Difference between revisions of "Part:BBa K2260002:Experience"
(→Applications of BBa_K2260002) |
(→Applications of BBa_K2260002) |
||
| Line 5: | Line 5: | ||
===Applications of BBa_K2260002=== | ===Applications of BBa_K2260002=== | ||
| − | An assay was conducted in order to measure the effect of our part, Phasin-HlyA, on PHB secretion. Triplicates of <i>E. coli</i> BL21 containing either Phasin-HlyA and PhaCAB or only PhaCAB (as a control) were placed in LB media containing 3% glucose, and allowed to grow for 48 hours. PhaCAB, the gene that produces PHB, ensured that equal amounts of PHB were produced in both our test and the control. The PhaCAB part came from Imperial College 2013, and can be found at https://parts.igem.org/wiki/index.php?title=Part%3ABBa_K1149052. At 24 hour intervals, live cells were separated from the media using differential centrifugation. This resulted in two fractions: a cellular fraction containing the live cells, and a secreted fraction containing the PHB that was released into the media, either through secretion or cell death. Before separation, CaCl<sub>2</sub> was added to agglomerate PHB particles together. | + | An assay was conducted in order to measure the effect of our part, Phasin-HlyA, on PHB secretion. Triplicates of <i>E. coli</i> BL21 containing either Phasin-HlyA and PhaCAB or only PhaCAB (as a control) were placed in LB media containing 3% glucose, and allowed to grow for 48 hours. PhaCAB, the gene that produces PHB, ensured that equal amounts of PHB were produced in both our test and the control. The PhaCAB part came from Imperial College 2013, and can be found at https://parts.igem.org/wiki/index.php?title=Part%3ABBa_K1149052. |
| + | |||
| + | At 24 hour intervals, live cells were separated from the media using differential centrifugation. This resulted in two fractions: a cellular fraction containing the live cells, and a secreted fraction containing the PHB that was released into the media, either through secretion or cell death. Before separation, CaCl<sub>2</sub> was added to agglomerate PHB particles together. | ||
| + | |||
| + | The fractions were then treated with with Triton X-100 in PBS to remove cellular debris, and washed to isolate PHB particles. The pellets were allowed to dry overnight, before being weighed and compared. The results appeared as follows: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/2/2b/Calgary2017_CorrectedSecretion.jpg"> | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/d/d1/Secretionfinal.JPG" | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 01:07, 2 November 2017
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_K2260002
An assay was conducted in order to measure the effect of our part, Phasin-HlyA, on PHB secretion. Triplicates of E. coli BL21 containing either Phasin-HlyA and PhaCAB or only PhaCAB (as a control) were placed in LB media containing 3% glucose, and allowed to grow for 48 hours. PhaCAB, the gene that produces PHB, ensured that equal amounts of PHB were produced in both our test and the control. The PhaCAB part came from Imperial College 2013, and can be found at https://parts.igem.org/wiki/index.php?title=Part%3ABBa_K1149052.
At 24 hour intervals, live cells were separated from the media using differential centrifugation. This resulted in two fractions: a cellular fraction containing the live cells, and a secreted fraction containing the PHB that was released into the media, either through secretion or cell death. Before separation, CaCl2 was added to agglomerate PHB particles together.
The fractions were then treated with with Triton X-100 in PBS to remove cellular debris, and washed to isolate PHB particles. The pellets were allowed to dry overnight, before being weighed and compared. The results appeared as follows:
<img src="
">
<img src="
"
User Reviews
UNIQd416b88a4d94bb7c-partinfo-00000000-QINU UNIQd416b88a4d94bb7c-partinfo-00000001-QINU