Difference between revisions of "Part:BBa K2449004"

Revision as of 03:49, 2 November 2017

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.

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.

T--SDU-Denmark--SDS-page_cep94A_medium_and_high_plasmid.jpg

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.

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).

Figure 3: BBa_2449004/cep94A on the pSB1C3 plasmid compared to the pSB3K3 plasmid.

Be aware that the generation time is reduced!

Figure 4: Cuvettes containing 2 mL of six different samples from the growth experiment after 72 hours. 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</font>

Conlusion

It is possible to make the E.coli live on cellobiose using this biobrick.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 994
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1072
Illegal NgoMIV site found at 2168
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 334

@@ Line 9: / Line 9: @@
-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 copi plasmid;<partinfo>pSB1C3</partinfo>.
+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; <partinfo>pSB1C3</partinfo>.
 <center>
 https://static.igem.org/mediawiki/2017/b/b2/T--SDU-Denmark--SDS-page_cep94A.jpg
 </center>
-<font size="2" style="text-align:center;"><b>Figure 1:</b>A SDS-PAGE of BBa_2449004/cep94A compared with a negative control .The ladder used was SeeBlue™ Plus2 Pre-stained Protein Standard</font>
+<center><font size="2" style="text-align:center;"><b>Figure 1:</b>A SDS-PAGE of BBa_2449004/cep94A compared with a negative control .The ladder used was SeeBlue™ Plus2 Pre-stained Protein Standard</font></center>
-It was further testet, if the protein could be expressed  on a medium copy plasmid;<partinfo>pSB3K3</partinfo> 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.
+It was further testet, if the protein could be expressed  on a medium copy plasmid; <partinfo>pSB3K3</partinfo> 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.
 <center>
 https://static.igem.org/mediawiki/2017/0/00/T--SDU-Denmark--SDS-page_cep94A_medium_and_high_plasmid.jpg
@@ Line 26: / Line 26: @@
 ===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).
+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).
 <center>
 https://static.igem.org/mediawiki/2017/d/d2/T--SDU-Denmark--SD_Growth_experiment.jpg
 </center>
-<font size="2" style="text-align:center;"><b>Figure 3:</b> BBa_2449004/cep94A on the pSB1C3 plasmid compared to the pSB3K3 plasmid.</font>
+<center><font size="2" style="text-align:center;"><b>Figure 3:</b> BBa_2449004/cep94A on the pSB1C3 plasmid compared to the pSB3K3 plasmid.</font></center>
 Be aware that the generation time is reduced!