1. UV Exposure Time (minutes)
Expression of the Arabidopsis ABC Transporter
Atwbc19 with a C-terminal GFP Fusion
Kayla Ruby Arroyo, Michelle Lang, Mentewab Ayalew
Spelman College Biology Department
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Methods
• PCR: Amplify Atwbc19 from pMDC32-Atwbc19
• Gel Extract: Purify Atwbc19 fragment
• Topo Cloning: insert into pENTR-D/TOPO vector to generate entry
clone (pENTR-D/TOPO-Atwbc19) for LR reaction
• LR Reaction: Generate pMDC85-Atwbc19 using LR clonase, entry
clone containing Atwbc19 insert, and destination vector pMDC85
(Figure 2).
• Restricted Digest: Identify correct clones using restriction digest
with Sac I and Spe I
• Transformation: Agrobacterium with pMDC85-Atwbc19 plasmid
isolated from E. coli
Figure 2 LR Reaction between pENTR-D/TOPO-Atwbc19 and
destination vector pMDC85 to generate pMDC85-Atwbc19.
Results
Conclusion and Future Work
• In conclusion, we successfully cloned the Atwbc19 tagged
with C-terminal GFP.
• Future studies consist of Atwbc19 with a C-terminal using
GFP fusion for the infiltration of tobacco leaves and stable
transformation of Arabidopsis plants. Expression levels will
subsequently be monitored by western blot.
Acknowledgments
This research was made possible with the sponsorship of
Spelman College, MBRS-RISE and the NSF.
Figure 3 PCR of Atwbc19. A 2,178
bp band was amplified for cloning in
the pENTR-D/TOPO vector.
Lanes
M-Promega 1kb ladder
1- No template
2- pMDC85-Atwbc19.
Figure 4 Double Digest of
pMDS85-Atwbc19 Clones
1,2,&5.
Lanes
M- Promega 1kb Ladder
1-Undigested Clone 1 DNA
2-Clone1 + SpeI
3-Clone1 + SacI/SpeI
4-Undigested Clone 2
5- Clone 2 + SpeI
6- Clone 2 + SacI/SpeI
7- Undigested Clone 5
8- Clone 5 + SpeI
9- Clone 5 + SacI/SpeI
M 1 2 3 4 5 6 7 8 9
3,000 bp
2,500
bp2,000bp
1,500 bp
1,000
bp
M 1 2
2,500 bp
2,000 bp
We successfully PCR amplified Atwbc19 (Figure 3) and
cloned it in the pENTR-D/TOPO vector). We subsequently
recombined it with pMDC85 (Karimi, et al. 2007) and obtained
several colonies. Three colonies were checked with restriction
digest ( Figure 4) and only two harbored the correct plasmid,
which showed the expected fragments at 9,951 bp, 1,851 bp,
and 1,249 bp.
Introduction
Purpose and Hypothesis
The Arabidopsis thaliana ABC transporter Atwbc19
confers kanamycin resistance to transgenic plants
(Mentewab, and Stewart 2005). Various versions of
Atwbc19 tagged with (GFP) were constructed for the
subcellular localization of the protein and to further
understand the mechanism of kanamycin resistance.
However, in these previously developed versions the
expression level of the protein was not high enough for
detection using confocal microscopy or western blot.
The goal of this project is to tag Atwbc19 with GFP
in the C-terminal region (Figure 1) and to increase
the amount of protein detected.
GFP
Figure1 Tagging Atwbc19 with GFP. GFP allows the
monitoring of the expression and subcellular localization
of Atwbc19
References: Karimi, Mansour, Ann Depicker, and Pierrer Hilson. "Recombinational Cloning with Plant Gateway Vectors." Update on Recombinational Cloning with Plant Gateway Vectors. 145. (2007): 1144-1154.
Mentwab, Ayalew, and Neal Stewart. "Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants." Nature Biotechnology. 23. (2005): 1177-1180.