Team:SDU-Denmark/Reproducibility
Scientific Reproduction
We have made it our obligation to follow the virtue of scientific reproduction. It is fundamental for the scientific method and is integrated in the scientific community.
Science began when people started wondering about the significance of their observations. Aristotle was wondering why the stone fell down when he dropped it from midair. He therefore developed a hypothesis about the essences in all things, which explained that the stone was moving towards its natural place - that the stone had a teleos (Ancient Greek: purpose, aim). Nowadays we laugh at that type of explanations but the essence of science still stands: explaining our observations KLAUSEN, S. H. 2014. Hvad er videnskabsteori. Akademisk Forlag..
Theories and hypotheses have virtues, which a good scientist should comply with, e.g.: to follow the rules of good reasoning, being coherent and explaining a phenomenon. A hypothesis or theory must always have implications that can be empirically tested for it to be of any use. To accept a hypothesis or theory one needs to show that it satisfies all demands - and then it needs to be tested by an objective scientist. This is called the demand of reproduction HEMPEL, C. G. 1966. Philosophy Of Natural Science. Foundations of Philosophy series. Princeton University..
Confirmation and Bacto-Aid
In our project we tried to follow several other iGEM teams’ protocols and methods for our work. We had success following the protocols from the iGEM teams of Imperial College London 2013, Tokyo Tech 2012 and Standford Brown 2015. We used their protocols in the make of PHB and some of their BioBricks. We had trouble with following 2015 UCLA's protocols for producing spider silk. At first we believed we had falsified their method – we certainly believed that we could not corroborate their hypotheses. In the end of the project we found that the things that had gone wrong was not really related to their protocols, but the components we had used in the work. The enzyme we used were of a different type than the one used by 2015 UCLA and our streptavidin beads had run past its expiration date.
This is a great example of there not being a final falsification – there was clearly something wrong with our auxiliary hypotheses. The auxiliary hypotheses were also the first place we started looking when the results did not arrive as planned – so having an overview of them helped us find that the mistakes perhaps were with them and not UCLA's protocols. We also found a mistake in our preliminary purification of PHB. The hypochlorite we used at the first had been stored wrong and the purification became visibly better when we used a different hypochlorite.
Scientific reproduction
Besides using UCLA's protocols, we also tested their BioBricks. We also tested existing BioBricks in the production of PHB. More information on the BioBricks can be found here. Testing existing BioBricks helps other iGEM teams in the coming years, because they will know that the BioBricks have been tested.
Unfortunately, we ran into problems when we tried their protocols for assembly of the silk genes. To do this the restriction sites are a crucial part of the silk gene sequences because they can overlap each other creating desired gene sequences. The genes were designed so that digesting with one enzyme would result in overhangs which would be able ligate. We tried to follow their protocol on digestion several times, however we did not succeed before we tried to digest for a longer time and at a different temperature. We found out that the enzymes we used were slightly different from the one UCLA used and this was the reason why we were not able to make a successful digestion at first.
According to their wiki, and the uploaded parts (K1763009 & K1763423) in Parts Registry, the genes named MaSp1 AB- and MaSp2 AB core sequence should contain two restriction sites for the enzyme BsaI, but we found, through SnapGene, that the sequences only contained a single restriction site at 108bp. The two genes could not be applied for Iterative Capped Assembly (ICA) as they had suggested.
When following UCLA's protocols, we were not able to assemble more than three genes corresponding to either one MaSp1 gene or MaSp2 gene (see more on the silk page). Even though we made adjustments to the protocols, we were still not able to make the four monomers, consisting of 12 fragments, which we aimed for. This could be due to the streptavidin beads, which are essential when performing ICA. We found out that the beads had passed their expiration date. Unfortunately, we first saw this mistake too late.
These examples show the importance of scientific reproduction. This is also what research ethics is about: presentation of results and methods. There is bound to be mistakes in the ongoing research and scientific reproduction helps determine these and tries to correct them. As stated above, a falsification does not entail that the competing hypothesis is verified - it just is not falsified.
