Difference between revisions of "Part:BBa K2449003"
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=ECUST_China 2019 characterization=
 
=ECUST_China 2019 characterization=
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In ECUST_China 2019 characterization, we used Cep 94A to degrade cellobiose to glucose and glucose-phosphate. To characterize the Cep 94A specifically, we performed SDS-PAGE and measured enzyme activity.
 
  
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SDS-PAGE revealed that Cep 94A was expressed successfully (about 91kDa). After that, we sonicated E.coli and extract Cep 94A, using DNS assay to measure the enzyme activity.
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===Usage and Biology===
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Cellobiose phosphorylase is an enzyme that catalyzes the chemical reaction cellobiose + phosphatealpha-D-glucose 1-phosphate + D-glucose. In ECUST_China 2019 characterization, we obtained the sequence of cellobiose phosphorylase from Part:BBa_K2449003. Unfortunately, this part was not contained in iGEM distribution kit, so we synthesized this gene (cep 94A) from Genewiz.
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===Characterization===
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In order to characterize the function of Cep94A specifically, we cloned the cep94A into p15a. Two aspects of characterization were condidered: the expression of Cep94A protein and the enzyme property.
 +
 
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Cep94A and the negative control (p15a) grew in 5mL LB (containing 0.1% Chl) at 37℃ for 12 hr and then was transferred to the 100 mL LB for enlarge culture. After almost 16 hours in 100mL LB, the Cep94A and p15a was collected to perform the SDS-PAGE. The result showed that Cep94A was actually expressed (about 91kDa) after induction.
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<html><div class="exper-com-box"><img style="height:400px;" src="https://static.igem.org/mediawiki/parts/2/2a/T--ECUST_China--Figure.1_SDS-PAGE_results_of_pIN1.jpg"> <br><span style="font-size: 14px;">
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<b>Figure 1.</b> SDS-PAGE results of Cep94A (about 91kDa)</span></div> </html>
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After verifying the expression of Cep94A, we decided to measure the Cep94A enzyme activity and evaluate the enzyme property to perform the quantitative experimental characterization. After induction for 16 hours, the cells were collected and resuspended in 10 ml of PBS buffer, afterwards sonicated to obtain 10 ml supernatant. The enzyme activity of Cep94A was measured by the DNS method: 2 μL cellobiose +1 μL enzyme solution + 17 μL citrate buffer (pH =4.8) was reacted at 50 ° C for 30 min. Then 30 μL DNS reagent was added, boiling for 10 min. The reacton mixture was diluted 5 times and transferred to 96-well microplate to measure the OD540 with a microplate reader.  
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<html><div class="exper-com-box"><img style="height:400px;" src="https://static.igem.org/mediawiki/parts/2/2a/T--ECUST_China--Figure.1_SDS-PAGE_results_of_pIN1.jpg"> <br><span style="font-size: 14px;">
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<b>Figure 2.</b> The DNS assay of Cep94A</span></div> </html>

Revision as of 01:37, 21 October 2019


cep94A

This is the so called cellobiose phosphorylase (Cep94A), which phosphorylates the cellobiose β-linkage resulting in its breakdown to D-glucose and α-D-glucose-1-phosphate. It is used in the following biobrick BBa_K2449004 For more info visit https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294459/

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 768
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 846
    Illegal NgoMIV site found at 1942
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 108


ECUST_China 2019 characterization

Usage and Biology

Cellobiose phosphorylase is an enzyme that catalyzes the chemical reaction cellobiose + phosphatealpha-D-glucose 1-phosphate + D-glucose. In ECUST_China 2019 characterization, we obtained the sequence of cellobiose phosphorylase from Part:BBa_K2449003. Unfortunately, this part was not contained in iGEM distribution kit, so we synthesized this gene (cep 94A) from Genewiz.

Characterization

In order to characterize the function of Cep94A specifically, we cloned the cep94A into p15a. Two aspects of characterization were condidered: the expression of Cep94A protein and the enzyme property.

Cep94A and the negative control (p15a) grew in 5mL LB (containing 0.1% Chl) at 37℃ for 12 hr and then was transferred to the 100 mL LB for enlarge culture. After almost 16 hours in 100mL LB, the Cep94A and p15a was collected to perform the SDS-PAGE. The result showed that Cep94A was actually expressed (about 91kDa) after induction.


Figure 1. SDS-PAGE results of Cep94A (about 91kDa)

After verifying the expression of Cep94A, we decided to measure the Cep94A enzyme activity and evaluate the enzyme property to perform the quantitative experimental characterization. After induction for 16 hours, the cells were collected and resuspended in 10 ml of PBS buffer, afterwards sonicated to obtain 10 ml supernatant. The enzyme activity of Cep94A was measured by the DNS method: 2 μL cellobiose +1 μL enzyme solution + 17 μL citrate buffer (pH =4.8) was reacted at 50 ° C for 30 min. Then 30 μL DNS reagent was added, boiling for 10 min. The reacton mixture was diluted 5 times and transferred to 96-well microplate to measure the OD540 with a microplate reader.


Figure 2. The DNS assay of Cep94A