Difference between revisions of "Part:BBa M36575:Experience"

Latest revision as of 20:56, 7 December 2015

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Stanford Location

Glycerol Stock

Barcode #: 0133027097

Plasmid Name: LasR

Antibiotic resistance: ampicillin

DNA 2.0 gene #: 232005

Organism expressed in: E. coli

Actuator

Applications of BBa_M36575

Used in conjunction with BBa_M36580 in BIOE44 at Stanford as an attempt to make a P. aeruginosa biosensor.

User Reviews

UNIQ7516e1791fc5ba64-partinfo-00000000-QINU UNIQ7516e1791fc5ba64-partinfo-00000001-QINU

This construct is likely not viable. In order to test whether this construct produces protein, we conducted two tests following Histidine-Tag purification that left us with three eluted samples. We first measured absorbance at 280 nanometers using the NanoDrop, which we followed with a more sensitive Bicinchoninic Acid Assay that indicates protein presence by colorimetric change. None of the three samples showed peaks at 280 nanometers when measured by the NanoDrop, using the elution buffer as a blank. This suggested that our samples contained a concentration of protein less than that normally detectable by the NanoDrop. We followed this with a BCA Assay.

We first put each of the three purified samples into the reagent mix for BCA, and all three turned a dilute blue (Figure A). Since a strong purple color suggests strong protein presence, the assay hinted slight LasR concentration or contamination from other proteins. Additionally, since the Histidine-Tag purification process is nickel-based, and BCA Assay is copper-based, the unexpected color change to a blue range instead of purple could be explained by the interaction of those metals together with solution (Figure B). Later we added just the elution buffer to the BCA reagents, and saw a color change to a similar dilute blue.

Image A. From left to right: Blank, Sample 1, 2, 3

Image B. From left to right: 0, 2, 5, 10, 20, 50 µL of Albumin, Sample 1, Sample 2, Sample 3, Buffer

In our second BCA Assay, we used 2 mg/mL Albumin to identify the protein concentration in our purified samples.From our experimental data, in comparison to the control with no albumin nor purified samples present, samples 1 and 3 seemed to have insignificant concentration levels. Sample 2, on the other hand, showed an absorbance level that fell in between those of when 5 µL and 10 µL of albumin were added to the reagents (Table 1). Since 50 µL of sample 2 were added to the reagent mix, the result suggested the sample had a concentration between 0.2 and 0.4 mg/mL.

Table 1. Listings of absorbances and protein concentrations.

We conducted the same Histidine-Tag purification process for a positive control, the p70417 plasmid-containing cells. The p70417 sample showed an absorbance level that exceeded that of sample 2. The high absorbance in both sample 2 and p70417 suggests that the high absorbance levels were not necessarily a result of LasR presence (Graph 1).

Graph 1. Absorbances of LasR and p70417 samples with BCA reagents.

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 ===Applications of BBa_M36575===
+Used in conjunction with BBa_M36580 in BIOE44 at Stanford as an attempt to make a ''P. aeruginosa'' biosensor.
 ===User Reviews===
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+This construct is likely not viable. In order to test whether this construct produces protein, we conducted two tests following Histidine-Tag purification that left us with three eluted samples. We first measured absorbance at 280 nanometers using the NanoDrop, which we followed with a more sensitive Bicinchoninic Acid Assay that indicates protein presence by colorimetric change.
+None of the three samples showed peaks at 280 nanometers when measured by the NanoDrop, using the elution buffer as a blank. This suggested that our samples contained a concentration of protein less than that normally detectable by the NanoDrop. We followed this with a BCA Assay.
+We first put each of the three purified samples into the reagent mix for BCA, and all three turned a dilute blue (Figure A). Since a strong purple color suggests strong protein presence, the assay hinted slight LasR concentration or contamination from other proteins. Additionally, since the Histidine-Tag purification process is nickel-based, and BCA Assay is copper-based, the unexpected color change to a blue range instead of purple could be explained by the interaction of those metals together with solution (Figure B). Later we added just the elution buffer to the BCA reagents, and saw a color change to a similar dilute blue.
+[[File:BCA Assay 1.jpg.png]]
+Image A. From left to right: Blank, Sample 1, 2, 3
+[[File:BCA Assay 2.png]]
+Image B. From left to right: 0, 2, 5, 10, 20, 50 µL of Albumin, Sample 1, Sample 2, Sample 3, Buffer
+In our second BCA Assay, we used 2 mg/mL Albumin to identify the protein concentration in our purified samples.From our experimental data, in comparison to the control with no albumin nor purified samples present, samples 1 and 3 seemed to have insignificant concentration levels. Sample 2, on the other hand, showed an absorbance level that fell in between those of when 5 µL and 10 µL of albumin were added to the reagents (Table 1). Since 50 µL of sample 2 were added to the reagent mix, the result suggested the sample had a concentration between 0.2 and 0.4 mg/mL.
+[[File:BCA Table.png]]
+Table 1. Listings of absorbances and protein concentrations.
+We conducted the same Histidine-Tag purification process for a positive control, the p70417 plasmid-containing cells. The p70417 sample showed an absorbance level that exceeded that of sample 2. The high absorbance in both sample 2 and p70417 suggests that the high absorbance levels were not necessarily a result of LasR presence (Graph 1).
+[[File:BCA Graph.png]]
+Graph 1. Absorbances of LasR and p70417 samples with BCA reagents.