Difference between revisions of "Part:BBa K2449004"

(→‎Expression of cep94A)
 
(9 intermediate revisions by 3 users not shown)
Line 9: Line 9:
  
  
−
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;<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>
 
<center>
 
https://static.igem.org/mediawiki/2017/b/b2/T--SDU-Denmark--SDS-page_cep94A.jpg
 
https://static.igem.org/mediawiki/2017/b/b2/T--SDU-Denmark--SDS-page_cep94A.jpg
 
</center>
 
</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 were 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 copi 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 bakteria.
+
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>
 
<center>
 
https://static.igem.org/mediawiki/2017/0/00/T--SDU-Denmark--SDS-page_cep94A_medium_and_high_plasmid.jpg
 
https://static.igem.org/mediawiki/2017/0/00/T--SDU-Denmark--SDS-page_cep94A_medium_and_high_plasmid.jpg
 
</center>
 
</center>
−
<font size="2" style="text-align:center;"><b>Figure 2:</b> 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</font>
+
<center><font size="2" style="text-align:center;"><b>Figure 2:</b> 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</font></center>
  
 
This SDS-PAGE shows that it is possible to express the protein on pSB3K3.
 
This SDS-PAGE shows that it is possible to express the protein on pSB3K3.
  
−
===Groth on cellobiose===
+
===Growth 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 <partinfo>BBa_K523014</partinfo>, that nether can live on cellubiose. The end result after 72 is shown in figure 4.
+
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>
 
<center>
−
https://static.igem.org/mediawiki/2017/d/d2/T--SDU-Denmark--SD_Growth_experiment.jpg
+
https://static.igem.org/mediawiki/2017/9/93/T--SDU-Denmark--Cep94-K3-reg.png
 
</center>
 
</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 reduce!¨
+
Be aware that the generation time is reduced!
  
 
<center>
 
<center>
 
https://static.igem.org/mediawiki/2017/1/1f/T--SDU-Denmark--Cellubiose_kuvette.jpg
 
https://static.igem.org/mediawiki/2017/1/1f/T--SDU-Denmark--Cellubiose_kuvette.jpg
 
</center>
 
</center>
−
<font size="2" style="text-align:center;"><b>Figure 3:</b>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</font>
+
<font size="2" style="text-align:center;">
 +
<center><b>Figure 4:</b> 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.  </center>
  
 
===Conlusion===
 
===Conlusion===
−
It is possible to make the ''E.coli'' live on cellubiose using this biobrick
+
It is possible to make the ''E.coli'' live on cellobiose using this biobrick.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 03:53, 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.

T--SDU-Denmark--SDS-page_cep94A.jpg

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).

T--SDU-Denmark--Cep94-K3-reg.png

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

Be aware that the generation time is reduced!

T--SDU-Denmark--Cellubiose_kuvette.jpg

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.

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