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kim lab

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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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