Results

Unfortunately, due to unsuccessful cloning, no measurements were obtained. (Despite the several protocols tested, no cloning have been confirmed. )

We do not know why the cloning did not work.

With the AQUA cloning, we frequently obtained red colonies, which is a strong indication that pSB1C3 with the RFP was integrated in E. coli. The issue here is that we used the linear plasmid provided by iGEM. The latter did PCR on pSB1C3 with RPF to provide us the linear plasmid. Some template should have stay in the linear plasmid that they sent us. To counter this phenomenon we used Dpn1 to digest the circular plasmid following the right protocol but it still remains some template.

AQUA cloning and enzymatic cloning were equally concerned about problem on analysis. As soon as colonies were present, we performed colony PCR which gave nothing and then when we did enzymatic digestions the results were inexplicable. It was no contamination because the negative control was always empty, meaning that the issue was not with the water, the primers or the buffers. The stripes were never the right length and yet we tried numerous enzymes and enzymes blends. It could be non specific hybridization but we verified with the ApE software and did not find any hybridation area. Even when a single enzyme was used and should have given us the length of the plasmid with the insert, sometimes several stripes were present. It was like there was a contamination or that two empty plasmid bound. We do not understand how it could be something else than our plasmid because it should have possessed the chloramphenicol resistance.

In a nutshell, the profiles did not correspond to the expected clones and the analysis by sequencing did not help to understand what was going on with these clonings.

Regarding the chemistry part of the project, it is a real shame that so many problems with the HPLC occured because it does not allow any conclusion. The issue with the acid hydrolysis is that initially it is used to produce sugars and that HMF is just an unwanted co-product. The quantity of HMF is very low and is insufficient to be quantified. Originally, we aim to use ionic liquids which are present in a significant amount of scientific articles but we learn that they are very expensive and they need chrome salts which are banned at the industry level. Since our purpose was to adapt our method in an industrial way, there was not interest in using ionic liquid even if they worked well.