Difference between revisions of "Part:BBa M50018:Experience"

Revision as of 02:43, 12 December 2016

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_M50018

This part was inserted into an expression vector plasmid by DNA 2.0 containing GFP to create the following complete plasmid:

Our completed sensor construct utilizes the Golden Gate cloning method to avoid extra DNA bases and improve recombination accuracy [8]. The sequence begins with the restriction site BsaI, followed by a GGGG sequence, the S32-P sequence, and ended with an AAAA sequence before a second BsaI restriction site. During production, our promoter was inserted into the commercial plasmid through use of the BsaI restriction enzyme. This enzyme created a gap in the existing plasmid just before the RBS, while also creating sticky ends on our promoter sequence that would allow it to fit into this gap. The added G and A sequences in our construct were necessary to create the correct sticky ends so our promoter fits perfectly into the existing plasmid with no extraneous bases. The rest of our final plasmid came from the expression vector, the E. coli Promoter cassette, which contained a Comet GFP sequence [9] whose expression we used to quantify promoter function.

we wanted to develop a heat-response curve quantifying GFP output. After transforming this plasmid into chemically competent E. coli, we selected colonies based on ampicillin resistance and produced 3 liquid cultures of biological replicates. 3 wells each of these cultures (for 9 total samples) were held in a spectrophotometer/plate reader while shaking for 4 hours, with the machine taking readings of CometGFP fluorescence and cell density (OD600) every 10 minutes). After normalizing fluorescence to cell density, we established the following curves at 25ºC, 37ºC, and 42ºC:

These graphs indicate that the slope of our observed GFP readings increased with temperature. For the S32-P bacteria, the rate of GFP production started at an increase of 1.84 arbitrary units/minute at 25°C, 2.21 at 37°C and finally 6.05 at 42°C. For the TS-Lim-P cultures, rates increased from 2.65 at 25°C to 3.66 at 37°C to 8.30 at 42ºC. This data demonstrates that there was a significant increase in rate of GFP production as temperature increased, which asserts our hypothesis that the σ32 promoter creates heat-sensitive protein expression. The fluorescence always increased linearly over time, which is supported by the high R2 values of our lines of best fit, all of which are near or above 0.9. As expected, the E.Coli control had little fluorescence resulting in a relatively flat line of best fit, and indicating a GFP production rate close to zero.

User Reviews

UNIQe1747b0a3996655f-partinfo-00000000-QINU UNIQe1747b0a3996655f-partinfo-00000001-QINU

@@ Line 5: / Line 5: @@
 ===Applications of BBa_M50018===
-This part was inserted into an expression vector plasmid by DNA 2.0 containing GFP, so we wanted to develop a heat-response curve quantifying GFP output.
+This part was inserted into an expression vector plasmid by DNA 2.0 containing GFP to create the following complete plasmid:
+[[File:S32-P plasmid map.jpeg|400px|thumb|center|Our completed sensor construct utilizes the Golden Gate cloning method to avoid extra DNA bases and improve recombination accuracy [8].  The sequence begins with the restriction site BsaI, followed by a GGGG sequence, the S32-P sequence, and ended with an AAAA sequence before a second BsaI restriction site.  During production, our promoter was inserted into the commercial plasmid through use of the BsaI restriction enzyme. This enzyme created a gap in the existing plasmid just before the RBS, while also creating sticky ends on our promoter sequence that would allow it to fit into this gap.  The added G and A sequences in our construct were necessary to create the correct sticky ends so our promoter fits perfectly into the existing plasmid with no extraneous bases.  The rest of our final plasmid came from the expression vector, the E. coli Promoter cassette, which contained a Comet GFP sequence [9] whose expression we used to quantify promoter function.]]
+we wanted to develop a heat-response curve quantifying GFP output.
 After transforming this plasmid into chemically competent E. coli, we selected colonies based on ampicillin resistance and produced 3 liquid cultures of biological replicates.
 wells each of these cultures (for 9 total samples) were held in a spectrophotometer/plate reader while shaking for 4 hours, with the machine taking readings of CometGFP fluorescence and cell density (OD600) every 10 minutes).  After normalizing fluorescence to cell density, we established the following curves at 25ºC, 37ºC, and 42ºC:
 [[File:S32-P Heat response curves.jpeg|900px|thumb|center|These graphs indicate that the slope of our observed GFP readings increased with temperature. For the S32-P bacteria, the rate of GFP production started at an increase of 1.84 arbitrary units/minute at 25°C, 2.21 at 37°C and finally 6.05 at 42°C.  For the TS-Lim-P cultures, rates increased from 2.65 at 25°C to 3.66 at 37°C to 8.30 at 42ºC.  This data demonstrates that there was a significant increase in rate of GFP production as temperature increased, which asserts our hypothesis that the  σ32 promoter creates heat-sensitive protein expression. The fluorescence always increased linearly over time, which is supported by the high R2 values of our lines of best fit, all of which are near or above 0.9. As expected, the E.Coli control had little fluorescence resulting in a relatively flat line of best fit, and indicating a GFP production rate close to zero.]]
 ===User Reviews===