Team:Arizona State/Notebook/Ernesto

LuxI Transformation · Benchling

LuxI Transformation

Made with Benchling
Project: Ernesto iGEM Notes
Authors: Ernesto Luna
Dates: 2016-06-09 to 2016-06-14
Thursday, 6/9/16
1.
Gathered 2 amp agar plates from the cold room (4C) and warmed them up in the incubator at 37.C.
2.
Prepared an ice bath to thaw out the frozen cells and maintain the plasmid DNA cold.
3.
Gathered 1 tube of BL21 cells from -80C storage and placed in the ice bath to thaw (~10 min).
4.
Gathered "tube 2" of concentrated LuxI plasmid from the refrigirator and placed in the ice bath..
5.
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.
A
B
1
LuxI TransformationLuxI Neg. (Control)
2
8uL dH20 2uL plasmid 40uL BL21 cells10uL dH20 40uL BL21 cells
Table1
*Note: mixing procedure carried out on ice
6. Took warm agar plates (2) from the incubator and labeled with "plasmid (or negative control), cell strain, Abx, initials, date".
7. Pipetted entire volume of each mixture onto each respective plate.
8. Added ~7-10 glass innoculation beads to each plate and swirled for a minute.
9. Disposed of innoculation beads in flask containing EtOH.
10. Placed plates upside down in the incubator and left over night.

LuxI Transformation

Made with Benchling
Project: Ernesto iGEM Notes
Authors: Ernesto Luna
Dates: 2016-06-09 to 2016-06-14
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).

LuxI Transformation

Made with Benchling
Project: Ernesto iGEM Notes
Authors: Ernesto Luna
Dates: 2016-06-09 to 2016-06-14
Tuesday, 6/14/16

Receiver Transformation in DH5AT

Made with Benchling
Project: Ernesto iGEM Notes
Authors: Ernesto Luna
Date: 2016-09-19
Monday, 9/19
Transform DH5As with F2620, LuxR, GFP+, and negative rec.
●
Add 2uL of F2620, LuxR, and neg rec. and 1uL of GFP+ (very concentrated) to 30uL of DH5AT competent cells
●
Incubate on ice for 10min
●
Heat shock at 42C for 35sec
●
Incubate on ice for 2min
●
Add 750uL of SOC media (RT) to each tube
●
Shake 37C for 30min
●
Plate 100uL on LB+Amp
●
Incubate overnight at 37C

Culture preparation for F2620 inductions

Made with Benchling
Project: Ernesto iGEM Notes
Authors: Ernesto Luna
Date: 2016-09-26
Monday, 9/26
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.
A
B
C
D
E
F
G
H
I
J
K
L
M
1
123456789101112
2
A200uL LB Amp200uL LB Amp200uL LB Amp100uL F2620 + 50uL neg. sender + 50uL LB Amp100uL F2620 + 50uL neg. sender + 50uL LB Amp100uL F2620 + 50uL neg. sender + 50uL LB Amp100uL GFP pos. + 50uL neg. sender + 50uL LB Amp100uL GFP pos. + 50uL neg. sender + 50uL LB Amp100uL GFP pos. + 50uL neg. sender + 50uL LB Amp100uL neg. rec. + 50uL neg. sender + 50uL LB Amp100uL neg. rec. + 50uL neg. sender + 50uL LB Amp100uL neg. rec. + 50uL neg. sender + 50uL LB Amp
3
B100uL F2620 + 50uL AubI + 50uL LB Amp100uL F2620 + 50uL AubI + 50uL LB Amp100uL F2620 + 50uL AubI + 50uL LB Amp100uL F2620 + 50uL LuxI + 50uL LB Amp100uL F2620 + 50uL LuxI + 50uL LB Amp100uL F2620 + 50uL LuxI + 50uL LB Amp100uL F2620 + 50uL EsaI + 50uL LB Amp100uL F2620 + 50uL EsaI + 50uL LB Amp100uL F2620 + 50uL EsaI + 50uL LB Amp100uL F2620 + 50uL LasI + 50uL LB Amp100uL F2620 + 50uL LasI + 50uL LB Amp100uL F2620 + 50uL LasI + 50uL LB Amp
4
C100uL F2620 + 10uL AubI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL AubI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL AubI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL LuxI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL LuxI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL LuxI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL EsaI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL EsaI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL EsaI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL LasI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL LasI + 40 uL neg. sender + 50uL LB Amp100uL F2620 + 10uL LasI + 40 uL neg. sender + 50uL LB Amp
5
D100uL GFP pos. + 50uL AubI + 50uL LB Amp100uL GFP pos. + 50uL AubI + 50uL LB Amp100uL GFP pos. + 50uL AubI + 50uL LB Amp100uL GFP pos. + 50uL LuxI + 50uL LB Amp100uL GFP pos. + 50uL LuxI + 50uL LB Amp100uL GFP pos. + 50uL LuxI + 50uL LB Amp100uL GFP pos. + 50uL EsaI + 50uL LB Amp100uL GFP pos. + 50uL EsaI + 50uL LB Amp100uL GFP pos. + 50uL EsaI + 50uL LB Amp100uL GFP pos. + 50uL LasI + 50uL LB Amp100uL GFP pos. + 50uL LasI + 50uL LB Amp100uL GFP pos. + 50uL LasI + 50uL LB Amp
6
E100uL GFP pos. + 10uL AubI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL AubI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL AubI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL LuxI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL LuxI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL LuxI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL EsaI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL EsaI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL EsaI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL LasI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL LasI + 40 uL neg. sender + 50uL LB Amp100uL GFP pos. + 10uL LasI + 40 uL neg. sender + 50uL LB Amp
7
F100uL neg. rec.+ 50uL AubI + 50uL LB Amp100uL neg. rec.+ 50uL AubI + 50uL LB Amp100uL neg. rec.+ 50uL AubI + 50uL LB Amp100uL neg. rec.+ 50uL LuxI + 50uL LB Amp100uL neg. rec.+ 50uL LuxI + 50uL LB Amp100uL neg. rec.+ 50uL LuxI + 50uL LB Amp100uL neg. rec.+ 50uL EsaI + 50uL LB Amp100uL neg. rec.+ 50uL EsaI + 50uL LB Amp100uL neg. rec.+ 50uL EsaI + 50uL LB Amp100uL neg. rec.+ 50uL LasI + 50uL LB Amp100uL neg. rec.+ 50uL LasI + 50uL LB Amp100uL neg. rec.+ 50uL LasI + 50uL LB Amp
8
G100uL neg. rec. + 10uL AubI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL AubI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL AubI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL LuxI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL LuxI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL LuxI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL EsaI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL EsaI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL EsaI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL LasI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL LasI + 40 uL neg. sender + 50uL LB Amp100uL neg. rec. + 10uL LasI + 40 uL neg. sender + 50uL LB Amp
9
H
Table1
Sender Cultures:
Inoculated 5, 15mL round bottom culture tubes with each respective sender. Grew at 37°C shaking overnight.
A
B
C
1
SenderTotal media volume needed for run (uL)Culture volume (mL)
2
Negative sender17403
3
AubI5402
4
LuxI5402
5
EsaI5402
6
LasI5402
Table2

LB agar plate induction of F2620 using LuxI

Made with Benchling
Project: Ernesto iGEM Notes
Authors: Ernesto Luna
Date: 2016-09-27
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

Made with Benchling
Project: Ernesto iGEM Notes
Authors: Ernesto Luna
Date: 2016-09-27
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.
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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).
A
B
1
ReceiverCulture volume (mL)
2
F26205
3
GFP +5
4
Negative receiver5
Table1
These cultures will be used tomorrow to grow from a 10% seeding-fresh media ratio to OD 600. This will take approximately 5 hours. Once the cultures reach OD 600 (log phase growth), we will use them as the receiver volumes in the 96 well plate F2620 inductions.

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