![](https://parts.igem.org/images/partbypart/icon_composite.png)
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 cep94A. 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 copy plasmid; pSB1C3.
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.
This SDS-PAGE shows that it is possible to express the protein on pSB3K3.
Growth 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. The cells were grown in M9 medium with 0.2 % cellobiose at 37°C for 72 hours with 40 rpm (NB: we noticed that growth in overnight cultures at low rpm reached higher ODs).
Be aware that the generation time is reduced!
</center>
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
None |