Difference between revisions of "Part:BBa K2027000"

Revision as of 10:46, 24 October 2016

Recombinant Apoptosis-Inducing Protein (L-lysine Alpha Oxidase)

This part is a native Scomber japonicus enzyme codon-optimized for Escherichia coli. The conjugated tag should allow for purification and visualization with anti-FLAG antibodies, visualization with Lumio™ Green, and purification with nickel columns. Tani et al. characterized this enzyme in their quest for synthesis of L-pipecolic acid from racemic lysine after nickel column purification, and we verified the function of our part in a similar way. More information can be found on the experience page for this part.

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 1004
Illegal BglII site found at 1032
Illegal BamHI site found at 767
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 25

Characterization

We were able to transform this part into E. coli and induce protein synthesis using IPTG. We extracted this protein by relying on its HIS-tag and using Ni-NTA resin columns.

We ran a SDS-PAGE gel and used Lumio-Green staining on the fractions we collected during the protein purification process to verify which fraction contained the isolated protein. In the gel below, the lanes, from left to right, become progressively cleaner and display a singular prominent band. The smear/spread may be due to extraneous cell content that remained in the cell lysate and that had not yet been removed.

After determining the protein existed in the elution buffer, we continued with a large-scale protein extraction and purification. With our final product, we wanted to measure its purity. Using the bicinchoninic acid assay (BCA) to determine the total concentration of protein in our final elution buffer, we found that our purified protein had a concentration of 149.317 mg/mL (shown below from Nanodrop 2000 software).

To characterize the construct's (L-lysine Alpha Oxidase) function, we induced a reaction between the purified protein samples (samples in equilibration buffer, wash buffer, and elution buffer) with racemic lysine. H2O2 is a side product of the reaction between lysine and L-lysine alpha oxidase. Following the reaction induction, we performed the Amplex Red hydrogen peroxide assay on the protein purification fractions. We had a standard curve of H2O2 of 0-10 uM H2O2 (before the addition of Amplex Red Working Solution). Based on the plate data, the elution buffer fraction displayed the most H2O2 activity compared to the other fractions. This was plausible since the elution buffer fraction contains the greatest and most purified amount of L-lysine alpha oxidase.

@@ Line 22: / Line 22: @@
 We were able to transform this part into <i>E. coli</i> and induce protein synthesis using IPTG. We extracted this protein by relying on its HIS-tag and using Ni-NTA resin columns.
-We ran a SDS-PAGE gel and used Lumio-Green staining on the fractions we collected during the protein purification process to verify which fraction contained the isolated protein. In the gel below, the lanes, from left to right, become progressively cleaner and display a singular prominent band.
+We ran a SDS-PAGE gel and used Lumio-Green staining on the fractions we collected during the protein purification process to verify which fraction contained the isolated protein. In the gel below, the lanes, from left to right, become progressively cleaner and display a singular prominent band. The smear/spread may be due to extraneous cell content that remained in the cell lysate and that had not yet been removed.
 https://static.igem.org/mediawiki/parts/b/b0/T--Stanford-Brown--rAIP_purity_page.png
-After determining which fraction the protein existed in, we continued with a large-scale protein extraction and purification. With our final product, we wanted to measure its purity. Using the bicinchoninic acid assay (BCA) to determine the total concentration of protein in our final elution buffer, we found that our purified protein had a concentration of 149.317 mg/mL (shown below from Nanodrop 2000 software).
+After determining the protein existed in the elution buffer, we continued with a large-scale protein extraction and purification. With our final product, we wanted to measure its purity. Using the bicinchoninic acid assay (BCA) to determine the total concentration of protein in our final elution buffer, we found that our purified protein had a concentration of 149.317 mg/mL (shown below from Nanodrop 2000 software).
 https://static.igem.org/mediawiki/parts/4/49/T--Stanford-Brown--raip_BCA_ss.png
-To characterize the construct's (lysyl oxidase) function, we induced a reaction between the purified protein samples with racemic lysine. H2O2 is a side product of hte reaction between lysine and lysyl oxidase.
+To characterize the construct's (L-lysine Alpha Oxidase) function, we induced a reaction between the purified protein samples (samples in equilibration buffer, wash buffer, and elution buffer) with racemic lysine. H2O2 is a side product of the reaction between lysine and L-lysine alpha oxidase. Following the reaction induction, we performed the Amplex Red hydrogen peroxide assay on the protein purification fractions. We had a standard curve of H2O2 of 0-10 uM H2O2 (before the addition of Amplex Red Working Solution). Based on the plate data, the elution buffer fraction displayed the most H2O2 activity compared to the other fractions. This was plausible since the elution buffer fraction contains the greatest and most purified amount of L-lysine alpha oxidase.