SBB11Ntbk-Joey's Angels

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Team 3 (Joey's Angels): Vini, Amy, Joey, Shane, Keith

Vinidhra Mani 16:10, 1 April 2011 (EDT)

Objective: To directly evaluate our promoter parts and see how they respond to various stresses. This will be accomplished by applying various chemical, mechanical and thermal stresses to promoters with the GFP gene directly downstream and measuring transcriptional activity of the promoter system through fluorescence.

Literature: The following link is for our main paper: Global responses of Escherichia coli to adverse conditions determined by microarrays and FT-IR spectroscopy

The following link is for the microarray data from our primary literature source: Microarray Data from our paper.

Materials:

  • Plasmid with constitutive promoter and GFP downstream (positive control)
  • Plasmid with promoter and no GFP downstream (negative control, WHITE CELLS)
  • Plasmids with promoters of interest, all with GFP downstream for our experiment
  • Saturated saline solution
  • 1M NaOH
  • 1N HCl
  • Ultrasonic bath
  • Hot water bath (45C)
  • Cold ice water bath (15C)
  • TECAN plates


Methods:
NOTES: For each experimental stress conditions and the negative global control

  • We given 34 stress promoters plus 1 constitutive promoter using E coli MG1061 strain
  • For all stress conditions, including global conditions, incubate at 37degC and shake at 200r/min, unless otherwise stated under “Experimental Conditions”
  • Ideally perform each experiment condition three independent times per promoter
  • We will first prepare the experimental samples as necessary and obtain a cell count by using OD methods. Subsequently, we will move on to test our controls and each of the various stress conditions.
  • For each experimental set, we will ideally have duplicates, possibly sets of 3. We would like to, time permitting, perform the experiment 3 independent times for each promoter as well.
  • Initially, we will begin by testing our positive and negative global controls, as detailed below. Once we have confirmed the results of our controls, we will move to the baseline experiment, and then each stress condition in the order and priority listed as time permits.
  • For every condition, unless detailed otherwise, we will incubate the samples at 37C and shake at 200r/min.
  • The idea is to attempt the incubation and growth procedures in the TECAN wells themselves. If this is not successful, we will incubate them in the blocks and subsequently aliquot them into the TECAN for measurement.



  • Each set of experiments will have data collection occurring at the following timepoints (subject to change based on growth in the baselines and controls): 10min, 30 min, 1hr, 2hr, 24hr, 48hr.
  1. Prepare experimental samples
    • Dilute to 1:100 from stock
    • Let cells grow (a few hours) to OD 600 between (0.4 and .6) and then do an LB blank
  2. Obtain and Analyze Two Global Controls
    • Two Global Controls (no experimental promoters):
    1. positive control: constitutive promoter (P_con) and GFP downstream
      • perform positive control experiment first
      • this is to ensure that we have a functioning GFP gene and methodology
    2. negative control: white cells (no GFP gene)
      • this is to set a “white cell” standard for our experiment, something we know will not or should not fluoresce
  3. Set the Baseline
    • For each promoter, we will put GFP downstream, as we are for each other stress condition, and place in regular LB broth at 37C without any applied stress and measure at timepoints
    • This will give us an indication of the background noise and how the starting points vary per promoter
  4. Induce Stress
    1. Chemical: SALINE sodium chloride (NaCl)
      • 5% NaCl in broth: with a total volume of 100ul of saturated saline, we would use 5ul of saturated saline solution and then add desired amount of cells and dilute as necessary
    2. Chemical: BASE sodium hydroxide (NaOH)
      • pH between 9 and 10
      • we will titrate/measure in LB for exact amount
      • pH the LB to specifications and add the cells to this growth broth
      • if this method does not work, we will have to add NaOH individually to the cells already in growth medium
    3. Mechanical: sonication bath
      • Put the samples in a test tube (in LB) in the ultrasonic bath
      • Total 5 min, take aliquots to measure at timepoints of 10s, 30s, 60s, 5min and stop.
    4. Thermal: heat stress (45 C)
      • Keep the growth medium and cells on a hot water bath maintained at desired temperature
    5. Thermal: cold stress (15 C)
      • Similar to heat stress, except it is necessary to keep a tab on the temperature more carefully, as we would need to create an ice bath ourselves, or place the samples in the deli, if there is one
    6. Chemical: ACID hydrochloric acid (HCl)
      • pH between 4 and 4.5 use same methods as base

Vinidhra Mani 15:21, 5 April 2011 (EDT)

Today we plated all 34 P_stress GFP promoters as well as the P_con GFP positive control onto Kanamycin resistant plates. We messed up at the beginning by using Spec resistant plates, but we ended up re-plating the ones that we messed up. We should be all ready to go tomorrow for picking our colonies and growing our bacteria.

Keith Licardo 13:08, 7 April 2011 (EDT)

Plating Cells

We received our cells (promoters with GFP upstream) in a 96-well block on Tuesday. The cells were then plated for Kan resistance. We used 12 plates overall. Each plate was divided three-ways to accomodate all 35 cells with different plasmids. After speaking with the GSIs, we decided not to do one of our negative controls. This control entailed measurement of green fluorescence on untransformed MG1610. The plates were grown overnight.

The colonies were picked the next day. Two colonies were picked for each type of transformed MG1610. Because 70 colonies would need to be grown, we decided to grow them in 24-well blocks. Three blocks were used.

5 ml of LB with Kan was used to grow the cells. The LB was first transferred on a 50-ml centrifuge tube to prevent contamination of the stock. 5-ml pipette tips were then used to transfer LB from this tube and into the blocks. The pipette tips were flame-sterilized on all axis to prevent contamination. The toothpicks used to transfer the colonies were similarly sterilized. The toothpicks were left on the blocks first and removed one at a time after the entire block was filled with colonies. The block was then covered with an adhesive sticker which allows for Oxygen to diffuse through. The blocks were left to incubate at 37 deg Celsius and shaken at 224 rpm in the incubation room in the fourth floor. The plates were covered with parafilm and left in the Anderson lab refrigerator.

The following spreadsheet points from which transformed cell line each well in the 24-well block came.

The spreadsheet for our first colony picking.  Our original cells from JCA came in a 96-well plate.  The first column in the spreadsheet tells you from which well in this original block the different promoters were placed in.  The fourth and fifth color-coded column tells you where the two colonies for each of the 35 promoters were placed in in the 24-well block.  Eg. A1 (column 4) from block 2 and B6 (column 5) from block 3 corresponds to promoter sfmC.  They share the same row.
The spreadsheet for our first colony picking. Our original cells from JCA came in a 96-well plate. The first column in the spreadsheet tells you from which well in this original block the different promoters were placed in. The fourth and fifth color-coded column tells you where the two colonies for each of the 35 promoters were placed in in the 24-well block. Eg. A1 (column 4) from block 2 and B6 (column 5) from block 3 corresponds to promoter sfmC. They share the same row.

Agar gel procedure. 7 April 2011

  1. Start melting any antibiotics (i.e. Kan/Amp/Spec) at 55 C. Antibiotics can be found in the fridge in the back near the incubator. Heat baths can be found on the counter at the right-side wall of the room.
  2. Add lb+agar powder to water in a sterile jar. The concentration is generally 40 mg powder per 1 L water. Jar's can be found in the cabinet at the front wall between the second door and the sink. Agar powder can be found on the shelf directly left of said cabinet. Scales/trays for measuring out the proper mass of powder will be next to the sink. Water may be taken from the sink. (NOTE: use the clean, non-industrial faucet on the left-hand side)
  3. Put desired amount of liquid lb+agar in container (22 mL for plate, unknown amount for strips). Location of plates is . Add appropriate amount of melted antibiotic to mixture and pour into plate/strips. Antibiotics are concentrated to 1000x (meaning add 1 uL antibiotic for every 1 mL lb+agar).
  4. Autoclave jar when done. The autoclave room is located on the third floor down the hall (turn right immediately after exiting the lab). Asking for assistance from a lab member/gsi is advised.

Shane

Keith Licardo 18:31, 7 April 2011 (EDT)

The 24-well blocks were shaken in the incubation room in the 4th floor. However, the blocks were designed to be placed in the shaker in the Andersen lab. Because of this, the blocks detached despite the tape used and one block placed near the edge of the shaker fell. This was block 3 which contained half of the second set of colonies. A new set of colonies was picked today to replace block 3.


Preparing Solvent-Based Stress Solutions, 11 April 2011

The following is an account of how Joey and I prepared the NaCl, NaOH, and HCl lb solutions for stressing promoter-transformed cells in the TECAN plates.

  • We prepared 3 conical vials for storage of said solutions. Joey added 9.5 mL, 10 mL, and 10 mL of lb+kan to the three vials and labeled them NaCl, NaOH, and HCl respectively.
  1. Saline Condition
    • Measured out around 3.6 g of NaCl (found on the and added it to 1 mL clean water from the sink in order to create a small amount of fully saturated salt water. Amounts were chosen based on information found online.
    • 0.5 mL of the salt water solution was added to the NaCl container (9.5 mL originally) in order to reach the "5%" concentration outlined by the paper.
    • Upon rereading the paper used for our information, Sergey then suggested that we use a concentration of 5 mg NaCl per 10 mg water for our salt condition, rather than the solution we had at the time. After some discussion, we decided to throw out the old salt solution and remake it to Sergey's specifications.
  2. Acid and Base Condition
    • We asked around the lab looking for NaOH and HCl. Confusion ensued. One of the people working there directed us to ask our GSI. We went to Sergey for help.
    • Sergey then began titering our acid solution using the pH meter and dilute HCl/NaOH solutions (all found by the sink) to our specified pH range (4-5). However, he overshot with the HCl and the solution was thrown out.
    • He then proceeded to titer our basic solution to our specified pH range (9-10) using the same equipment while Joey proceeded to measure out a new vial of lb for the acidic condition.
    • Sergey successfully titered our basic solution and then proceeded to retry titering the acid solution. The final solutions reached pHs of 3.96 and 9.9, as marked on their respective vials.
  • The three completed solutions were stored in the BioE 115 fridge in b144 in the "student" shelf.

Shane

First Stress Condition: NaCl, 12 April 2011

The following is an account of my setting up of the NaCl stress condition and subsequent data collection using the TECAN machine.
Gathering materials

  • The NaCl/NaOH/HCl LB solutions were stored in the fridge in b144, which was locked, so I had to wait for someone (Professor Anderson) to unlock the room for me. *Note: For future reference, never store stuff in b144. Use the fridges in the Anderson lab instead.* I then retrieved the 96 well block from the warm room on the fourth floor. The rest of the equipment (96-well TECAN plate, boat, multichannel, pipette tips, etc.) came from around the Anderson lab.

Preparing the plate

  • To start, I pipetted 10 uL from each well in the 96-well block containing our bacteria with promoters (71 wells in all: 2 for each of 34 promoters + 2 for the positive control + 1 mistake) into the corresponding wells in the 96-well TECAN plate. I poured the entire NaCl LB solution into the boat and pipetted 90 uL into each occupied well in the TECAN plate plus an additional empty well as a blank.

Taking the data

  • After making sure the TECAN machine and corresponding computer were on and connected, I followed the procedure for collecting data as prescribed by the video sent out by the Anderson lab (see http://www.youtube.com/watch?v=mQJVjaiW-J0 for more specifics). I made no changes to any of the settings. I did click through one popup notification in order to proceed with the procedure.

Shane

Keith Licardo 13:23, 19 April 2011 (EDT)

Team 3: "Joey's Angels"
Keith, Vini, Joey, Shane, Amy

Shane transferred 1 mL from each of the 24-well blocks into a 96-well block. The block was sealed with a sterilized cover that allowed for oxygen to diffuse. This was grown overnight without any shaking but was left in the warm room. The coordinates where each promoter is placed in the 96-well block are listed in the spreadsheet below.

Acidic Condition

  • The acidic solutions were made by Shane and Joey. 10 uL of the confluent Kan LB was added to 90 uL of acidic Kan LB into the 96-well Tecan plate. This was run using the overnight program for the Tecan. OD and GFP was measured.
  • The pre-conditioned LB was first added into the 96-well block using the multi-tip pipette followed by the cells.
  • Flame-sterilization technique was followed to prevent contamination during the overnight Tecan measurements.

Heat Shock Condition

  • Relaxed sterilization technique for the heat and cold conditions.
  • 100 uL from the confluent Kan LB was added to 900 uL in a 96-well block specifically for the heat condition. The block was the one added to the heat bath (47 deg C) 10 minutes prior to the Tecan measurement. The data points were for 0, 10, 30, 90, and 180 minutes. OD and GFP was measured.
  • The heat bath was set up in the water bath machine in the Anderson lab. Water was added so that the bath was half-full. It took around 15 minutes for the temperature to go up to 47 deg C.
  • The 96-well block was simply put in the bath. The block floated in the water and was surrounded by pipette holders to prevent from tipping over. The block was sealed with a cover that allowed for oxygen to diffuse. It would soak water.
  • OD and GFP were measured. To do this, the overnight Tecan program was used but only for 1 cycle. Overall, there were five spreadsheets of data obtained from the Tecan. Tecan plates were re-used to avoid using 10 plates.
  • The Tecan plates were clean by first rinsing with dW then rinsing twice with 70% EtOH then finally with water. It was then left upside-down to dry on kimwipes. Upon measurement, the bottom of the plates were wiped with the kim wipes.
  • NOTE: The same Tecan plates were used for both the cold and hot condition.

Cold Shock Condition

  • 100 uL from the confluent Kan LB was added to 900 uL in another 96-well plates specifically for the cold condition. The block was the one added to the cold bath (15 deg C) 10 minutes prior to the Tecan measurement. he data points were for 0, 10, 30, 90, and 180 minutes. OD and GFP was measured.
  • The cold bath was set up in a bucket. Water was added first. Ice from the autoclave room was added piece by piece until the temperature went down to a constant 15 deg C. Too much ice would result in 0 deg C. Around a quarter of a cup of ice was added to a bucket an inch deep in water. A thermometer was left to ensure a constant temperature.
  • The 96-well block was simply put in the bath. Although the block could float, there was only enough water to keep the block from floating. The block was sealed with a cover that allowed for oxygen to diffuse. It would soak water.
  • OD and GFP were measured. To do this, the overnight Tecan program was used but only for 1 cycle. Overall, there were five spreadsheets of data obtained from the Tecan.

The confluent cells from the three 24-well blocks were transferred into a 96-well block.  The spreadsheet lists which promoters are in the 96-well block and from which well in the 24-well block it came from.  There were 35 promoters including the constitutive promoter.  2 colonies were picked for each, resulting in 70 colonies overall incubating in the 96-well block.
The confluent cells from the three 24-well blocks were transferred into a 96-well block. The spreadsheet lists which promoters are in the 96-well block and from which well in the 24-well block it came from. There were 35 promoters including the constitutive promoter. 2 colonies were picked for each, resulting in 70 colonies overall incubating in the 96-well block.

Keith Licardo 14:51, 21 April 2011 (EDT)

Data Analysis

Here are the data in a compressed zip file. Overall, there were six conditions that were measured with the Tecan: negative control, saline, acid, base, cold, and heat. The negative control, saline, acid, and base were measured overnight using the Tecan overnight program. However, the heat and cold conditions were measured using five single time points at 0, 10, 30, 90, and 180 minutes. For each of those time points, the heat and cold cultures were measured with the overnight program for the Tecan but only for one cycle. This was chosen because the OD and the GFP were measured simultaneously with one cycle and in one excel spread sheet.

The analysis protocol is outlined as follows:

  • Analyze data only for 0, 10, 30, 90, and 180 minutes.
  • The data for all the conditions including the negative control should be normalized.
    • To normalize: [GFP(promoter)-GFP(LB without cells)] / OD
  • The negative control will act as the reference.
  • For each condition and for each promoter in that condition, get the ratio of the normalized fluorescence to the reference fluorescence.
  • Compile the ratios of each promoter for each stress conditions.
    • Compare this compilation to the microarray data. Do our data match the microarray data?


Literature: The following link is for our main paper: Global responses of Escherichia coli to adverse conditions determined by microarrays and FT-IR spectroscopy

AMY Li 15:21, 26 April 2011 (EDT)

The following link contains a zip file of the ratios between the various stress conditions and the reference condition. These are in xls format.
Ratios between the various stress conditions and the Reference Condition

Keith Licardo 20:21, 30 April 2011 (EDT)

All cells and solutions made were either destroyed or discarded. Shane and Joey removed the 24-well and 96-well blocks from the shaker in the Anderson lab, bleached it, and left next to the sink to be autoclaved. The same was similarly done for the 96-well block from the incubator room in the fourth floor.

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