Part:BBa_K2449004
cep94A controlled by LacI promoter
cep94A is a cellobiose phosphorylase, which efficiently phosphorylates the cellobiose at its β-linkage, resulting in the degradation of cellobiose to D-glucose and α-D-glucose-1-phosphate.
Exprestion of cep94A
There were conducted an assay to see the expression of the protein coded by Cen94A. It has a weight on 92,7 kDa, it is easely seen on the SDS-PAGE as seen on figure 1 This is on the high copi plasmid;pSB1C3.
Figure 1:A SDS-PAGE of BBa_2449004/cep94A compared with a negative control .The ladder used were SeeBlue™ Plus2 Pre-stained Protein Standard
It was further testet, if the protein could be expressed on a medium copi plasmid;pSB3K3 as seen in figure 2. It would be necessary to have on another plasmid with an other origin of replication, if two plasmids have to transformed into the same bakteria.
Figure 2: BBa_2449004/cep94A on medium copy plasmid pSB3K3 and on the high copy plasmid pSB1C3. The ladder used were SeeBlue™ Plus2 Pre-stained Protein Standard
This SDS-PAGE shows that it is possible to express the protein on pSB3K3.
Groth on cellobiose
The gene cep94A coding for cellobiose phosphorylase could make E.coli live on cellubiose on pSB1C3, this is shown in figure 3. The experiment shows it is not possible to use pSB3K3 plasmid. It is also tested agains BBa_K523014, that nether can live on cellubiose. The end result after 72 is shown in figure 4.
<img src="https://static.igem.org/mediawiki/2017/2/21/T--SDU-Denmark--SD_Growth_experiment.svg" width="330px">
Figure 3:The four figures all have time in hours on the x-axis and log(OD600) on the y-axis. All the experiments were conducted at once in the same water bath and the data shown is an average of three different samples, taken every eight hours. (A) The eight curves shown in this figure signifies all the different BioBricks supposed to degrade cellobiose, as well as a negative control. (B) The BBa_2449026/SS+Cep94A BioBricks ability to grow with cellulose as sole carbon source. (C) BBa_2449004/cep94A on the pSB1C3 plasmid compared to the pSB3K3 plasmid. (D) BBa_2449004/cep94A compared to BBa_523014/bglX.
It is al
Figure 3:Cuvettes containing 2 mL of six different samples from the growth experiment from figure Z3. The cuvettes contains from left to right, BBa_2449904/cep94A in cellobiose media, BBa_2449904/cep94A with no carbon source, a negative control in cellobiose media, a control with no carbon source, BBa_523014/bglX in cellobiose media and BBa_523014/bglX with no carbon source
Conlusion
It is possible to make the E.coli live on cellubiose using this biobrick
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 994
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1072
Illegal NgoMIV site found at 2168 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 334
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