1	Jacqueline Farst
2	Background Major Vector-Borne Diseases of Cattle Worldwide
3	Anaplasmosis
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6	Kocan, K. M., Ewing, S. A., Hair, J. A. and S. J. Barron (1984) American Journal of Veterinary Research 45: 1800-1807
7	Background What is Actin??
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10	AAAP sequences
11	Objective To clone and express rAAAP Antibody Production - Confirm target - Purify inclusion appendages Biological activity in vitro
12	Rationale Defining the role(s) of AAAP in A. marginale transmission could lead to a new way to control it and similar pathogens Interference with transmission of zoonotic pathogens would improve public health Control of vector-borne diseases in food animals would improve the global food supply
13	Steps to Meet Objectives 1.) Design PCR (polymerase chain reaction) primers. 2.) Perform PCR reactions 3.) Perform cloning reactions 4.) Purify plasmid DNA 5.) Verify product sequences 6.) Express the protein 7.) Confirm with Western Blot 8.) Purify inclusion appendages to identify components
14	Primer Design
15	Primer Design
16	Performing PCR Reactions PCR reactions are very important in this project. The first step was to extract DNA from the bovine red blood cells, then check the DNA concentration. Next the PCR reactions were performed to produce an amplicon.
17	How PCR works…
18	Performing PCR Reactions The AAAP Illinois product is at 1250 base pairs. This product was then used in the cloning reaction.
19	Cloning Reaction Invitrogen’s pBAD – TOPO Cloning Reaction
20	Cloning Reaction After the Topo reactions were completed, LB plates, with ampicillin, were inoculated
21	Cloning Reactions This shows the inoculated plates growing colonies of E. coli with the AAAP insert included in them.
22	Screening Clones with PCR
23	Cloning Reaction After the colonies were grown on plates, there was a random selection of single colonies that were inoculated into LB broths, and incubated overnight. Following incubation, PCR was performed on the broths to ensure correct orientation.
24	Purifying Plasmid DNA QIAprep Spin Miniprep Kit.
25	Sequencing The Plant Microbe Genomic Center sequenced the plasmid and reported their findings.
26	Continue Sequencing
27	Work in Progress Sequence analysis Express the protein Confirm protein with Western Blot Purify inclusion appendages to identify components
28	Experience This summer internship helped me realize that I do want to pursue a career in research. Not only do I find the work very interesting, but I had a lot of fun working closely with lab mates.
29	Thank you to ALL Lab mates!