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Applications of BBa_M50051

Stanford Location

Plasmid Name: LsrR Repressor Protein

DNA 2.0 Gene #: pD444-CH

Organism: E. coli

Device Type: Actuator

Glycerol Stock Barcode: 0133027118

Box Label: BIOE44 F16

Data

Figure 1: Images of the Western Blot with BBa_M50051(Sections D and E) 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 LsrR 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 LsrR 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 4: Graph of the growth rate based on the change in optical density readings at 600nm over time of the LsrR transfected E.coli strain induced at 0 μM and 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_M50051, we looked at well sections D and E (BBa_M50051 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 70-80 kilodaltons (the anticipated length) region in sections D and E, 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.

The LsrR gene when induced produces LsrR which appears to significantly reduce the rate of growth of the cells. This could be because the gene is working properly and is inhibiting the entry and degradation of AI-2, but it could also be that the introduction of the plasmid is detrimental to the cells. The error bars in the change in growth are statistically different even at 0 IPTG, so this needs to be better understood before a conclusive statement is made about the functionality of this construct. With this information though we found that AI-2 could provide possible control through protein 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.

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