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Understanding expression of Agrobacterium rhizogenes proteins (GALLS) required for gene transfer to plants. PowerPoint Presentation
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Understanding expression of Agrobacterium rhizogenes proteins (GALLS) required for gene transfer to plants. By Chris Brown Dr. Walt Ream’s Laboratory Microbiology department. Background.

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slide1

Understanding expression of Agrobacterium rhizogenes proteins (GALLS) required for gene transfer to plants.

By

Chris Brown

Dr. Walt Ream’s Laboratory

Microbiology department

background
Background

Agrobacterium damages at least 1.4 million dollars worth of agriculture per year in California and Oregon alone.

slide3
Agrobacterium is the only known prokaryote to transfer genes to a eukaryote.

Agrobacterium is used throughout microbiology to transfer DNA into plant cells

Agrobacterium tumefaciens attached to a plant cell. Image by Martha Hawes

slide4

http://www.nature.com/nature/journal/v433/n7026/images/433583a-f2.2.jpghttp://www.nature.com/nature/journal/v433/n7026/images/433583a-f2.2.jpg

http://cms.daegu.ac.kr/sgpark/microbiology/agrobacterium.jpg

  • Agrobacterium tumefaciens and A. rhizogenes infect wounded plants and transfer plasmid DNA (T-DNA) and virulence (Vir) proteins into plant cells.
fig 1 2 galls
Fig. 1.2 GALLS

Understanding the GALLS proteins

GALLS Full Length

GALLS-FL

Repeat 1

Repeat 2

Repeat 3

GALLS-CT

GALLS C Terminal

research question what causes the lower quantity of galls fl relative to galls ct

GALLS

FL

170

130

CT

100

Research Question:What causes the lower quantity of GALLS-FL relative to GALLS-CT ?

Hypothesis:

Two Causes

1. mRNA degradation

2. Codon bias

slide7

Transcription of GALLS gene

Repeat 1

Repeat 2

Repeat 3

GALLS-CT probes

Sense probe

GALLS-FL probes

Antisense probe

D

C

A

B

Ratio of Antisense to Sense RNA levels

Ratio of Sense RNA levels

slide8

Facts about Codon Bias

  • Codon UUA is found in .075% of leucines in GALLS
  • CUA is found in 5% of leucines in GALLS
  • Most common leucine in GALLS is CUC
  • GALLS contains 8 of these rare codons
  • 7 CUA 1 UUA
  • GALLS-FL upstream of GALLS-CT contains 6 of the 8 rare codons including the single UUA
fig 1 2 location of rare leucine codons
Fig. 1.2Location of rare leucine codons

GALLS-FL

Repeat 1

Repeat 2

Repeat 3

CUA (606 codon)

CUA (1347 codon)

UUA (152 codon)

CUA (1139 codon)

CUA (495 codon)

CUA (740 codon)

GALLS-CT

CUA (83 codon)

CUA (254 codon)

slide10

Method for Overcoming Codon Bias

loxP site

Over expression of tRNA gene corresponding to CUA

  • Located and amplified tRNA gene from Agrobacterium rhizogenes genome using Polymerase Chain Reaction (PCR)
  • Inserted amplicon of tRNA corresponding to leucine CUA into pCR4-TOPO® vector
  • Inserted loxP site into plasmid

PCR product

pCR4-TOPO®

slide11

Method for Overcoming Codon Bias cont.

  • Recombined the plasmid containing rare leucine tRNA gene corresponding to CUA with broad-host-range plasmid (compatible with
  • A. tumefaciens) via Cre-Lox recombination
slide12

Cre-Lox

3`GTATGT 5`

5`CATACA 3`

3`GTATGT 5`

5`CATACA 3`

3`GTATGT 5`

3`GTATGT 5`

5`CATACA 3`

5`CATACA 3`

5`CATACA 3`

5`CATACA 3`

3`ACATAC 5`

3`ACATAC 5`

5`TGTATG 3`

5`TGTATG 3`

3`GTATGT 5`

3`GTATGT 5`

5`TGTATG 3`

5`CATACA 3`

3`GTATGT 5`

5`CATACA 3`

3`GTATGT 5`

5`CATACA 3`

5`TGTATG 3`

5`CATACA 3`

slide13

Recombined the plasmid containing rare leucine tRNA gene corresponding to CUA with broad host range plasmid (compatible with A. tumefaciens) via Cre-Lox recombination

Cre protein Mediated

pCR4-TOPO vector

tRNA gene (CUA)

loxP site

slide14

Method for Overcoming Codon Bias Cont.

  • Transformed cointegrate (broad-host-range/tRNA containing plasmid) into A. tumafaciens
  • Expressed gene and determine protein yield
slide15

Method for overcoming Codon Bias Part 2

  • Replace rarest leucine codon (UUA) with most common leucine codon (CUC)
  • Create mutation in TTA (UUA) codon 152 of Galls from Leu UUA to Leu CUC using overlap extension Polymerase Chain reaction

UUA (154 codon)

PCR product

GALLS-CT

slide16

B

A

3`

5`

3`

5`

1. RXN with primers A & C

C

D

A

5`

3`

3`

5`

C

1. RXN with primers B & D

B

5`

2. Combination of DNA from Reactions in step one with out primers (self priming)

3`

D

5`

5`

3`

5`

5`

RXN with Primers A & D

A

5`

3`

3`

5`

D

slide17

Method for overcoming Codon Bias Part 2C

  • Insert PCR product with leucine mutation into vector containing the flanking sequence of Galls

Vector

Vector

Galls Flanking

PCR product

Galls Flanking

ΔUUA (154 codon)

PCR product

GALLS-CT

slide18

Method for overcoming Codon Bias Part 2C

  • Insert entire plasmid containing Galls-Δleu154 (UUA CUC) into an A. tumafaciens compatible vector
  • Transform into A. tumafaciens
slide19

Results of tRNA over expression

FL

170 kD

130 kD

CT

GALLS-FL

Repeat 1

Repeat 2

Repeat 3

-

+

tRNA

CUA (1347 codon)

CUA (1139 codon)

CUA (606 codon)

CUA (495 codon)

CUA (740 codon)

GALLS-CT

CUA (83 codon)

CUA (254 codon)

results of codon 152 leu uua to cuc mutation
Results of Codon 152 (Leu UUA to CUC) Mutation
  • Transformation of DNA unsuccessful
  • Co-transformation yielded all colonies containing only plasmid the not containing GALLS
  • Possible toxicity to A. rhizogenes
future work
Future Work
  • Use a regulated non- Agrobacterium promoter to allow control of GALLS-FL production
acknowledgements
Acknowledgements

HHMI

COS Cripps Scholarship Fund

Dr. Kevin Ahern

Larry Hodges

Dr. Walt Ream