Brown iGEMinternational genetically engineered machines competition

August Update

1/55

Brown iGEM 2007

• Lead Sensor

• Tristable Switch

• iGEM Jamboree on November 4th at MIT

2/55

Lead Sensor

Introduction

Speaker: Deepa Galaiya

3/55

Lead Detection

Signal Amplification

Fluorescent Output

Lead

General Design

Lead Detection

Signal Amplification

4/55

5/55

Lead Sensor

Lead Detection

6/55

PbrR691 Coding Region

PbrA Coding Region

Non-Coding Region

In Ralstonia metallidurans:

7/55

PbrR691 Coding Region

PbrA Coding Region

Non-Coding Region

RBS

RBS

Lead Promoter

PbrR691 Promoter

8/55

PbrR691 Coding Region

PbrA Coding Region

Non-Coding Region

3 versions

6 versions (Neils method)

3 versions

1 version

2 versions

15 total

9/55

Planned Ligations

- All 15 parts into BioBrick plasmid- pTet to PbrR691 alone- All promoters and PbrR691

combinations to LuxI- All promoters and PbrR691

combinations to GFP- pTet-PbrR691 to promoters-LuxI- pTet-PbrR691 to promoters-GFP

10/55

Completed Ligations

- All 15 parts into BioBrick plasmid

- pTet to PbrR691 alone (done today!)

- All promoters and PbrR691 combinations to LuxI

- All promoters and PbrR691 combinations to GFP

- pTet-PbrR691 to promoters-LuxI

- pTet-PbrR691 to promoters-GFP11/55

Results

• 15 parts into Biobrick gelled and sequenced

• PbrR691/promoter combinations in presence of lead nitrate give no GFP production and no AHL production compared to control.

12/55

Discussion & Future Plans

PbrR691 most likely not expressed in combination parts. (Transcription factors needed?).

Promoter not abandoned yet!

Constitutive expression under pTet possible. Neils’ group was able to overexpress protein in E Coli under

IPTG control.

13/55

Lead Sensor

The Amplifier

Speaker: Jeff Hofmann

14/55

T9002

J37015 (The Amplifier)

Differences: Positive Feedback Loop Stronger Ribosome Binding Site

15/55

How did we measure this?

GFP fluorescence – average control GFP fluorescence Cell Density

16/55

Expected: Amplifier produces more GFP than T9002

Results: T9002 produces far more GFP than Amplifier(G

FP –

Con

trol

) /

Cell

Den

sity

Time (hours)

17/55

Expected: Direct relationship between AHL input and GFP output

Results: Indirect relationship between AHL input and GFP output

18/55

More AHL = Less GFP!

J37015 GFP output at 5 Hours

0

1000

2000

3000

4000

5000

6000

0 0.1 nM 1 nM 10 nM 100 nM 1 uM 10 uM 100 uM 1 mM

[AHL]

(GFP - control) / Cell Density

5 Hours

J37015 GFP output at 5 Hours

0

1000

2000

3000

4000

5000

6000

0 0.1 nM 1 nM 10 nM 100 nM 1 uM 10 uM 100 uM 1 mM

[AHL]

(GFP - control) / Cell Density

5 Hours

19/55

Why does this happen? Possible wrong promoter GFP is further away from promoter in J37015

T9002

J37015 (The Amplifier)

20/55

Why does this happen? Possible wrong promoter GFP is further away from promoter in J37015

T9002

J37015 (The Amplifier)

21/55

Lead Sensor

Sequencing

Speaker: Rohan Maddamsetti

22/55

Sequencing

• Importance of Sequencing

• Sequencing the Amplifier

• Sequencing the Lead Parts

• Where do we go from here?

23/55

Tri-Stable Switch

Speaker: Kyle Schutter

24/55

A

B C

The Tristable Switch

A switch with three distinctand inducible states.

25/55

Achieving Tri-stability

State A

State C

State B

Output A

Output B

Output C

Input A

Input CInput B

26/55

The Switch

pBADpBAD TetRTetRLacILacI

LacILacI

TetRTetRAraCAraC

AraCAraC

pLacpLac

pTetpTet

The Architecture as planned

C

A

B

27/55

The Switch

pBADpBAD TetRTetRLacILacI

LacILacI

TetRTetRAraCAraC

AraCAraC

pLacpLac

pTetpTet

The Architecture as planned

L-arabinose

28/55

The Switch

pBADpBAD TetRTetRLacILacI

LacILacI

TetRTetRAraCAraC

AraCAraC

pLacpLac

pTetpTet

The Architecture as planned

IPTG

29/55

The Switch

pBADpBAD TetRTetRLacILacI

LacILacI

TetRTetRAraCAraC

AraCAraC

pLacpLac

pTetpTet

The Architecture as planned

anhydrotetracyclineanhydrotetracycline

30/55

• Parts in the registry only allow two stable states and a third inducible state

• pBAD promoter is attached to gene

pBADpBAD TetRTetR LacILacI

LacILacI

TetRTetRAraCAraC

AraCAraC

pLacpLac

pTetpTet

pCpCAraCAraC

Architecture ReDesigned

31/55

pBADpBAD TetRTetR LacILacI

LacILacI

TetRTetRAraCAraC

AraCAraC

pLacpLac

pTetpTet

pCpCAraCAraC

Architecture ReDesignedL-arabinose

32/55

pBADpBAD TetRTetR LacILacI

LacILacI

TetRTetRAraCAraC

AraCAraC

pLacpLac

pTetpTet

pCpCAraCAraC

Architecture ReDesignedL-arabinose

But there is a lot of araC in the system33/55

pBADpBAD TetRTetR LacILacI

LacILacI

TetRTetRAraCAraC

AraCAraC

pLacpLac

pTetpTet

pCpCAraCAraC

Architecture ReDesigned

The system will fall into whichever of the

other two states is stronger.34/55

Characterization

• Registry not as well characterized/reliable as expected

– Rumor that araC gene has promoter region inside it

– All repressors LVA tagged: fast degradation leads to poor repression

– AraC connected to pBAD promoter

35/55

iGEM Characterization Protocol

• Starting to formulate Characterization protocol for Promoters and Repressors

– Promoters: relative, how “on” or “off”

– Repressors: cooperativity, binding constant

36/55

Tristable Switch

Creating the Parts

Speaker: Adam Emrich

37/55

3 Major Steps to Create Parts

• Transformation of DNA, to make more DNA

38/55

The 3 Major Steps

Transformation

Restriction

Ligation

39/55

Purposes of each Step

Transformation: Increases the amount of DNA.

Extraction: Removes DNA from cells.

Restriction: Cuts DNA, to prepare it for Ligation.

Ligation: Attaches cut DNA, to create new genetic parts.

40/55

Step 1: TransformationPurpose:

To create more DNA.

Method:

1. Insert DNA into specially prepared Competent Cells.

2. Plate out cells, allow to grow overnight.

3. Create overnight culture.

4. Extract DNA. 41/55

Step 2: RestrictionPurpose:

To cut DNA from Step 1, in preparation for Step 3: Ligation.

Method:

1. Insert DNA into a vial.

2. Add buffer and restriction enzymes, incubate 4-6 hours.

3. Heat inactivate enzymes. 42/55

Step 3: Ligation

Purpose:

To attach DNA from Restriction step together, resulting in new Genetic Parts.

Method:

???

We do not have an exact method for this yet.

43/55

Protocol Determination

Transformation: ~Early July

Competent Cell Preparation: ~Early August

Ligation: ?

44/55

Step 3: Ligation ProtocolOur ligation success rate is about 10%.

We are currently running experiments to determine a protocol that works at a higher success rate.

45/55

Plan for the Future1. Establish a working protocol for Ligations

2. Work on project during semester

3. Assemble a Bi-Stable Switch by the Jamboree

46/55

47/55

The Next Step

• Preparing for November 4th, MIT

• Continue progress

• Build

• Characterize

48/55

Next Year

• Master protocols earlier

• Transformation to Ligation

• Problem-solving skills

49/55

New Technologies

• Automated Assembly

• Synthesize all DNA

• New Equipment - Plate Reader

50/55

Student Led

• iGEM Student Group (Fall ‘07)

• 2008 Applications judged by 2007 Team

• Be Selective

• Goal: 50 Applications

51/55

Sponsors

Thank you

52/55

53/55

John Cumbers

Alex BrodskyTayhas Palmore

Gary Wessel

Multidisciplinary Lab(Kathy, Shirley, Faye, Phil, Sarah)

CCMBMCB

MPPB Department of Physics Division of Engineering

54/55

Brown iGEMinternational genetically engineered machines competition

August Update

55/55