Notebook

March

Brainstorming

Project ideas:
Pests
Bacterial Pest attractor (engineer bacteria to produce a substance, such as a pheromone or smell, that attracts and kills pests such as insects)
Natural pesticides (engineer bacteria to produce a substance that repels or kills pests, such as those that harm crops, that can later be implemented into plants)

Water/Environment:
Desalination of water (engineering bacteria to get rid of salt in salt water in order to make drinkable water from sea water)
Getting rid of estrogen mimicking compounds in water (engineering bacteria to degrade them or sequester them)
Waste treatment (engineering bacteria that can be integrated into waste water treatment)
CFCs (engineer bacteria to produce metabolites that break down chlorofluorocarbons—compounds that contribute to the degradation of the ozone layer)

Health
Stomach ulcers (creating a medication containing engineered bacteria to specifically target and kill Helicobacter pylori – the organism that causes stomach ulcers)
Diabetes (engineered bacteria as Islet cells to produce insulin)
Allergies/Immune system (engineer bacteria to produce antihistamines or alter epitopes)
Bone density (engineer bacteria to produce and secrete calcium and other compounds to help heal broken bones or to prevent osteoporosis)

Process Improvement
Oil fractionation catalyst (engineering bacteria to improve the separation of crude oil into valuable fraction and waste fraction)
Nitrate fixation (engineer bacteria to improve nitrogen fixation so not as much fertilizer is needed)

Kill Switches (engineering bacteria to undergo induced or programmed cell death in order to control the organism)

The chosen project

After dividing in to a few groups based on which project was preferred, each group made a presentation on the proposed project. In the end, we decided to go with the glucose sensing project.

Project outline

Possible things to consider

Glucose and insulin
-Detection of blood glucose levels (glucose-specific promoter?)
-Insulin production release (measure/regulation of production?)

Immune response
-Possibly use "bactoblood" strain from Berkeley 2007

Kill switch
-Control and regulation?

Delivery system
-Intravenous?
-NASA Biocapsule?

April

We divided the team into sub-groups. Each group assumed responsibility for a different aspect of the project.

Sub-groups:

-Wiki and Mascot Design

-Biosafety

-Human Practices

Wiki and Mascot Design:

This group is responsible to obtaining and uploading all necessary information to the wiki. This includes team pictures and biographies, experiments and results, and the projects of the other sub-groups. This will be a priority over mascot design.

Biosafety:

This group must demontrate the safety protocols that the Lethbridge team practices in the lab.

Human Practices:

This group is responsible for letting the poublic know what our team is and what we are doing. This can be done creatively. The point it to raise awareness for iGEM, synthetic biology and diabetes.

May

May 8, 2012: Tansformation of Parts from Kit Plate and Growing Cells from Glycerol Stocks

Transformation of parts from kit plate:

-B0034 in pSB1A2
-K093005 in pSB1A2
-J23100 in J61002 (Amp)
-E1010 in pSB2K3

Overnight cultures were also made of:

-pMA-T K33109 (HST-1)
-pSB1AK3 B0014

May 9, 2012: Miniprep and Restriction of Parts

Overnight cultures from May 8th were miniprepped and restricted in order to determine sizes.

Master mix was prepared by mixing the ingredients together in order, as shown above. 20µL worth of master mix was combined with 5µL of plasmid DNA (1-5).

-pMA-T BBa_K331009 (Heat-stable toxin signal sequence)
-pSB1AK3 BBa_B0014 (Double terminator)

Restrictions were incubated at 37˚C for 30mins then stored at -20˚C.

Colonies were also picked. Overnight cultures were made of :

-E1010 pSB2K3
-J23100 J61002 (Amp)
-K93005 pSB1A2
-B0034 pSB1A2

May 10,2012: Miniprep and Restriction of Parts

Overnight cultures from May 9th were miniprepped and restricted in order to determine sizes

Master mix was prepared by mixing the ingredients together in order, as shown above. 20µL worth of master mix was combined with 5µL of plasmid DNA (1-7).

-J23100 (J61002)
-J23100 (J61002)
-B0034 (pSB1A3)
-B0034 (pSB1A3)
-E1010 (pSB2K3)
-E1010 (pSB2k3)
-K093008 (pSB1A2)

Restrictions were incubated at 37˚C for 30min then stored in -20˚C.

May 11th: Electrophoresis of restricted parts from May 10th

Purpose: confirm existence of the following parts:

-J23100 (J61002)
-B0034 (pSB1A3)
-E1010 (pSB2K3)
-K093008 (pSB1A2)

We also grew up J04500 in pSB1AK3 for assembly

PCR of RFP (E1010) into fusion standard

(adding extra bp to get into fusion standard)

Add 17.8 uL to 8 tubes with Pfu polymerase 0.2 uL Template DNA 2 uL Put tubes on gradient function program Pfu test with various annealing temperatures:

Annealing gradient (degrees celsius):
-49.2
-51.6
-53.0
-54.5
-55.9
-57.3
-58.8
-61.2

• only 54.5 worked

May 12th

Assembly of promoter, RBS with ss and RPF with double terminator

Method: Restriction, gel extraction, & ligation

Restriction:

-J04500

-K331009

-E1010 (fusion)

-B0015

Gel extraction

We made a 1% TAE agarose gel and ran restrictions for 1 hour @ 120V

Gel extracted the DNA

Ligation:

Ligated together J04500 & K331009

Ligated together E1010 fusion & B0015

Transformation of ligated products from May 12th

May 13th

Transformation of ligated products from May 12th

2uL ligation mix to 20uL DH5ɑ, incubate on ice for 30 mins

Heat shock 45sec in water bath at 42 degrees celcius

Incubate on ice 5 mins

Add 400uL SOC media; resuspend

Shake for 1h @ 37 degrees

200uL culture onto plate

Incubate @ 37 degrees for appx. 16h

May 14th

Picking Cells from May 13th transformation

Cells grew on 2 plates, 2 colonies were picked

Picked cells were put into 5ml LB media with kanamycin

Cells did not grow

May 15th

Cells did not grow from May 14th transformation

May 19th: PCR of E1010 and J23100 (Mlc primers)

E1010

-Add 49.5uL to 4 PCR tubes and add 0.5uL Taq polymerase and 1uL template DNA to each

-E1010 (fusion) primer annealing temp – 55.5˚C

J23100:

-Add 49.5 uL of MM to 4 PCR tubes and add 0.5 uL Taq polymerase to each tube

-J23100-Mlc primer annealing temp- 58.0˚C

-Ran 1.5% agarose TAE gel at 90V for 45 mins

-E1010 expected size: 700 bp

-J23100 expected size: 215 bp

-Didn’t work

May 20: Redo may 19 PCR of E1010 (fusion) and J23100-mlc

Alterations to PCR:

-2uL MgCl2

PCR cycle:

-First Denaturation: 3 mins @ 94˚

-Denaturation: 15 sec @94˚

-Annealing: 30sec E1010: 54.5˚ J23100: 55.0˚

-Elongation: I min 30 sec @ 72˚

-Final elongation: 7 mins @72˚

-Add 49.5 uL of MM to 4 PCR tubes

-Add 1uL template DNA

-Add 0.5 uL Taq DNA polymerase to each

-Didn’t work (again!)

May21st: PCR of J23100 and E1010 repeat again!

-For both J20100 and E1010*

-Put 45.9 uL of MM into x PCR tubes

-Add 3 uL of template DNA

-PCR PFU test #4

-Ran on 1.5% agarose gel at 120V for 1hour

PCR protocol:

-First Denaturation: 2 mins @ 95˚

-Denaturation: 30 sec @95˚

-Annealing: 30sec E1010: 54.5˚ J23100: 55.0˚

-Elongation: I min 30 sec @ 72˚

-First elongation: 7 mins @72˚

-*changed to 30 cycles

May 22, 2012: PCR of J23100-Mlc

PCR Parameters:

Initial denaturation 95˚C 3mins 30 cycles of:

-denaturation 95˚C 30s

-annealing (various) 30s

-elongation 72˚C 1min

Final elongation 72˚C 7mins

PCR Samples:

PCR product expected size: 214 bps

1.5% agarose gel

Ran at 120v for 1 hr

May 23, 2012: Ligation of PCR products into pGEM Vector

1) prefix –J23100-mlc-spe cut site (Red tubes) (sample 3 from May 22nd PCR)

2) Prefix-(fusion)-E1010-suffix (Black tubes) (sample 1 from May 23rd PCR)

Contents:

-2x Rapid Ligation Buffer 5µL

-pGEM-Tor pGem-T Easy Vector 1µL

-PCR Product 3 µL

-T4 DNA Ligase 1µL

-Miliq H2O 1 µL

-Ligate at room temp for 1h 15 mins

• One of each sample ligated overnight @ 4˚c

• 2 of each ligated at room temperature for approx. 1hr

May 23, 2012 Transformation of J23100-Mlc and E1010 fusion

For each ligation mix:

-Added 2µL of ligation mix ( E1010 pcr + pGEM; J23100- mlc + pGEM) to 20.0µL of DH5α)

-Incubated on ice for 30mins

- 45secs in water bath @ 42˚C

- immediately incubated on ice for 5mins

-Added 400µL of SOC media; resuspended

-Put in shaker for 1hr @ 37 ˚C

-200µL cultured onto plate(4 plates, Amp)

-Incubate @ 37 ˚C for approximately 16hrs

May 24, 2012: Transformation of PCR Products in pGEM Vectors in DH5ɑ

Sample 3 from PCR product ligation

For each ligation mix:

-Added 2µL of ligation mix ( E1010 pcr + pGEM; J23100- mlc + pGEM) to 20.0µL of DH5α

-Incubated on ice for 30mins

- 45secs in water bath @ 42˚C

- immediately incubated on ice for 5mins

-Added 400µL of SOC media; resuspended

-Put in shaker for 1hr @ 37 ˚C

-200µL cultured onto plate(4 plates, Amp)

-Incubate @ 37 ˚C for approximately 16hrs

May 25, 2012: QIA Spin Miniprep of J23100-mlc (A: colony 1), J23100-mlc (B: colony 2)

-Put 1.5mL of transformed cells in a microcentrifuge tube; centrifuge and repeat until cells from entire tube are in the microcentrifuge tube.

-Resuspend pelleted bacterial cells in 250uL Buffer P1

-Add 250uL Buffer P2, mix by inversion

-Add 350uL Buffer N3, mix by inversion

-Centrifuge for 10 mins @ 13000 rpm

-Put supernatant into the QIAprep spin column

-Centrifuge for 60s at 14000 rpm, discard flow through

-Add 750uL buffer PE and centrifuge for 60s at 14000 rpm

-Centrifuge again to get rid of remaining buffer

-Add 50uL of Buffer EB to spin columns, let stand for I min, centrifuge for 1 min at 14000 rpm

May 29, 2012: Transformation of E1010 fusion in pGEM in DH5ɑ

For each ligation mix:

-Added 2µL of ligation mix ( E1010 pcr + pGEM; J23100- mlc + pGEM) to 20.0µL of DH5α

-Incubated on ice for 30mins

- 45secs in water bath @ 42˚C

- immediately incubated on ice for 5mins

-Added 400µL of SOC media; resuspended

-Put in shaker for 1hr @ 37 ˚C

-200µL cultured onto plate(4 plates, Amp)

-Incubate @ 37 ˚C for approximately 16hrs

May 31, 2012: Restriction ofE1010 psB2k3, E1010 fusion pGEM, J20100-mlc pGEM, CK331009 psEk3, E1010 p5B2k3

• NEED PROTOCOL AND VOLUMES FOR GEL. GEL TABLE IS IN THE POWERPOINT

June

June 1, 2012: Restriction of J23100-Mlc, K093005, B0014 (samples A and B), J04500, K331009 (samples A and B), E1010 Fusion (samples A and B)

• NEED PROTOCOL, TABLES ARE IN POWERPOINT, NEED TO LABEL AS SAMPLES A AND B