Difference between revisions of "Part:BBa K314100:Experience"

WashU_StLouis_2015

Our team worked closely with Vanderbilt University’s iGEM team over the summer to help them troubleshoot issues they had been having with this promoter. There were concerns that the promoter had nonhomogeneous expression despite its identity as a constitutive promoter. Additionally, Vanderbilt asked us to test another part, R0010, under the same conditions. We took their parts and induced them with IPTG to determine their effectiveness. We ran a total of 6 induction experiments, three using our lab’s induction protocol and three using their lab’s induction protocol. The parts were transformed and induced in DH10B as well as MG1655. The specific protocols we used can be found under the “Materials and Methods” section of the wiki.

Our protocol required the growth of a stationary phase culture to begin the experiment. After leaving a tube of culture sitting in the incubator overnight, we took that culture out the following day and diluted it in a new culture to an OD of 0.1. We grew that culture for another 2 hours. Afterwards, we prepared 8 serially diluted IPTG concentrations in corresponding mixtures of LB and antibiotic. Then, having pipetted those solutions to different columns in a 96 well plate, we pipetted the newly grown cultures into their separate rows. We incubated 3 of these plates for 7 hours.

Additionally, we also used their protocol as a means to test the validity of the two parts. Similar to our protocol, stationary phase cultures were grown overnight. The following morning, those cultures were pipetted into their allocated rows with prepped, diluted inducer concentration corresponding to the appropriate column. The fluorescence values we measured followed no strict patterns and greatly differed from plate to plate. Since the deviations we obtained for each point were significant, the results from this experiment were inconclusive.

Methods: WashU iGEM Induction Protocol

-Pipette 4 mL sterile LB/CM into four 14 mL culture tubes, R0010-RFP (in BL21 and MG1655) and K314100-RFP (in BL21 and MG1655). Also pipette 4 mL sterile LB to two 14 mL cultures tubes, wild type BL21 and MG1655 -Place the cap on the culture tubes to the first position to allow aeration. -Place the tubes into the incubator and allow them to grow overnight (16 hours) at 37 deg C and 250 RPM Next day... -Make frozen stock for all cultures grown -Measure OD at 600 nm in cuvettes for each culture (OD O/N) (900 uL water and 100 uL culture well mixed) -Dilute cultures to OD of 0.1 in a total of 4 mL fresh LB/antibiotic (just LB for the BL21 and MG1655) in new 14 mL culture tubes -Volume of overnight culture = (0.1*4)/(OD overnight) -Volume of LB/antibiotic (just LB if negative control) = 4mL - V overnight -Incubate 2 hours at 37 deg C and 250 rpm -While waiting for the incubation to complete, label eighteen 15 mL conicals, cm-1 through cm-9 and wt-1 through wt-9 to create 5x dilutions. -Add 10 mL LB and 10 μL cm to conical cm-9. -Add 8 mL LB and 8 μL cm to conicals cm-1 through cm-8. -Add 10 mL LB to conical wt-9. -Add 8 mL LB to conicals wt-1 through wt-8. -Calculate volume to take from the 0.5 M IPTG stock to achieve the max [IPTG] with the following equation. -Vstock = (10 mL total in tube)*(2.5mM IPTG)*(0.6 mL total in deep well plate)/((500 mM IPTG)*(0.5 mL inducer/LB/ antibiotic in deep well plate) -Add 60 μL of 500 mM IPTG to conicals cm-9 and wt-9. -Vortex both conicals. -Transfer 2 mL of conical cm-9 to conical cm-8, and transfer 2 mL of conical wt-9 to conical wt-8. -Vortex conical cm-8 and wt-8. -Transfer 2 mL of conical cm-8 to conical cm-7, and transfer 2 mL of conical wt-8 to conical wt-7. -Vortex conical cm-7 and wt-7. -Repeat for conical cm-6 through cm-2 and conical wt-6 through wt-2. -Do NOT transfer anything into conical cm-1 or conical wt-1. -Pour each conical (cm-1 through cm-9 and wt-1 through wt-9) into its own trough – one at a time. With the multi-channel pipette, transfer 500 μL per well into the deep well plates. -After the diluted cultures have grown 2 hours, pour each into its own trough. Using the multi-channel pipette, transfer 100 μL per well into the correct rows. -Cover the deep well plates with breathable membranes. -Incubate 6-8 hours at 37°C and 250 rpm. To measure... -Centrifuge at 3000 g for 15 minutes. Remember to balance with another plate. -Discard supernatant. -Resuspend all cells in 200 μL 1x PBS. -Transfer 200 μL of wells to a black 96-well plate. -Measure with a Tecan(get proper product name) set to 580 nm excitation and 610 nm emission for mRFP, absorbance at 600 nm and the gain set to 100. -Divide fluorescence by absorbance per well. -Subtract average fluorescence/absorbance of corresponding wild type strain from the fluorescence/absorbance of the strains with the fluorescent protein. -Calculate average fluorescence/absorbance and standard deviation based on the entire population for each strain at each inducer concentration. -Plot average fluorescence/absorbance versus inducer concentration for each strain.

Vanderbilt Induction Protocol

-Pipette 4 mL sterile LB/CM into four 14 mL culture tubes, R0010-RFP (in BL21 and MG1655) and K314100-RFP (in BL21 and MG1655). Also pipette 4 mL sterile LB to two 14 mL cultures tubes, wild type BL21 and MG1655 -Place the cap on the culture tubes to the first position to allow aeration. -Place the tubes into the incubator and allow them to grow overnight (16 hours) at 37 deg C and 250 RPM Next day... -Make frozen stock for all cultures grown -Measure OD at 600 nm in cuvettes for each culture (OD O/N) (900 uL water and 100 uL culture well mixed) -Label eighteen 15 mL conicals, cm-1 through cm-9 and wt-1 through wt-9 to create 5x dilutions -Add 10 mL LB and 10 uL cm to conical cm-9 -Add 8 mL LB and 8 uL cm to conicals cm-1 to cm-8 -Add 10 mL LB to conical wt-9. -Add 8 mL LB to conicals wt-1 through wt-8. -Add 50 μL of 500 mM IPTG to conicals cm-9 and wt-9 (conical concentration will be 2.5 mM IPTG) -Vortex both conicals. -Transfer 2 mL of conical cm-9 to conical cm-8, and transfer 2 mL of conical wt-9 to conical wt-8. -Vortex conical cm-8 and wt-8. -Transfer 2 mL of conical cm-8 to conical cm-7, and transfer 2 mL of conical wt-8 to conical wt-7. -Vortex conical cm-7 and wt-7. -Repeat for conical cm-6 through cm-2 and conical wt-6 through wt-2. -Do NOT transfer anything into conical cm-1 or conical wt-1. -Pour each conical (cm-1 through cm-9 and wt-1 through wt-9) into its own trough – one at a time. With a multi-channel pipette, transfer 600 μL per well into the deep well plates. -Pour each overnight culture into its own trough. Using the low volume multi-channel pipette, transfer 0.6 μL per well into the correct rows. -Cover the deep well plates with breathable membranes. -Incubate 24 hours at 37°C and 250 rpm. To measure... -Centrifuge at 3000 g for 15 minutes. Remember to balance with another plate. -Discard supernatant. -Resuspend all cells in 200 μL 1x PBS. -Transfer 200 μL of wells to a black 96-well plate. -Measure with a Tecan(get proper product name) set to 580 nm excitation and 610 nm emission for mRFP, absorbance at 600 nm and the gain set to 100. -Divide fluorescence by absorbance per well. -Subtract average fluorescence/absorbance of corresponding wild type strain from the fluorescence/absorbance of the strains with the fluorescent protein. -Calculate average fluorescence/absorbance and standard deviation based on the entire population for each strain at each inducer concentration. -Plot average fluorescence/absorbance versus inducer concentration for each strain.