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Contents 1 Ethics 1.1 Public Health 1.1.1 PEALS 1.1.2 Agriculture 1.1.3 Scientists 1.1.4 The Public 1.2 Environmental Safety- Plants 1.3 Environmental Safety- Genome Shuffling 1.4 Environmental Safety- BioBricks 1.4.1 L-form switch Biobrick 1.4.2 HBSU-xFP 1.5 Future 1.6 References

Ethics

Public Health

Synthetic biology is a rapidly emerging field, but it is still in a very early stage of its development. It is vital to encourage debate with relevant stock holders and the general public in order to investigate the ethics that arise from this research. Therefore, in this section we aim to discuss the ethics relevant to our project.

PEALS

On the 1st of August, our team had a discussion with the executive director, Professor Janice McLaughlin from Policy, Ethics and Life Sciences (PEALS) Centre regarding ethics and biosafety. She helped us identify the relevant sock holders to our project and brought our attention to some topics that we may wish to explore further.

Agriculture

One of our project sub themes involves inserting L-forms in to plants, visualising them and considering their benefits. These plants include crop plants, therefore this topic may raise concerns regarding agriculture and farming. We are interested in the opinions, ideas or concerns that an agriculture representative may have regarding this topic.

Scientists

The Public

Environmental Safety- Plants

Our team is suggesting the use of L-forms instead of normal bacteria or genetically modifying plants to provide benefits to plants. This is because we believe that using L-forms will be safer and have fewer ethical issues, as discussed here. However there are still safety and ethical issues to consider.

Our BioBrick for turning B. subtilis cell walls on and off could mutate, the cells regaining their walls regardless of the presence of xylose. However, it would be reasonably simple to produce a similar mutant, in which part of or the entire murE operon has been removed. As our BioBrick controls the expression of this operon the effect should be the same. It would not be feasible for random mutations to mask this, as functions of multiple genes essential to peptidoglycan synthesis would have to be replaced.

The murE operon contains the gene Spovd. Although involved in peptidoglycan synthesis, the product is involved in sporulation. Could our BioBrick have a further effect, inhibiting the ability of B. subtilis to sporulate? We are currently considering methods of exploring this. Sporulated bacteria are very difficult to destroy, able to survive for long periods of time in near-extreme conditions. Our L-forms burst in the harsh conditions which induce sporulation.

Establishing whether our bacteria can survive in the wild is only the first step when considering the safety of releasing plants containing our L-forms into the environment. Could DNA released by burst L-forms be incorporated by other bacteria? If so, what effects could this have? What would happen to animals that ingest plants containing our L-forms?

Although there are naturally occurring L-forms that appear to survive (and indeed subsist) in the human body, our L-forms do not survive in the pH and osmotic conditions found in the stomach and digestive tract. It should also be stressed that our L-forms should be harmless, as the B. subtilis we have modified is. However there are ways in which cell wall free bacteria could be harmful, if they were able to survive harsh osmotic conditions. It has been speculated that L-forms are difficult for the immune system to detect, having lost many of the antigens found on the cell wall. Furthermore, many popular antibiotics, including penicillin, work by targeting the cell wall. L-forms are therefore resistant to these, which would make them quite difficult to kill if not for their inherent fragility.

Environmental Safety- Genome Shuffling

The fusion of two cells of the same species causes genetic recombination to occur. This technique has started to be explored in bacteria as a method of directed evolution, as discussed here.

Genome fusion is interesting as it causes recombination over the whole genome, rather than a specific gene, and the recombination occurs randomly across random segments of the genome. This means that we don’t necessarily know, or need to know, the genes responsible for a chosen process to ‘improve’ said process. Furthermore, we won’t know why the improvements have occurred without further, optional, study. Is this important? Could we unknowingly be creating potentially harmful bacteria?

Yes. One could assay already harmful bacteria for increased pathogenesis, for ability to survive against immune system components. Research has even been done in a similar area where viruses were evolved to reproduce as quickly as possible (though the viruses quickly lost the ability to infect cells). Thankfully there are incredibly stringent rules for obtaining and working with pathogenic bacteria.

But could we accidently produce dangerous bacteria? Admittedly many of the traits desirable in ‘useful’ bacteria- sturdiness, rapid replication- are also those which cause increased pathogenesis. It should be noted that we are proposing the fusion of bacteria of the same species. Whilst the base composition of our ‘progeny’ may be unique, the genes will be same. There will always risks, a protein gaining a novel product, a gene switched off, but these are not risks that are unique to our project- they apply to any kind of breeding. However, as the many de novo negative mutations that arise even from human reproduction, we do need to be careful.

There are risks with not understanding how or why our experiments have modified the bacteria on a genetic level. If adverse effects are expressed de novo by our bacteria, it may be difficult to understand what has caused them- as it could be due to modifications in any number of the organism’s genes- our process can modify all of them!

Environmental Safety- BioBricks

To convert B. subtilis 168 to L-forms that express HBsu-xFP we transformed them with different BioBricks, using plasmids as vectors. The BioBrick could theoretically pass from these cells into other bacteria. Could the genes be expressed, and would this be dangerous?

First, our L-forms also currently contain antibiotic resistance markers. These are necessary as they allow us to grow the L-forms up without risk of contamination. However, if released into the wild these genes could pass into other, pathogenic bacteria, reducing the effectiveness of various antibiotics in medicine. Antibiotic resistance is already a major problem facing healthcare! Therefore not only is releasing bacteria containing antibiotic resistance conferring genes dangerous, it is also illegal. If we aimed to release our L-forms into the environment, we would first need to remove these markers.

As we are using B. subtilis as a chassis our BioBricks can not be expressed unless integrated into the bacterial genome. This requires the genome to have an area of homology with the plasmid, so homologous recombination can take place. If one of these vectors did get into a bacteria with no region of homology, it may be able to replicate in the cell but would have no gene expression, and so would be harmless. Human, or any animals cells thankfully do not have any of the homology regions we used and so there is no risk of us taking up this DNA. Even if we did, the chance of this happening would be miniscule.

L-form switch Biobrick

The L-forms switch BioBrick is on a pSB1C3 integration plasmid, which has homology to pbpb and murE. Therefore the BioBrick would be able to integrate into the genome of any organisms that contains these regions. A NCBI Nucleotide Blast of the murE homology region gave no species other than B. subtilis that contained regions with over 85% homology (although a number of bacteria have 100% homology for pbpb, both regions need high homolgy for integration). This suggests that this BioBrick could only be integrated into this organism.

If the BioBrick is expressed, it will cause the bacteria to lose it's cell wall. As mentioned in Environmental Safety- Plants this should merely cause the bacteria to quickly lyse, and shouldn't have any negative effects.

The BioBrick also contains a cat gene, which confers bacteriostatic chloramphenicol resistance. As discussed above, this would give any organism that takes up and integrates the BioBrick resistance to chloramephenicol. This should not be too troublesome as on B. subtilis can take up the BioBrick, and this organism is harmless so there shouldn't be a need to kill it with antibiotics.

HBSU-xFP

The HBSU-xFP BioBrick was submitted to the iGEM repository on a pSB1C3 plasmid. It is not under the control of a promoter, so cannot be expressed even if it were integrated into a bacterial genome. This should also not be possible as the plasmid does not have a region for integration. The plasmid does have a chloramphenicokl resistance marker on it however.

However we initially had the HBSU-xFP BioBrick on a pMUTIN4 plasmid. This plasmid has the BioBrick under the control of an IPTG inducable promoter, and also contains a lacI gene. Therefore even if the plasmid integrates the Biobrick will only be expressed if the transformed bacteria is exposed to IPTG (or lactose if the bacteria produces beta-Galactosidase). If the BioBrick is expressed, it could affect sporulation as HBsu is involved with making the DNA more compact before sporulation. As our BioBrick will lead to higher levers of HBsu in the cell, it may make the bacteria sporulate more easily, though this is purely conjecture. The BioBrick is also very large- ~10000bp so it may disrupt local genes.

The pMUTIN4 plasmid does have a single integration site with homology to amyE, so could integrate into the genome of any organism that contains this gene. It also has two bactericidal antibiotic resistance markers: ery, which is expressed in B. subtilis and amp, expressed in E. coli. In E. coli the the plasmid will not integrate but this is not necessary for amp expression.

Future

References