Team:Arizona State/Notebook/Ernesto
Notebook:Ernesto Luna
LuxI Transformation
Thursday, 6/9/16
- Gathered 2 amp agar plates from the cold room (4C) and warmed them up in the incubator at 37.C.
- Prepared an ice bath to thaw out the frozen cells and maintain the plasmid DNA cold.
- Gathered 1 tube of BL21 cells from -80C storage and placed in the ice bath to thaw (~10 min).
- Gathered "tube 2" of concentrated LuxI plasmid from the refrigirator and placed in the ice bath.
- Prepped 2 eppendorf tubes (1.5mL), one for the LuxI transformation, and another for a control. Mixed as follows and kept on ice for 10 mins.
- Took warm agar plates (2) from the incubator and labeled with "plasmid (or negative control), cell strain, Abx, initials, date".
- Pipetted entire volume of each mixture onto each respective plate.
- Added ~7-10 glass innoculation beads to each plate and swirled for a minute.
- Disposed of innoculation beads in flask containing EtOH.
- Placed plates upside down in the incubator and left over night.
LuxI Transformation
Friday, 6/10/16
Followed up to check for growth. 3 small cultures grew in the LuxI plate and displayed a faint,red color. Disposed of negative control plate (no growth), and parafilmed LuxI plate to store in the cold room (4C).
Receiver Transformation in DH5AT
Monday, 9/19
Inoculated and grew cultures for each of the senders that I will be using to induce F2620. Due to well amount limitations on the 96 well plate, we are only inducing with 4 senders at a time. In this first run, we are only inducing with AubI, LuxI, LasI, and EsaI media. Below is a table of the 96 well plate organization (scroll to the sides to see everything). Intended to run the 8hr induction tomorrow, but I read Cassandra's protocol wrong and forgot to grow receiver cultures for seeding. Will be doing everything in triplicate and will use sender media concentrations of 10% and 50% in well volumes of 200uL. It is important to note that all senders are in Bl21 and all receivers are in DH5aT.
Inoculated 5, 15mL round bottom culture tubes with each respective sender. Grew at 37°C shaking overnight.
LB agar plate induction of F2620 using LuxI
Tuesday, 9/27
Goal: produce some qualitative data of an F2620 induction using LuxI. Inoculated 2 lb agar plates with LuxI sender and F2620 + receiver controls (LuxR, GFP+, neg. receiver). Inoculated by dabbing the agar plate after picking colonies from each source plate. Placed LuxI in the middle and each receiver at varying distances around it in a cross shape. Also set up a control plate with the same design, except I did not inoculate with LuxI., Also grew 5mL overnight cultures of LuxI, F2620, LuxR, GFP+, and negative receiver. Will inculate tomorrow in LB Amp plate with LuxI in the middle and F2620 and each control at varying distances from it in a cross shape. Will inoculate by pipetting drops of the overnight cultures in their respective positions (most likely 20uL LuxI and 5-10 uL of each receiver). Will also set up a control plate without LuxI in the middle.
Sender media filtration and Receiver cultures
Tuesday, 9/27
Objective: to filter out the HSL media from AubI, LuxI, LasI, and EsaI cultures to use for induction of F2620. Removed inoculation pipette tip from each culture tube and centrifuged in the large centrifuge for 10min @ 3.6x10 RPM. Extracted supernantant media with a pipette and then through the syringe with a 45um nylon filter (different syringe and filter for each collection). Collected all filtered media (~2-3mL each) in fresh, clean tubes and placed in 4°C room for use in induction tomorrow. Will have to rerun this part of the F2620 inductions since ideally we don't want the HSLs to wait too much since we don't know much about their stability. For now, will continue the inductions using this media, but will run again without waiting a day and compare. Hopefully, this yields quantitative insight on HSL stability based on OD GFP readings.
Since I didn't grow any reciever cultures yesterday, I grew them today. Inoculated 3, 15mL round bottom culture tubes with each respective receiver (F2620 and induction controls).
