SBB11Ntbk-Joey's Angels

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File:Paper.pdf

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

Joey's Angels (Vini, Amy, Keith, Joey, Shane)

Protocol:

Team 3: Vini, Amy, Joey, Shane, Keith

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.

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

a. 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

b. 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

c. 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.

d. Thermal: heat stress (45 C) -Keep the growth medium and cells on a hot water bath maintained at desired temperature

e. 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

f. 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)

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

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.

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.