Difference between revisions of "Part:BBa K3349011"

Latest revision as of 15:35, 25 October 2020

Pectin Lyase PelC-GFP

The PelC Pectin Lyase is originally from Paenabacillus amylolyticus. This particular pectin lyase is more specific to methylated pectin or homogalacturonan substrates rather than polygalacturonic acid [1]. This protein also works with both PelB (BBa_K3349001) and Pnl (BBa_K3349001) in this organism to effectively break down pectin. PelC has an optimal activity at 55°C and a pH of 10. The Km in in terms of 80% methylated substrates is 0.41 +/-0.06mg/ml. Calcium is a cofactor for this protein and is therefore necessary within the buffer conditions [1].

A GFP reporter was added onto the C-terminus. As well a C-terminal his tag was added to the GFP for nickel affinity purification.

pnl modelling

Figure 1: Restriction digest of PelB and PelC DNA from the pUC57 plasmid using EcoRI and PstI enzymes run on a 1% agarose gel. The red boxes show the band corresponding to that of our DNA constructs. PelC-GFP in lane 4 shows similar results as well as an unexpected band at around 1500bp. We are not sure why this is occurring as the there are no illegal cut sites within the sequence.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 901
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 287

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3349011 short</partinfo>
-The PelC Pectin Lyase is originally from <i>Paenabacillus amylolyticus</i>. A GFP reporter was added onto the C-terminus.
+The PelC Pectin Lyase is originally from <i>Paenabacillus amylolyticus</i>. This particular pectin lyase is more specific to methylated pectin or homogalacturonan substrates rather than polygalacturonic acid [1]. This protein also works with both PelB (BBa_K3349001) and Pnl (BBa_K3349001) in this organism to effectively break down pectin. PelC has an optimal activity at 55°C and a pH of 10. The Km in in terms of 80% methylated substrates is 0.41 +/-0.06mg/ml. Calcium is a cofactor for this protein and is therefore necessary within the buffer conditions [1].
+A GFP reporter was added onto the C-terminus. As well a C-terminal his tag was added to the GFP for nickel affinity purification.
+<html>
+<img src= "https://2020.igem.org/wiki/images/c/c9/T--Lethbridge_HS--insertdigest2020.png" alt="pnl modelling" style="width:500px;height:700px;">
+</html>
+<b>Figure 1: </b> Restriction digest of PelB and PelC DNA from the pUC57 plasmid using EcoRI and PstI enzymes run on a 1% agarose gel. The red boxes show the band corresponding to that of our DNA constructs.
+PelC-GFP in lane 4 shows similar results as well as an unexpected band at around 1500bp. We are not sure why this is occurring as the there are no illegal cut sites within the sequence.
 <!-- Add more about the biology of this part here