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.
Expression of cep94A
An assay was conducted to to see the expression of the protein encoded by cen94A. It has a weight of 92,7 kDa, it is easily seen on the SDS-PAGE as can be 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 was SeeBlue™ Plus2 Pre-stained Protein Standard
It was further testet, if the protein could be expressed on a medium copy 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 bacteria.
Figure 2: BBa_2449004/cep94A on medium copy plasmid pSB3K3 and on the high copy plasmid pSB1C3. The ladder used was 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 can make E.coli live on cellobiose on pSB1C3, this is shown in figure 3. The experiment shows it is not possible to use pSB3K3 plasmid. It was also tested again BBa_K523014, that nether can live on cellobiose. The end result after 72 is shown in figure 4.
Figure 3: BBa_2449004/cep94A on the pSB1C3 plasmid compared to the pSB3K3 plasmid.
Be aware that the generation time is reduced!¨
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 cellobiose 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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