Team:Arizona State/Notebook/Brittany

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Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Date: 2016-06-06
Monday, 6/6/16

My First Entry

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Dates: 2016-06-07 to 2016-06-11
Tuesday, 6/7/16
Here's a sample experiment to show you how notebook entries work.
Use tables with formulas to make a master mix calculation. Edit the number of reactions to see it in action!
A
B
C
1
Number of Reactions80
2
ComponentAmount (uL/tube)Master Mix (uL)
3
Water161280
4
10x Buffer2160
5
Enzyme180
6
Master Mix Total191520
7
Template DNA1
8
Reaction Total20
Table1

Sub project (with Brady)

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Date: 2016-06-07
Tuesday, 6/7/16
"Gas Chromatography/Mass Spec, etc. (investigate on campus resources, protocols for sample prep, best growth phase for HSL production, HSL stability, etc."
find articles with HSL stabability
find a MALDI machine / similar
Idea
- create protocol/method how to extract/ purify HSL molecule from LB media in order to take the mass spec of it on MALDI.
- this would then yeild the concentration/ strength of the HSL molecule that is being used in the crosstalk.
- the stabability would be further tested by the degradation of the HSL molecule after being added to the Reciver bacterial population.

My First Entry

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Dates: 2016-06-07 to 2016-06-11
Wednesday, 6/8/16
Insert new days to organize your entry (and see it on the Home calendar). Use checkboxes to plan out your day:
Combine above reaction in tube
Incubate at 37°C for an hour
Run gel
Extract band and save

Short Transformation

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Dates: 2016-06-08 to 2016-06-09
Wednesday, 6/8/16
Transformation
1.
Took out competent cells out from the -80↓C which thawed on the ice for a couple minutes.
2.
Took the Lb+ ampicillin resistant agar pre-made plates from the cold room (4↓C).
3.
Took the EsacI from the 20↓C inside fridge.
4.
Placed plates into a 37.9 ↓C incubator.
a.
For the purpose of not shocking the bacteria once transformation is plated.
5.
Took two 1.5 mL epindorph tubes (always in ice)
6.
10uL of H2O added to each tube
a.
one positive control [labeled as EsacI]
b.
one negative [labeled EsacI neg.]
7.
EsacI positive- 1 ul of EscaI and 30uL of the competent cells.
8.
Negative – just 30 uL of the competent cells
A
B
1
EsacI10 ul water + 1ul EsacI+30ul of competent cells
2
EsacI negative10 ul water +30ul of competent cells
Table1
12. Cells were incubated in ice for 10 minutes
13. The LB agar+amp plates were taken out and labeled.
14. transformation is plated.
15. Glass beads are added to the plates.
16. Shake in a figure 8 formation so that the cells spread all throughout the plate.
17. Plates are incubated until the next day.

My First Entry

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Dates: 2016-06-07 to 2016-06-11
Thursday, 6/9
Link to other Benchling files by dragging them in from the file browser at the left.

Short Transformation

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Dates: 2016-06-08 to 2016-06-09
Thursday, 6/9
Spot Plates
grow culures+incubate

Spotting Plates

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Date: 2016-06-09
Thursday, 6/9
Spotting Plates
1.
Took transformed colony plate out of the incubator.
a.
(tiny colonies)
2.
Took out a new LB+agar+amp res. Plate out of the cold room.
a.
Incubated plates while prepping liquid media/labeled plates.
3.
Circled and labeled colonies on plate. [1-4]
4.
Took out 4 10mL tubes, poured 4mL of LB media.
5.
Took out the clean, incubated plates out.
a.
Labeled + made a grid.
6.
Using a 2-20ul pipette tip each individual colony was scratched off the pre-existing plate, and moved to the new plate.
a.
Pipette tip was discarded into the tube with 4mL of LB –liquid media.
7.
Tubes were placed in incubator/shaker overnight.
8.
New plate was placed into the 37
incubator.
9.
Old plate was placed in the cold room.

Sub project

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Date: 2016-06-09
Thursday, 6/9
Brief outline of a Potential Protein Purification+ Mass Spec. Protocol
- the HSL media would have to be placed in a buffer exchange to switch the media.
- then placed in HPLC to be purified (find out if the molecule is a protein to use correct machine)
- HPLC needs a max and a min absorbance cxn (for settings) [look that up in articles? or past exp.]
- HPLC check reasonable absorbance rates online
- Then use lyopholizer to further purify the sample. [ the machine dries out all the volatile compound leaving just the pure protein sample.
- MALDI take a mass spec of the sample.
- sample must be in H20 or a certian buffer.
- find out what type of matrix can be used for the sample to "fly" best into the machine. [article]
LAST: save results from HPLC + MALDI

My First Entry

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Dates: 2016-06-07 to 2016-06-11
Friday, 6/10
Drag and drop an image from your desktop to view it inline and take notes about it:
pAS329_ligation_check.png
thumbnail
Lane 1: DNA ladder Lane 2: undigested Lane 3: digested vector Lane 4: insert DNA Lane 5: ligation tube

Plasmid prep/DNA CXN/ Gel Electro

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Project: BrittanyiGEM2016
Authors: Brittany Flores
Date: 2016-06-10
Friday, 6/10
Plasmid Prep
1.
Took out the pipette tips out of the tubs before spinning them down
2.
Spun down the media for 10 minutes at max (13.5 rpm).
3.
Decanted the supernant
4.
Re-suspended the pellet with resuspension buffer, ( 200uL) and place into a new 1.5 mL Eppendorf tube
5.
lysis the cells -  with 200 ul of the lysis solution.
a.
mixing the contents by gently inverting the tube 6-8 times.
I.
lysing of the cells shouldn’t take longer than 5 minutes.
6.
Neutralization of the cells by adding 350 uL of the neutralization buffer.
a.
mixing by invention of the tube 4-6 times.
7.
The solution is centrifuged at 12,000 rpm for 10 minutes for the “sticky” to remain as a pellet at the bottom of the mixture.
8.
prepare a column where, 500uL of the column preparation solution is added to the tubes/ columns
a.
tubes are spun down at 12,000 rpm for 1 minute to clean the column.
9.
Then the cleansed lysate cells (supernatant) is pipetted into the columns.
10.
Centrifuged at 12,000 rpm for 1 minute.
a.
discard the followthrough.
11.
Add 750uL of the wash solution into the column.
a.
centrifuge at 12,000rpm for 1 minute
I.
decant the flow through
b.
centrifuge at 12,000 rpm for 1 minute.
12.
Place a the column in a new tube.
13.
Add 50 uL of elution solution
a.
centrifuge at 12,000 rpm for one minute.
DNA concentration
~Did a nanodrop version of the DNA concentration
tubes 1,2,3,4
88, 84, 72, 78 ng/uL
which means that i got about 20% yield…. according to previous DNA can should be 400ng/uL
Gel electrophoresis
in order to check to see if there was any DNA plasmid in the transformation. and if anything was recovered during lysis of the cells and plasmid prep.
●
1 % agarose gel.
●
1 ladder, and a postive control, the rest were the samples.
●
Results: no plasmid…

My First Entry

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Dates: 2016-06-07 to 2016-06-11
Saturday, 6/11
Insert code blocks to keep your scripts right next to your notes and results.
def calculate_score(input_bases, matched_bases): mismatches = [] for i, (b, ob) in enumerate(zip(input_bases, matched_bases)): if b != ob: mismatches.append(i)

Mass Spec

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Date: 2016-06-15
Wednesday, 6/15
- So if the HSL molecule comes from the LB+ampicilin then by spinning the media down the pellet would be the wanted cells.
- then the pellet can be transferred into a smaller tube, and then the cells can be lysed.
- once the cells are lysed then they undergo a LLE procedure.
- and the HPLC can further purify ...ect...

Traditional Transformation protocol

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Date: 2016-06-15
Wednesday, 6/15
The DNA used was LUXI LUXR EcaI and a negative control
We'll be following the traditional transformation protocol described for chemically competent cells.
1.
Heat 4 LB+Amp plates in 37C incubator
2.
Add 30uL of DH5alpha turbo cells to each tube with appropriate sample (2uL of LuxI, LuxR, EsaI, water)
3.
Incubate on ice for 10min
4.
Heat shock at 42C for 35sec
5.
Incubate on ice for 2min
6.
Add 750uL of SOC media (RT) to each tube
7.
Shake 37C for 1hr
8.
Plate on LB+Amp (Spin down at max for 1min, remove 200uL of supernatant, resuspend and plate 100uL)
9.
Incubate overnight at 37C

Plate inductions

Made with Benchling
Project: BrittanyiGEM2016
Authors: Brittany Flores
Date: 2016-06-16
Thursday, 6/16
Plate induction- slow version (higher yield) (source-goldbio.com)
1.
From a relatively fresh plate, pick a colony and grow overnight at 30°C (or 37°C) in 1-2 ml LB+Antibiotic in a 15 ml tube on a rotator or shaker.
2.
Dilute 1:50 (1:100 if 37°C overnight) in 2 ml LB+Antibiotic and grow 3-4 hours at 37°C in 15 ml tube in a rotator.
3.
Prepare 1 ml LB+Antibiotic+1mM IPTG in a 15 ml conical and prewarm to 37°C about 10 minutes before use.
4.
After 3-4 hours remove 1 ml from tubes at 37°C and place in labeled 1.5 ml tubes. Centrifuge at maximum speed for 30 seconds at RT and remove supernatant. Freeze pellet at -20°C until
needed. THIS IS THE UNINDUCED CONTROL.
5. Add 1 ml LB+Antibiotic+1mM IPTG (prewarmed to 20°C) to 15 ml tube and incubate rotating or shaking at 20°C for 12-16 hours. This will get the final volume back to 2 ml and the final concentration of IPTG to 0.5mM
6. After 12-16 hours, transfer 1 ml from induced sample to labeled 1.5 ml tubes and centrifuge at maximum speed for 30 seconds at RT and remove supernatant. Freeze pellet at -20°C until needed. THIS IS THE INDUCED SAMPLE.
7. Sample preparation for SDS-PAGE: Add 100 μl of 1X Loading Buffer with 1% BME to uninduced and induced samples. Vortex the samples for 10 seconds to 1 minute or until there are no clumps of bacteria. Boil the samples for 3 to 5 minutes. Centrifuge at maximum speed for 30 seconds at RT and load 5-25 μl (usually 10 μl) depending on gel (amount of protein, size of pellet, Western, etc.).

Cloning Inducible Promoter

Introduction

Our quorum sensing receivers consist of two parts: the transcription factor and the inducible promoter. The transcription factor senses HSLs in the system and if present, binds with the inducible promoter to then activate GFP. For our project, we have four more receivers without inducible promoters: Las, Bta3, Exp, and Cer.

Materials

  • Receiver backbone
    • Inducible promoter part
      • Restriction enzymes
        • Ligase
          • Fast Digest Buffer (10x)
            • Ligase Buffer

            Procedure

            • Digest backbone
            1. ad
            1. asdf
            • Digest insert (or PCR, if necessary)
            1. 1.

            Success!!

            Made with Benchling
            Project: BrittanyiGEM2016
            Authors: Brittany Flores
            Date: 2016-06-17
            Friday, 6/17
            Today we tested the DNA concentrations and then ran a 1% agarose gel to confirm the plasmid.
            A
            B
            C
            D
            E
            1
            		Concentrations (ug/ul) 1Concentrations (ug/ul) 2Concentrations (ug/ul) 3Concentrations (ug/ul) 4
            2
            		LUXI323.288424.057284.423319.464
            3
            		LUXR84.94852.58549.47795.52
            4
            		ESAI99.133171.416145.724152.674
            Table1
            Figure 1 : Gel Images from a 1% agarose ; LuxI, LuxR, EsaI
            LauxI.LuxR.EsaI-final.jpg
            thumbnail
            Figure 1:

            Sequencing LuxI,R,EsaI

            Made with Benchling
            Project: BrittanyiGEM2016
            Authors: Brittany Flores
            Date: 2016-06-20
            Monday, 6/20
            Sequenced LuxI, LuxR, EsaI
            - In "Brittany's Box" [-20^C freezer] I chose the first clone
            In reference to: "Figure 1 : Gel Images from a 1% agarose ; LuxI, LuxR, Esau" [06/17/2016] Lane's 2
            A
            B
            C
            1
            		TemplatePrimer
            2
            		1LuxISenderF
            3
            		2LuxISenderR
            4
            		3LuxRRecieverF
            5
            		4LuxRRecieverR
            6
            		5EsaISenderF
            7
            		6EsaISenderR
            Table1
            Table 2: Amounts used per PCR reaction tube, that was sent to BIOB sequencing lab.
            A
            B
            1
            		0.5ulPlasmid
            2
            		1.0ulprimer
            3
            		8.5ulH2O
            Table2
            Document needed for sequencing & account number
            SOLS_Sequencing_Form_.pdf

            Sub project_promoters/primers

            Made with Benchling
            Project: BrittanyiGEM2016
            Authors: Brittany Flores
            Date: 2016-06-20
            Monday, 6/20
            Need --> inducible promoter for : LasR, Bta3R, CerR, and Expo (for Sin)
            "Ryan has the promoter sequences noted in his spreadsheet.
            Inverted repeats:"
            Cer -
            A
            1
            		AAGGTATCCATGCTAGGAATTCCTGCTTTGTATCGGCCCGCAGGTTGTGTATCAGAAAGAGGCACGGCATCAGCGCGGCAGCAATCCGCAGGTGTAGTGACGCAACGTCAGGCACGGCTCCCAAAGGTGTTTGGAACGTGCTAAATAAGCAGGACGGCTGCGCGGTGGCCGGGGGGAGCCGGCACACTAGTCATGTCGTACCCATACCTT
            Table1
            Bta3 -
            A
            1
            		AAGGTATCCATGCTAGGAATTCCACGCGCGCGACGCTAGAACGGCCGCGTGCGTCGCCGGTCAACCTCCCAAGTTTCGTGGTAACGCTCACCTCCGCGCTGACGATAGGCTGCGATCTCTTTGCTCACTAGTCATGTCGTACCCATACCTT
            Table2
            Exp -
            A
            1
            		AAGGTATCCATGCTAGGAATTCCCTGTCTCACCCCTTTCGGGTATCGGAATTGGCGCCGCAACGGCACGATGCGGCAATGCGTTGCGTCTTGAACCGACGGCAAGGATGATCTTCATCCACCTTGCGTGCCGGCAAAGCCGCGGGTGACGGCCTTTGCGTTCTTCCCAAAAAACGCGGTAGACGGGGCGGATCAGCGAGAAGGATCATCCGATACCATGGGAGGGTTGGCGGGATGATTCATCGTTAGCTATCTTGATGCCTCGACTCCGAGTTTTTGCAGGTTTCCTGCAAGACCGGGCGTGATATGAAGAATTATCGCGGTTGCAACATGAATGGCTTCTGAACGGCGTATTCACATGGCCTGGCGGGTTGCGCGGCGCGTTTTTGATTTGAGGATGCTCTACACTAGTCATGTCGTACCCATACCTT    
            Table3
            LasR, Bta3R, CerR, and Expo (for Sin). For all of these, I have the MRV + transcription factor cloned. They are missing the inducible promoter part.
            I’ve tried LasR_MRV + pLas for a long time, and I am still in the process of troubleshooting it. For the others, I could not find the inducible promoters parts. Ryan has gBlock tubes for these inducible promoters in his box, but they are empty? If you would like to take a look, they are in rows 9 and 10 in the QS Receiver cloning box (right side of the -20 freezer).
            --> did he order primers?
            amplify and clone!!! :D
            amplify the primers with pcr, and ligation -- long transformation and sequencing.

            Sequencing Results

            Made with Benchling
            Project: BrittanyiGEM2016
            Authors: Brittany Flores
            Date: 2016-06-22
            Wednesday, 6/22
            023_B05_BM1.doc
            024_A05_BM2.doc
            025_H07_BM3.doc
            026_G07_BM4.doc
            027_F07_BM5.doc
            028_E07_BM6.doc

            Cloning Inducible Promoter

            Introduction

            Our quorum sensing receivers consist of two parts: the transcription factor and the inducible promoter. The transcription factor senses HSLs in the system and if present, binds with the inducible promoter to then activate GFP. For our project, we have four more receivers without inducible promoters: Las, Bta3, Exp, and Cer.

            Materials

            • Receiver backbone
              • Inducible promoter part
                • Restriction enzymes
                  • Ligase
                    • Fast Digest Buffer (10x)
                      • Ligase Buffer

                      Procedure

                      • Digest backbone
                      1. ad
                      1. asdf
                      • Digest insert (or PCR, if necessary)
                      1. 1.

                      Mass Spec Protocol W.I.P.

                      Made with Benchling
                      Project: BrittanyiGEM2016
                      Authors: Brittany Flores
                      Date: 2016-07-18
                      Monday, 7/18

                      Mass Spec CONT.

                      Made with Benchling
                      Project: BrittanyiGEM2016
                      Authors: Brittany Flores
                      Date: 2016-07-19
                      Tuesday, 7/19

                      Mass Spec CONT.

                      Made with Benchling
                      Project: BrittanyiGEM2016
                      Authors: Brittany Flores
                      Date: 2016-07-20
                      Wednesday, 7/20

                      Mass Spec CONT.

                      Made with Benchling
                      Project: BrittanyiGEM2016
                      Authors: Brittany Flores
                      Date: 2016-07-21
                      Thursday, 7/21

                      HSL- redo

                      Made with Benchling
                      Project: BrittanyiGEM2016
                      Authors: Brittany Flores
                      Date: 2016-07-26
                      Tuesday, 7/26

                      HSL - Brady

                      Made with Benchling
                      Project: BrittanyiGEM2016
                      Authors: Brittany Flores
                      Date: 2016-07-27
                      Wednesday, 7/27
                      Redo MASS Spec
                      500ul of HSL + 1000uL of LB
                      LLE --> ethyl acetate 4 times ( aqueous layer)
                      rotary evaporation for 30 minutes.
                      resuspension in methanol about 400ul
                      waiting on the HPLC

                      HPLC

                      Made with Benchling
                      Project: BrittanyiGEM2016
                      Authors: Brittany Flores
                      Dates: 2016-08-04 to 2016-08-08
                      Thursday, 8/4

                      HPLC

                      Made with Benchling
                      Project: BrittanyiGEM2016
                      Authors: Brittany Flores
                      Dates: 2016-08-04 to 2016-08-08
                      Friday, 8/5
                      ask patrick if he has a made metrix ? research how to make the matrix

                      HPLC

                      Made with Benchling
                      Project: BrittanyiGEM2016
                      Authors: Brittany Flores
                      Dates: 2016-08-04 to 2016-08-08
                      Monday, 8/8
                      mass spec

                      MALDI

                      Made with Benchling
                      Project: BrittanyiGEM2016
                      Authors: Brittany Flores
                      Date: 2016-08-22
                      Monday, 8/22
                      - reflector mode
                      - find NMR machine ISTB 1 , contact info

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