Difference between revisions of "Part:BBa M50050:Experience"

Latest revision as of 07:52, 12 December 2016

General Information

Applications of BBa_M50050

Standardized growth control for bacteria species that have pathways for sensing and responding to Autoinducer-2.

Stanford Location

Plasmid Name: LuxS Synthase Plasmid

DNA 2.0 Gene #: pD441-CH

Organism: E. coli

Device Type: Actuator

Glycerol Stock Barcode: 0133027189

Box Label: BIOE44 F16

Data

Figure 1: Images of the Western Blot with BBa_M50050(Sections A and B) compared to wild-type control (Sections C and F

Figure 2: Graph of the growth curves based on optical density readings at 600 nm over time of the LuxS transfected E. coli strain and the Cupid Pink (Pink) control strain at two different induced IPTG concentrations. A) Strains were induced at an in-solution IPTG concentration of 0 μM IPTG B) Strains were induced at an in-solution IPTG concentration of 1000 μM IPTG

Figure 3: Graph of the growth rate based on the change in optical density readings at 600 nm over time of the LuxS transfected E. coli strain and the Cupid Pink (Pink) control strain at two different induced IPTG concentrations. A) Strains were induced at an in-solution IPTG concentration of 0 μM IPTG B) Strains were induced at an in-solution IPTG concentration of 1000 μM IPTG

Data Analysis and Conclusions

All figures show comparisons between the LuxS transfected bacteria, a wild-type control strain, and a similar IPTG-inducible E. coli strain with a T5 promoter (Pink).

Western Blot

Based on our analysis of our Western blot, we concluded that we were successful in transfecting our E. coli strain with the correct plasmid and gene sequence. For BBa_M50050, we looked at well sections A and B (BBa_M50050 transfected E. coli at 0 μM and 1000 μM in-solution concentration respectively) compared to the wild-type controls (C and F). What we see is that there are bands of proteins located at the 40 kilodaltons (the anticipated length) region in sections A and B, but not in the control sections C and F.

Cell Density Tests

The cell growth analysis examines the response of the cells to varying amounts of IPTG, and therefore, varying amounts of enzymes. However, since adding a strong ribosome binding site to the bacteria could potentially affect growth rate, a control p76002 or Cupid Pink provided by the Teaching Lab, which has a strong ribosomal binding site and fluoresces in responses to IPTG, was included to compare the growth rates of the experimental line to. An untransformed E. coli culture, hereby referred to as control, was also used to represent homeostatic growth rate.

Based on the data collected from our overnight cell density tests, we can conclude that the over-expression of LuxS increases cell growth during the exponential growth phase, but does not result in any vastly different final cell density concentrations. This increase in cell growth is greater than the growth seen in cupid pink with a T5 inducible promoter, however the cell growth with LuxS is around the same as Wild Type E.coli. With this information we find that AI-2 through these protein pathways could have possible control of growth, and there may be future studies in expanding upon these pathways.

Experimental Procedures

Western Blot

Western Blots were performed on transformed bacteria to verify the expression of LuxS and LsrR, and on the untransformed E. coli cells, control, to account for any background. To do so, cultures of each strain were grown overnight at 37 degrees Celsius and split into two aliquots, each induced with either 0 μM and 1000 μM IPTG. Samples were then taken at 0, 4, 6, and 22 hours post-induction to measure the protein levels over time. To normalize the protein levels, 8*108 cells were taken from each samples, and the samples were immediately pelleted and frozen to prevent the cells from synthesizing anymore. After the samples have been collected, the bacteria were lysed with 10% SDS and boiled at 100 degrees Celsius for 10 minutes to denature the proteins. Sodium dodecylsulfate polyacrylamide gel electrophoresis was performed on the samples to separate the proteins based on molecular weight. The proteins were transferred onto a PVDF membrane and blocked with Pierce Fast Block for 5 minutes to stop nonspecific interactions and probed with anti-his antibody diluted at 1:1000 in Blotto for 1 hour. Afterwards the membrane was washed with TBS for 5 minutes three times and incubated in the 1:1000 goat-anti-mouse HRP conjugated secondary antibody. A 1:10 dilution of chromogenic substrate, Opti-4CN was then added and then membrane was imaged.

Cell Density Tests

To perform this tests, cultures of LsrR, LuxS, Cupid Pink, and control, were grown overnight in LB with kanamycin for LuxS and Cupid Pink, ampicillin for LsrR and no antibiotics for the control. The following day, the cultures were diluted to an OD600 of 0.1 and aliquots were taken and induced with 1000 μM, 500 μM, 200 μM, 100μM, 10 μM, 1μM and 0μM IPTG. 200μL of the aliquots were then transferred into a 96 well plate along with samples of the media with and without antibiotics to account for any background measurements. The plate was placed into the plate reader overnight, where it was shaken and incubated at 37°C, and set to measure the cell density every half hour for 12 hours. This protocol was done 5 times.

User Reviews

UNIQf39fec8274f90fcc-partinfo-00000000-QINU UNIQf39fec8274f90fcc-partinfo-00000001-QINU

@@ Line 1: / Line 1: @@
 __NOTOC__
-This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
+==General Information==
-how you used this part and how it worked out.
+===Applications of BBa_M50050===
+* Standardized growth control for bacteria species that have pathways for sensing and responding to Autoinducer-2.
+===Stanford Location===
+Plasmid Name: LuxS Synthase Plasmid
-===Applications of BBa_M50050===
+DNA 2.0 Gene #: pD441-CH
+Organism: E. coli
+Device Type: Actuator
+Glycerol Stock Barcode: 0133027189
+Box Label: BIOE44 F16
 ==Data==
-<html>
+[[File:BBa_M50050_and_BBa_M50051_Western_Blots.png|thumb|500px|center|Figure 1: Images of the Western Blot with BBa_M50050(Sections A and B) compared to wild-type control (Sections C and F]]
+[[File:BBa_M50050_-_LuxS-Pink_OD600_0_IPTG.png|thumb|500px|testing|center|Figure 2: Graph of the growth curves based on optical density readings at 600 nm over time of the LuxS transfected E. coli strain and the Cupid Pink (Pink) control strain at two different induced IPTG concentrations. A) Strains were induced at an in-solution IPTG concentration of 0 μM IPTG B) Strains were induced at an in-solution IPTG concentration of 1000 μM IPTG]]
+[[File:BBa_M50050-DeltaOD600_LuxS-Pink_0_IPTG.png|thumb|500px|center|Figure 3: Graph of the growth rate  based on  the change in optical density readings at 600 nm over time of the LuxS transfected E. coli strain and the Cupid Pink (Pink) control strain at two different induced IPTG concentrations. A) Strains were induced at an in-solution IPTG concentration of 0 μM IPTG B) Strains were induced at an in-solution IPTG concentration of 1000 μM IPTG]]
+==Data Analysis and Conclusions==
+All figures show comparisons between the LuxS transfected bacteria, a wild-type control strain, and a similar IPTG-inducible E. coli strain with a T5 promoter (Pink).
+===Western Blot===
+Based on our analysis of our Western blot, we concluded that we were successful in transfecting our E. coli strain with the correct plasmid and gene sequence. For BBa_M50050, we looked at well sections A and B (BBa_M50050 transfected E. coli at 0 μM and 1000 μM in-solution concentration respectively) compared to the wild-type controls (C and F). What we see is that there are bands of proteins located at the 40 kilodaltons (the anticipated length) region in sections A and B, but not in the control sections C and F.
+===Cell Density Tests===
+The cell growth analysis examines the response of the cells to varying amounts of IPTG, and therefore, varying amounts of enzymes. However, since adding a strong ribosome binding site to the bacteria could potentially affect growth rate, a control p76002 or Cupid Pink provided by the Teaching Lab, which has a strong ribosomal binding site and fluoresces in responses to IPTG, was included to compare the growth rates of the experimental line to. An untransformed E. coli culture, hereby referred to as control, was also used to represent homeostatic growth rate.
+Based on the data collected from our overnight cell density tests, we can conclude that the over-expression of LuxS increases cell growth during the exponential growth phase, but does not result in any vastly different final cell density concentrations. This increase in cell growth is greater than the growth seen in cupid pink with a T5 inducible promoter, however the cell growth with LuxS is around the same as Wild Type E.coli. With this information we find that AI-2 through these protein pathways could have possible control of growth, and there may be future studies in expanding upon these pathways.
+==Experimental Procedures==
+===Western Blot===
+Western Blots were performed on transformed bacteria to verify the expression of LuxS and LsrR, and on the untransformed E. coli cells, control, to account for any background. To do so, cultures of each strain were grown overnight at 37 degrees Celsius and split into two aliquots, each induced with either 0 μM and 1000 μM IPTG. Samples were then taken at 0, 4, 6, and 22 hours post-induction to measure the protein levels over time. To normalize the protein levels, 8*108 cells were taken from each samples, and the samples were immediately pelleted and frozen to prevent the cells from synthesizing anymore.
+	After the samples have been collected, the bacteria were lysed with 10% SDS and boiled at 100 degrees Celsius for 10 minutes to denature the proteins. Sodium dodecylsulfate polyacrylamide gel electrophoresis was performed on the samples to separate the proteins based on molecular weight. The proteins were transferred onto a PVDF membrane and blocked with Pierce Fast Block for 5 minutes to stop nonspecific interactions and probed with anti-his antibody diluted at 1:1000 in Blotto for 1 hour. Afterwards the membrane was washed with TBS for 5 minutes three times and incubated in the 1:1000 goat-anti-mouse HRP conjugated secondary antibody. A 1:10 dilution of chromogenic substrate, Opti-4CN was then added and then membrane was imaged.
-<img src="BBa M50050-DeltaOD600 LuxS-Pink 0 IPTG.png">
+===Cell Density Tests===
-<html/>
+To perform this tests, cultures of LsrR, LuxS, Cupid Pink, and control, were grown overnight in LB with kanamycin for LuxS and Cupid Pink, ampicillin for LsrR and no antibiotics for the control. The following day, the cultures were diluted to an OD600 of 0.1 and aliquots were taken and induced with 1000 μM, 500 μM, 200 μM, 100μM, 10 μM, 1μM and 0μM IPTG. 200μL of the aliquots were then transferred into a 96 well plate along with samples of the media with and without antibiotics to account for any background measurements. The plate was placed into the plate reader overnight, where it was shaken and incubated at 37°C, and set to measure the cell density every half hour for 12 hours. This protocol was done 5 times.
 ===User Reviews===