Team:TU Darmstadt/Human Practices
Integrated Human Practices
Introduction
One important part of iGEM is to discuss the project idea and
its implementation with society and experts to get new
impressions. This progress is summarized in the Human
Practices of which we draw feedback that helped us to design
our project "The Real MVP". To collect as many impressions and ideas as possible
we set out and started our outreach, talking with fascinating
people from all different fields of society and science.
Although not everyone understood every project detail, the
discussions throughout the year were always interesting and
intriguing. We really learned a lot through our Human Practices
(HP) work, both inside and outside the lab. For instance, we
learned that architects use modular building blocks to design
a bridge and that the toolbox system is used in many different
areas.
If you want to know more, you can have a look in our part of the
integrated Human Practices by clicking on the buttons below.
Also, we want to invite you to have a look at our big report where
you can accompany us on our journey through the year with iGEM
and see our Human Practices progress. For this just click on
"Click me"-button!
For more information on our integrated HP please click below.
Down below you can see the first steps of our journey throughout the last year. Feel free to click on the picture and read our full report!
The first key word which was mentioned in nearly every
conversation was "communication". Communication with other
disciplines, with
experts
and
society
in general is really important to get some new ideas and
impressions which we included in the development of our
project.
In particular, experts like
Dr. Jörg Mampel and Prof. Sibylle Gaisser stressed that it is important to leave the
science bubble and educate, because this would help
synthetic biology to become an everyday topic and reduce
fears in society.
For example, talking to Dr. Schülke and Prof. Bailer made us
think about safety aspect and the toxicity of our
Virus-like particles (VLPs). All the experts suggested
methods for analyzation and purification to us, which we
included in our lab work. Another often mentioned aspect
was the potential use for a industrial application and the
biggest topic was the modification degree and the
modularity of our VLPs. All these main points appeared
often during our Human Practices work and this led us to
our final project. In the following you can read how we
integrated the main points, which resulted from the
communication with others.
Modularity has always been the key of our project idea. It
poses many advantages in all different fields of life as
we learned during the conversation with the architects of
the
Digital Design Unit. Our system was inspired by the
concept of modularity.
The question that was raised as we started our work with
Virus-like particles (VLPs) was: How do we actually
implement this modularity? The concept of
sortase-mediated modification
was the first and most important aspect of our project, but
would it be possible to reach even better modularity?
Experts, like Dr. Stefan Schülke and Prof. Susanne Bailer,
suggested the modification degree.
Dr. Schülke from the
Paul-Ehrlich-Institut
stated that it is important to functionalize the particles
so that they present as many fusion proteins as possible
but not that many that the integrity of the particle is
disturbed. Later Prof. Bailer mentioned that they were
already thinking about adjusting the
degree of modification, but have not tried it yet. We
see this as a very
important aspect in the development of our modular system.
In the talk with Prof. Santi from the DAFNE we learned that
there is a quite simple method to adjust the
modification ratio. He explained to us that we could solve the
problem by cloning the
coat protein
(CP) with a LPETGG-Tag
and without one, so we have taken the first step to make
sure, that not all coat proteins get functionalized. We
used this in our wetlab and cloned the coat protein with
and without the tag to generate VLPs where only some
fusion proteins can be connected to the particle via
Sortase A7M.
Because of the laborious purification process for in vivo
produced VLPs we could not test modification ratios on
VLPs themselves but rather tried to generate data about
the expression levels of our promoters in dependence of
the inducer concentration to develop strategies for
adjusting the
modification
ratio of in vivo production.
Therefore, we started using a dual expression system.
All in all, the results we obtained from reporter
protein expression suggest that it is possible to adjust
the expression ratio of the included ORFs. The ideas of
the experts contributed therefore significantly to our
vision of "The Real MVP”. We can imagine that a future
system of dual expression plasmids containing tagged and
untagged CP could serve as a suitable platform to produce
different VLPs in vivo.
During our lab work we thought of how to analyze whether
the product consists of correctly
assembled Virus-like particles (VLPs). Nearly all of our
experts told us that
ultracentrifugation and the following electron microscopy
would be a good method for that. We employed their
expertise and used their methods to purify and analyze the
VLPs. Their input boosted our confidence, as we were not
sure in the beginning if these methods were still
state of the art.
Regarding advanced VLP purification, Prof. Bailer told us
that an
industrial production
would require various cleaning steps to make sure that no
cell-material might cause cross-reactions when the VLPs
are injected into the human body. In addition, we would
have to get rid of the excess proteins. We were interested
about methods to separate the assembled VLPs from
unassembled proteins or our sortase and the proteins which
were not connected to the particles. According to Prof. Luca Santi and Dr. Chiara Lico a purification via size
exclusion could be a good idea, so we tried this method
with our in vivo generated VLPs. The suggested
purification steps led to intact VLPs. Dr. Chiara Lico and
Dr. Stefan Schülke also recommended us to use DLS (dynamic
light scattering) to analyze the size and purity of our
particles. We got the opportunity to visit the Paul-Ehrlich-Institut and be
present while a DLS was performed with our particles. We
were able to see that modified particles are larger than
unmodified ones.
To conclude, the advices from the experts were very
helpful and made us aware of the demanding downstream
processes in the industry.
When thinking of possible applications, we thought about
how we could achieve a production in an
industrial scale
. This is important so that the costs and the effort are
low enough the production pays off for a company. Prof. Luca
Santi told us that it would be necessary to produce and
purify the Virus-like particles (VLPs) in a large scale.
In addition, Dr. Chiara Lico mentioned that one of the
benefits of VLPs is that they can be easily and
economically produced also at large scale due to their
self-assembly. So, we decided to fulfill this requirement.
Therefore, we designed an automated self-inducing
bioreactor to somehow
simulate a large-scale production. With this
system it is also possible to control the induction ratio
what might be important according to the setting of the
modification
degree.
Unfortunatley, assessing our MVP production quantitavely
was not possible to a satisfying degree. However, Prof.
Bailer mentioned that VLP quantification generally is
complicated, as there is no sophisticated method to
date.
Considering that our particles could be used in health
care, among other things, we wondered how to deal with new
therapeutics. She
told us that every new application has to get a new
approval. That would cause more bureaucracy for the
companies. However, some other experts like Prof. zur
Hausen and Dr. Stefan Schülke told us that in case of the
modularity our system could save costs and effort. The
reason for the lower effort and costs is, that some
standard tests could be done only once and lead to many
different applications.
During our Human Practises work many people mentioned the
aspect of the toxicity of the particles. We were often
referred to the
Paul-Ehrlich-Institut (PEI) to have a closer look at
this topic. There we talked to Dr. Schülke.
During the conversation he confirmed that Virus-like particles
(VLPs) without modifications are usually not very
immunogenic because they only consist of the virus shell
but lack the immune-activating viral genetic material
packaged within the particles.
When we visited the PEI we also did an
endotoxin test
and the results showed us that we would need more
purification steps until an application in the human body
will be possible, as a production in
E. coli produced a high rate of endotoxins.
However, with pure proteins and
in vitro assembly, the endotoxin levels were
lower, showing the potential of additional purification
steps.
Dr. Schülke mentioned the potential of VLPs for passive
immunization against potentially fast-acting diseases. We
concluded that our focus on vaccines as a possible
application was justified.
Additionally, Prof. Bailer mentioned that VLPs as particles do not need
adjuvants, which could cause
problematic side effects. She explained that due to the surface structure of the
particles there is also
the possibility to expose many antigens what makes them potentionally highly
effective. Dr. Schülke also
mentioned that
there are already existing VLP-based vaccines authorized
for human usage, for example against infection with the
human papillomavirus (HPV) or Hepatitis B. Being
interested in those already existing vaccines, this input
led us to contact the
Nobel laureate Prof. zur Hausen
later on.
Biosafety is an immensely important topic to us, as we used genetically modified
organisms (GMOs) in our
laboratory work. These organisms, containing antibiotic resistances, should not
be released into the
environment. People outside of our science bubble often do not know about our
practices in the lab,
about our use of GMOs, or about our safety practices. Therefore, they may develop
fears towards synthetic
biology. We experienced this at the
"Hessentag", where we were able to talk to diverse group
of people who all agreed on the need for strict safety
policies regarding our work.
This impression was further confirmed in conversation with the
schools
we visited and with Prof. Sibylle Gaisser, a professor of ethics and biosafety.
Beyond our
considerations on safety, we learned about the aspects of safety in the context
of an industrial
application. This not only concerns the aspect of toxicity, but also the
biosafety in our lab. We
therefore decided to discuss this topic extensively, as you can see on our
safety page.
As our VLPs are designed as a modular platform, to be modified to one’s liking,
safety problems
inherently appear. We were concerned about the possibility of dual use of our
MVPs, and adamant about
wanting to reduce such misuse. In our conversation with
Prof. Bailer, of the Fraunhofer Institute in Stuttgart,
she reassured us that she does not see the extreme misuse
of our VLPs like bioterrorism as a substantial problem of our project.
Nonetheless we took up
Prof. Gaisser´s idea of establishing a safety form for our modular platform. After some
research, we came up with the following sheet that addresses the main concerns like
the proposed use,
the protein sequence, and possible risks for humans or the environment. We
exemplified the form with
our collaboration
project with the
iGEM Freiburg
team. The safety form for our VLPs requires the user to state
their intentions,
therefore helping to prevent misuse, as proposed projects may be assessed before
starting.
If you can not imagine how this could look like, please click here.
