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Function Identification of gene pZJM103 from Trypanosoma brucei through TAP tagging and Identification of Binding Partners Kimberly Brewer Governor’s School for Science and Mathematics: SPRI Dr. Jim Morris Clemson University Trypanosoma • Trypanosoma brucei are parasitic protozoa • One strain of Trypanosoma brucei, transmitted to humans by tsetse flies. Trypanosoma brucei rhodesiense is • Trypanosoma brucei brucei is killed by the cause of Trypanosomiasis, the trypanolytic chemicals in human serum disease better known as African thus it is not dangerous and is the Sleeping Sickness trypanosoma used in the lab. • Trypanosoma brucei have antigenic variation, a process in which the parasite can change its surface coat to make it able to survive in the host, whether it be a mammal or an insect. Red Blood Cell Background courtesy of EYE of Science.com Life Cycle of Trypanosoma Brucei • The metacyclic form of the trypanosome is injected under skin of a mammal by Tsetse fly. • The metacyclic form transforms into the trypomastigote form and divide through binary fission in the interstitial spaces at the bite site causing a chancre due to build-up of biological waste. • The flagellated form enters the bloodstream through the lymphatic system and eventually enter the central nervous system. • The tsetse fly ingests a blood meal and becomes infected with a short, stumpy form. • They develop into procyclic trypomastigotes in the midgut of the fly and divide for 10 days. • The organisms migrate to the salivary glands and turn into epimastigotes • They then divide and transform into metacyclic trypanosomes, the infective stage for mammals. Trypanosomiasis • 3 Types of Trypanosoma brucei that cause • Symptoms (continued) Trypanosomiasis – Sleeplessness -- T.b. brucei – Stiff neck • Cattle disease (does not infect humans) – Depression – T.b. gambiense – Anemia • Western and Central Africa – Focal seizures • Incubation longer (weeks or months) • Involvement of CNS after months or years – Tremors – T.b. rhodesiense – Palsies • Classical “African Sleeping Sickness” – Coma • Eastern and Southern Africa – Death • 2-3 week incubation • Diagnosis • CNS involvement in 3-4 weeks Staining blood smear with Wright’s or • Trypanosomiasis Giemsa stain – Infects individuals in 36 Sub-Saharan African PCR countries WHO tests – There are over 300,000 case reported annually • Treatment – 66,000 deaths annually from African Sleeping Suramin for non-CNS sickness Melarsoprol (arsenical) for CNS – Infection occurs due to a bite from an infected tsetse fly Difluoromethylornithine or Eflornithine for T.b. gambiense with CNS • Symptoms of Trypanosomiasis Replacement for Melarsoprol • Chancre at bite site not effective against T.b.rhodesiense • Rash Pentamidine isethionate for non-CNS • Severe Headache TAP (Tandem Affinity Purification) Tagging • TAP tagging means fusing a TAP Tag to the target protein and introducing it into the host. • TAP tags are very tolerant to all buffer conditions • Using this method, it should be possible to purify 103 and identify its binding partners . This in turn could be used to identify the function of the gene. Reproducing pHD 918 • A transformation into DH5∂ with Clone 1 Clone 2 Clone 3 pHD 918 from Christine Clayton • Pick 3 separate clones • Plasmid purification of clones Insert • Restriction Digest to DH5∂ determine if pHD 918 which was taken up by the DH5∂ had any mutations Clone 1 HpaI BamHI – Using HindIII and BamHI TAP Tag DH5∂ – Using XbaI XbaI 556 bp XbaI • Clone 1 took up the correct pHD 918, clones 2 & 3 did not • Clone 1 was prepared into a Insert glycerol stock for later use Original PCR of 103 • PCRs with primers to add HpaI and HindIII restriction sites were successful – The first PCR, with a 50˚ annealing temperature, produced dimmers Expected product – The second PCR, with a 55º (103) annealing temperature, produced a promising band. Column Dimers purified and used as the template in a PCR at 52° annealing. It did not produce a band. There was no DNA in the column Product purification product because of (103) a problem with the columns. dimers Successful PCR of 103 using HpaI and HindIII primers • PCR was run at a 54° annealing and the products were Column Purified • Another PCR was run at 57° Dimers annealing and no products were produced • It was determined that a mistake was made in the original 55° PCR so it was repeated and the results were kept as the most successful PCR over a 56° PCR which produced a smaller band Gel purification • A gel purification of the remaining products of the 55º PCR reaction was done. • It was ligated into pGEMEZ and transformed into DH5∂ • No results could be formulated due to a mistake in incubation. • The pGEMEZ did not pick-up the expected plasmid so the process was repeated Cloning into pGEMEZ • Purified 103 from minipreps from scrapes of glycerol stocks were cloned into pGEMEZ • The products of the afore mentioned ligation were transformed into DH5∂ and grown-up. The ones that grew were miniprepped • A restriction digest attempting to obtain a 2.1kb band was run on the minipreps using EcoRI and the transformation was discovered to have failed. Conclusions • In conclusion, the TAP tag has still not been inserted onto the gene but the gene has been replicated and transformed. The next step is to try to place the tag on the pZJM 103.
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