Difference between revisions of "Part:BBa K2598027:Experience"

Revision as of 07:31, 21 October 2019

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K2598027

2019 US AFRL CarrollHS

This part was characterized as a part of composite part"J18912-bFMO" <h>J18912-bFMO</h> We created three overnights that contained 3 mL of LB Broth, 3 µL of the antibiotic Kanamycin (50 ng/µL) and E.coli DH5α cells containing J18912-bFMO and three overnights that contained 2.4 mL of LB Broth, 600 µL of Tryptophan (a precursor to indole), 3 µL of the antibiotic Kanamycin (50 ng/µL) and E.coli DH5α cells containing J18912-bFMO) to compare the functionality between the two plasmids, and one overnight that only contained 3 mL of LB Broth only to serve as our blank.

Figure 1: Hsu, T. (2018) Employing a biochemical protecting group for a sustainable indigo dyeing strategy. Nature Chemical Biology, 14, 258. Retrieved from: https://www.nature.com/articles/nchembio.2552

To measure indigo production, we performed an extraction protocol on these overnights to extract indigo using DMSO and measured the absorbance on the plate reader. To convert the raw data, we made an indigo standard curve (shown below) and graphed the data below:

As shown in the graph above, there was a greater yield of indigo in the overnights that contained additional tryptophan.

Cell-Free Experiments

After we measured the overnights, we moved on to optimizing our system in cell-free. We set up cell-free reactions and graphed the results below.

Unfortunately, the only “response” we saw in cell-free were reactions with indole, which we believe was due to the indole crashing out of solution. Although we saw indigo production in cells, we did not see similar results in cell-free which will require further optimization.

User Reviews

UNIQ1e5c10d66c6f58b4-partinfo-00000000-QINU UNIQ1e5c10d66c6f58b4-partinfo-00000001-QINU

@@ Line 6: / Line 6: @@
 ===Applications of BBa_K2598027===
 ===2019 US AFRL CarrollHS===
-This part was characterized as a part of composite part
+This part was characterized as a part of composite part[https://parts.igem.org/Part:BBa_K3145003:Experience "J18912-bFMO"]
+<h>J18912-bFMO</h>
+We created three overnights that contained 3 mL of LB Broth, 3 µL of the antibiotic Kanamycin (50 ng/µL) and E.coli DH5α cells containing J18912-bFMO and three overnights that contained 2.4 mL of LB Broth, 600 µL of Tryptophan (a precursor to indole), 3 µL of the antibiotic Kanamycin (50 ng/µL) and E.coli DH5α cells containing J18912-bFMO) to compare the functionality between the two plasmids, and one overnight that only contained 3 mL of LB Broth only to serve as our blank.
+[[File:BFMOpathway.png|650px|frame|center|Figure 1: Hsu, T. (2018) Employing a biochemical protecting group for a sustainable indigo dyeing strategy. Nature Chemical Biology, 14, 258. Retrieved from: https://www.nature.com/articles/nchembio.2552''']]
+[[File:J18912bFMOovernights.png|650px|center|''']]
+To measure indigo production, we performed an extraction protocol on these overnights to extract indigo using DMSO and measured the absorbance on the plate reader. To convert the raw data, we made an indigo standard curve (shown below) and graphed the data below:
+[[File:Indigostandardcurve.png‎|650px|center|''']]
+[[File:J18912bFMO1.png‎|400px|left|<p align="justify">'</p>]]
+[[File:J18912bFMO+trp.png|400px|center|<p align="justify">'</p>]]
+[[File:J18912bFMOovenrights.png|650px|center|''']]
+As shown in the graph above, there was a greater yield of indigo in the overnights that contained additional tryptophan.
+===Cell-Free Experiments===
+After we measured the overnights, we moved on to optimizing our system in cell-free. We set up cell-free reactions and graphed the results below.
+[[File:T7mutbFMOCFS.png|650px|center|''']]
+Unfortunately, the only “response” we saw in cell-free were reactions with indole, which we believe was due to the indole crashing out of solution. Although we saw indigo production in cells, we did not see similar results in cell-free which will require further optimization.
 ===User Reviews===