acquired in the hospital, about 75% are associated with the use of urinary catheters, tubes inserted into the bladded through the urethra to drain urine which in 1 in 4 hospitalised patients end up receiving.

Bacteria like to live as sessile communities adhered to surfaces instead of isolated planktonic individuals. These immobilised bacterial communities protect themselves by creating biofilms (what we typically call bacterial slime), jelly-like coatings comprising a mixture of long carbohydrates, proteins, and DNA molecules. In the context of UTIs, such biofilms are responsible for causing inflammation as well as providing a safe haven in which the bacteria can replicate and mature. Biofilms have accumulated within their matrix enzymes that destroy antimicrobials; this helps protect embedded bacteria against our immune system as well as administered antibiotics. Biofilms associated with UTIs can form readily on both the surface of our living cells and that of the inserted catheters, and result in infections that are largely recurrent and chronic, even after antibiotic administration.

Given that antibiotic resistance in UTI-causing bacteria is such a significant problem that it warrants even multiple specific mentions in WHO's recent antimicrobial resistance global report, the administration of prolonged-courses of antibiotics in attempt to treat biofilm-protected UTIs would of course be ill-advised. Clinical resistance towards even last-resort antibiotics like carbapenems and tigecyclines have surfaced, and at present there has yet to be any antibiotics capable of degrading and destroying bacterial biofilms released for clinical use.

Inspired by how UTIs, both their own clinical significance and with respect to the bigger picture of modern antibiotic resistance, and with bacterial biofilms as a tangible, well-characterised target in mind, our team has decided to pursue the creation of a synthetic biological system that can detect the presence of bacterial biofilms and subsequently destroy both the biofilms and the offending bacteria. To achieve that aim, our system would have to be able to perform the following tasks autonomously:

1. Identify the occurrence of biofilm production and other virulent behaviour by pathogenic bacteria through the detection of signals that they use to communicate
2. Release biofilm-degrading proteins into the infected environment to break down the biofilm
3. Release antimicrobial proteins into the infected environment to specifically kill bacteria without the use of antibiotics.

Enzymatic degradation of biofilms, especially the E. coli biofilms which we are interested in is a subject that has been extensively researched on, and various iGEM teams in past years have taken on projects related to biofilm degradation (eg Groningen 2014, Indonesia 2014, Lyon-INSA 2012, UBC 2010). Most studies however involved the lysis of the enzyme-synthesizing bacterial chassis after the synthetic biological production of biofilm-degrading enzymes, either for the isolation and purification of said enzymes or for direct one-off release of enzymes into the extracellular environment for therapeutic purposes.

Our team is interested in not only a one-off pathogen-responsive antibiofilm/antimicrobial-releasing system as described above, but also a design with an enzyme secretion system that can exert its antibiofilm/antimicrobial effect without killing the chassis cell. This design would be especially useful in prophylactic applications, whereby these engineered antibiofilm bacteria can, for example, be immobilised on the surface of medical catheters or implants where the formation of biofilms are highly undesirable and autonomously secrete enzymes that destroy the pathogens' protective biofilms as well as the pathogenic bacteria themselves.

We will house our designed antibiofilm/antimicrobial system within E. coli K12, which is an extensively-studied, safe-to-use standard model organism widely adopted in both microbial genetic research as well as industrial applications. To achieve the design aims outlined above, we will need to functionalize our K12 with the following biological components:

1. Promoters that can trigger protein synthesis in response to the quorum sensors of pathogenic bacteria. In our case, we are targeting pathogenic P. aeruginosa and E. coli.
2. Antibiofilm enzymes, specifically those that can hydrolyse the biofilms of P.aeruginosa and E. coli.
3. Antimicrobial enzymes that can kill P. aeruginosa and E.coli.
4. Secretion tags which mark the synthesised enzymes for secretion from the bacteria.
5. Lysins that destroy our K12 to allow the release of the synthesised enzymes in a one-off, high concentration pulse.

We will be discussing the technical details of these parts mentioned here as well as some important underlying biochemical concepts in our next post, so please do stay tuned!

THE RISE AND FALL OF SHEWY

The project Shewy has its name from Shewanella, a bacteria that seemingly can carry out electrosynthesis. Based on the paper "Towards Electrosynthesis in Shewanella", we decided to further investigate the scheme for electron transfer in Shewy and to look at reversing it so that the bacteria were passing electrons into their environment.

The aim: generate reductive power

As much as this project seemed ambitious, there were numerous applications for the reductive power of Shewy. By fixing carbon dioxide with protons from water, and sending electrons from Shewy straight into oxygen, bacteria could possibly evolve oxygen, meaning that the bacteria could be used in oxygen evolving devices in spacesuits or rockets.

Mabel and Fred came up with the idea that by using reverse electron transfer we could send electrons to some other compound that we want to reduce. For instance, if the electrode is on and we are driving the electron transport chain in the reverse direction, we would add an enzyme that breaks down the fumarate (original electron acceptor) and then direct the electrons to whatever we want to reduce.

The main application we had in mind with Shewy was the synthesis of biopolymers. The idea was to use reverse electron flow to fix CO₂ to lactate and then polymerise lactic acid to form PLA, which is one of the main plastics used in 3D printing. Printing circuits was another application of the Shewy idea. If the metals could be selectively reduced in an small, controlled space, they could be "printed" to generate circuits. By removing the proton gradient generating components in Shewy, and inserting bacteriorhodopsin, a focused light source would cause metal to be reduced in a controlled space.

In light of these ideas, we spent much of the first half of the Easter term reading around reversed electron flow. However, although Shewy is a biochemically challenging and interesting project, further research showed that it might need a lot of work if we were to render it into synthetic biology. It seemed that at least a hundred enzymes were involved in the reduction of oxygen, we needed to know inputs and outputs of various complexes, and should develop a way to specifically relate the project with engineering aspect crucial to synthetic biology. While these discussions were ongoing, Leon found a research group that had been working on a similar project and whose funding has been reduced to zero. Having done lots of research without making a lot of progress, we decided that it could take longer than this summer to narrow down the project, let alone do the lab work required.

Our Shewy space travel nose-dived in a meeting we had at the start of March, during which we realised we needed to take a step back and reassess our original ideas. George had previously worked in a clinic treating urinary tract infections (UTIs) and drew everyone's attention to how significant a problem they are. UTIs are caused by uropathogenic bacteria that invade the cells lining the bladder and form biofilms. It was here that our focus turned, particularly as this was in accordance with our survey results and linked in well with the microbiome topic that had been popular previously. This meeting marked a change of direction for our project, meaning we had to return straight back to the drawing board. We were then faced with the question:

How could synthetic biology be used to better solve a problem that science and medicine is currently failing to address?

A BACKSTORY

Bacteria do mean business; however, before there was any notion of what business this may be and how we could incorporate it into our project, we first had to establish a team. Last year, the Oxford iGEM team had to set up everything from scratch as the University had never entered the competition before. Their project did very well, winning them a gold medal in Boston. It was last year's team which began the process of starting iGEM 2015. Once they had sparked interest, they set up some preliminary meetings so that we could start to finalise a team, and come up with a strategy for what we would do over the Christmas vacation.

During the vacation, we surveyed the general public so that we could find out their opinions on synthetic biology, what we could do to improve its profile, and how they wanted synthetic biology to help them. The responses we got to our surveys were truly diverse and, at times, reflected the public's lack of understanding about what synthetic biology can and cannot do. Duke received a response suggesting that we use synthetic biology to stop racism. While we're confident in synthetic biology's huge potential, we weren't sure whether it extended quite this far. There were also numerous suggestions that we "create" more energy, showing to us that perhaps more effort is needed to educate the public on the first law of thermodynamics.

Despite these scientifically unsound suggestions, we gathered that the public would love to see synthetic biology being put to use in the areas of environment and medicine, particularly in relation to global warming and antibiotic resistance. We each presented on an idea based on our surveys and voted on the top 6 topics. These were SPACE as proudly presented by Sam, using the human microbiome (which later evolved into our actual project) by Raffy, metal extraction by Leon, desalination by Duke, biological readouts in contact lenses and plants by Will and Lychee respectively.

Given Sam's great enthusiasm for space, we started exploring it in greater detail as a team. We tried combining some of the other ideas with space exploration and discussed different ways that synthetic biology could help with the long journey to the next planet/system/galaxy. This is where we also started exploring Fred's idea of using Shewanella oneidensis ("Shewy") as a long term food or energy source in space. It was interesting during these early stages of the project, for the biochemists as least to puzzle over some unfamiliar aspects of biochemistry and see how we could incorporate them into our project. However, at times we were too bio heavy, perhaps lacking a focus on the engineering aspects of what we're trying to achieve. Instead of approaching things from an engineer's perspective, targeted on making a design that works with as few original ideas as possible (to loosely quote Freeman Dyson), we fully went down the biochemist's route of 1) a wacky idea, 2) lots of wacky idea discussion and finally 3) does this actually work?

Outcome: half a term spent on Shewy...

THE HUNT: BACTERIA STYLE

Bacteria mean business. The swarm growing from the left, Myxococcus xanthus, hunt their prey by spreading as what scientists call a biofilm aka "bacterial slime".

Biofilms can form virtually anywhere. Not only are they responsible for dental plaque, the muck on the inside of fish tanks and the slime on recently fresh fruit, they are also involved in up to 80% of all the microbial infections that we acquire.

They are responsible for causing urinary tract infections (UTIs), the most common infection acquired at hospital. Streptococcus pneumonia make biofilms and cause pneumonia. Biofilm infections lead to chronic wounds. Most permanent indwelling medical devices, such as joint prosthetics and heart valves, will have a biofilm community growing on them. Biofilms are behind a whole host of medical, industrial and environmental problems that are very costly and technically challenging to remedy.

In nature, the majority of bacteria are not free-living; instead they are found associated in these biofilms, ingenius living environments made by the microorganisms to aid their growth - and their assault on other living things.

Such an environment serves as a shelter from which the bacteria can launch their attack, whether it be on the urinary tract, the lungs of a cystic fibrosis patient or the various plants and pipelines for aquaculture. And such an environment also serves as a hide-out: there is currently no commercial antibiotic that specifically targets bacterial biofilms.

It's time to start a war on bacterial biofilms and, with the increasing prevalence of drug-resistant strains, a completely novel approach is essential.

This is where synthetic biology and the International Genetically Engineered Machine (iGEM) Competition join the story. Synthetic biology is about harnessing the tools that evolution has already developed for us. All we need to add is a little imagination!

THE ENGINEER'S RATIONALISATION OF LIFE

Synthetic biology takes the processes designed by nature and categorises them into standardised systems, allowing bacteria to be designed, built and tested, like how you would build a car.

It's about combining the skills of an engineer and engineering principles with molecular biologists and systems biology thinking. The nature of synthetic biology is interdisciplinary, drawing on a number of scientific fields to provide engineering solutions to some of our biggest problems.

It's about appreciating the complexity of living systems and then sorting them into useable parts, parts that can be built back up into new and exciting tools - tools that will rid of pollution, enhance food production, produce energy, do the housework and, in our iGEM project, destroy biofilms, kill pathogenic bacteria and provide a novel approach to treating microbial infections.

It's about selecting a feature of an organism, identifying its DNA, cataloging it and engineering it into a biological chassis. The approach is beautifully simple: the designs of biology applied to the logic of engineering and then put back into nature.

It's about a lot more than bacteria. Synthetic biology is the engineer's rationalisation of life: where we once aimed just to explain the world, synthetic biology now gives us the capacity to improve it.

HELLO WORLD!

Welcome! We are the 2015 Oxford iGEM team.

What's iGEM all about?

iGEM is a socially-driven research competition in the field of synthetic biology. Participating teams of undergraduates from all over the world work throughout the summer with the aim of using synthetic biology to create solutions to major social or environmental problems. As such, the competition is just as much about "beyond the lab" work in the form of outreach and human practices as it's about the actual technology derived from our lab research. A summer of hard work culminated in the presentation we'll give at the iGEM Jamboree, which will be happening in Boston at the end of September.

What problem are we interested in?

Antimicrobial resistance is an increasingly serious threat to global public health and is an issue in all partts of the world, and one of the ways through which bacteria confer themselves protection against antimicrobial drugs is the growing of biofilms. Biofilms, or "bacterial slime", are responsible for a whole host of medical, industrial and environmental problems that are very costly and technically challenging to remedy. The scale of the problem is huge, with up to 80% of all infections involving the formation of a biofilm. Some examples where biofilms pose especially big an issue are urinary tract infections (UTIs), catheter and implant infections, dental plaque formation as well as infections in cystic fibrosis patients. In industry and infrastructure, biofilms are also the main culprit behind the fouling of various plants and pipelines for aquaculture, water treatment, and food production.

Both antimicrobial resistance and the other problems associated with biofilm formation are big issues in their own right but are especially problematic when they're combined. The bacteria, already constantly evolving to afford themselves more innate resistance against antibiotics, produce biofilms as protective layers that shield them from the drugs even more comprehensively.

How are we proposing to solve the problem?

There is currently no commercial antibiotic that specifically targets bacterial biofilms, but researchers have identified a range of bacterially-derived biomolecules that degrade and destroy biofilms. As such, our project involves the engineering of bacterial strains to make them produce and secrete enzymes that can destroy the pathogenic bacteria and the biofilms they make. The beauty of the antibiofilm agents we plan to use is that they have been shown not to induce resistance in the target bacteria, meaning that having them continually produced at a low level will not be nearly as bad as with traditional antibiotics. Our system is applicable to a whole host of biofilm environments and with a simple design that can be used in multiple sectors, we hope to get a step further in providing a novel approach to treating microbial infections.

In terms of product formulation and design, we hope to ultimately arrive at a functional proof-of-concept e.g. an enzyme-secreting infection-clearing catheter or a modular system that continuously and cheaply cleans out pipelines, and to achieve that we have a 16-strong interdisciplinary team comprising 1 engineer, 2 physicists, 2 chemists, 2 biologists, and 9 biochemists working on different aspects of the project in parallel.

How can you find out what we're up to?

This blog will chart our progress at each step of the way, from the initial conceptualisation of the project, to the execution of our experimental work in the lab and subsequently our flying out to Boston to attend and present out project at the Jamboree. Our team's Wiki page, which will contain all the technical details of our project will also be online in due course. What's more, we'll be hosting events in Oxford related to our project and Synthetic Biology as a whole, information about which you can find on the blog.