Team:CGU Taiwan/Notebook

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Notebook





May 2016

  • Week 1 (5/1~5/8)

    4/25 Receive the kit from iGEM.
    4/27 Meet with Dr. Chang via skype to discuss the main idea of iGEM project, patent of leishmania and materials.
    5/4 Receive the pX63-Hyg from Dr. Chang and dissovle the pX63-Hyg from the filter paper with 100 μl TE buffer.
    5/5 Transform the pX63-Hyg into DH5α to amplify the plasmid.
    5/6 Fail to transform the pX63-Hyg and try again the transformation with another source of competent cell.
    5/7 Still fail to transform the pX63-Hyg.

    Week 2 (5/9~5/15)

    5/10 Try again the transformation with another protocol.
    5/11 Still fail to transform the pX63-Hyg.
    5/15 Ask for another piece of filter paper containing the pX63-Hyg.

    Week 3 (5/16~5/22)

    5/22 First meet with Dr. Chang in NTMU to discuss the whole procedures of iGEM project.

    Week 4 (5/23~5/31)

    5/28 Receive the DC2.4 and J774A1 cells from NTMU and amplify the cells in CGU.
    5/29 Meet with Dr. Chang to learn about the culture of leishmania, DC2.4 and J774A1 cells in NTMU.
    5/31 Cryopreserve the DC2.4 cells





June 2016

  • Week 1 (6/1~6/5)

    6/6 Receive another piece of filter paper containing pX63-Hyg from Dr. Chang.

    Week 2 (6/6~6/12)

    6/13 Dissolve the filter paper with 100 μl TE buffer overnight.
    6/14 Transform the plasmid into DH5α.

    Week 3 (6/13~6/19)

    6/15 Get four colonies from the plate and amplify in each 4ml Amp+-LB liquid.
    6/16


    1.Store the bacteria in -80 oC and mini extract the DNA of bacteria.
    2.Restriction enzyme check of pX63-Hyg by BamHI restriction enzyme.
    3.Successfully get the pX63-Hyg.

    Week 4 (6/20~6/26)

    6/23 Transfer four strains of leismania to CGU from NTMU.
    6/24 Amplify and cryopreserve four strains of leishmania.
    6/26 Cryopreservation of four strains of leishmania.2.G418, tunicamycin selection of leishmania (double transfectants).

    Week 5 (6/27~7/3)

    6/28

    1.Receive DNA oligos from IDT and dissolve them in ddH2O.
    2.MasterMix digestion of 5’UTR-Hyg, HA, 3’UTR, OVA1-OVA2, HA-3’UTR and linear pSB1C3 following iGEM protocol.

    6/29

    1. Preparation of competent cells (DH5α).
    2. Ligate 5’UTR-Hyg, HA, 3’UTR, OVA1-OVA2 and HA-3’UTR with linear pSB1C3. (First time)

    6/30

    1. Check the quality of competent cells by transformation of pRF and ddH2O.
    2. Ligate 5’UTR-Hyg, HA, 3’UTR, OVA1-OVA2 and HA-3’UTR with linear pSB1C3. (Second time)
    3. Transformation of the ligation samples from 160629.

    7/1

    1. Confirm the efficiency and authenticity of competent cells made from 160629.
    2. Cryopreservation of four strains of leishmania selected by drug.
    3. DNA mini preparation of pSB1C3-5’UTR-Hyg/HA/3’UTR and check the insert authenticity by PCR.

    7/2

    1. Restriction enzyme check of pSB1C3-5’UTR-Hyg/HA/3’UTR by EcoRI and PstI.
    2. Digestion of OVA1, OVA2 and linear pSB1C3.

    7/3

    1. Transformation of the plasmid from the plate 1-B in order to get thepSB1C3 backbone and easily to ligate other inserts.
    2. Successfully maintain four strains of leishmania and get pSB1C3-5’HYG/HA/3’UTR.




July 2016

  • Week 1 (7/4~7/10)

    7/4

    1. Cryopreservation of four strains of leishmania selected by drug.
    2. Ligate OVA1-OVA2 with linear pSB1C3. 3.DNA mini preparation of the plasmid from the plate 1-B.

    7/5

    1.Enzyme digestion of the plasmid from the plate 1-B by EcoRI and PstI.
    2.Transformation of the ligation samples from 160704.

    7/6

    1.Enzyme digestion of the plasmid from the plate 1-B by EcoRI and PstI.
    2. Transformation of the ligation samples from 160704.

    7/7

    1. Design OVA primers.
    2. 2.3 intron sequence primer design.

    Week 2 (7/11~7/17)

    7/11 Receive OVA primers. Dissolve them in ddH2O
    7/12

    1.Perform OVA PCR by high fidelity polymerase.
    2. Digest pX63-HYG and HA with BamHI.
    3. Purify OVA PCR product by gel extraction.
    4. Digest OVA and pSB1C3 with EcoRI, PstI and SapI.

    7/13

    1. Purify digested pX63-HYG and HA with gel extraction.
    2. Purify digested OVA and pSB1C3 with gel extraction.
    3. Ligate pX63-HYG and HA, pSB1C3 and OVA.
    4. Transform the pX63-HYG-HA and pSB1C3-OVA ligation products.

    7/15

    1. Perform OVA PCR by high fidelity polymerase.
    2. Digest pX63-HYG and HA with BamHI.
    3. Digest OVA with EcoRI, PstI and SapI.

    7/15

    1. Purify digested pX63-HYG and HA with gel extraction.
    2. Purify digested OVA and pSB1C3 with gel extraction.
    3. Ligate pX63-HYG and HA, pSB1C3 and OVA.

    Week 3 (7/18~7/24)

    7/18 Transform the pX63-HYG-HA and pSB1C3-OVA ligation products from 160715.
    7/19

    1. Re-transform pX63-HYG-HA and pSB1C3-OVA ligation products from 160715.
    2. Ligate pSB1C3 and OVA.

    7/20

    1. Seed pX63HYG-HA colonies. The sequencing result is incorrect.
    2. Check pSB1C3-OVA colony by colony PCR.

    7/21

    1. Digest OVA with EcoRI, PstI and SapI.
    2. Check pX63HYG-HA colony by colony PCR.

    Week 4 (7/25~7/31)

    7/25 Ligate pSB1C3 and OVA.
    7/26 Transform pSB1C3-OVA ligation product.
    7/27

    1. Receive ALA from Taipei Medical University.
    2. Check pSB1C3-OVA colony by colony PCR.

    7/28

    1. Sequencing of pSB1C3-OVA
    2. Digest pSB1C3-OVA with BamHI.

    7/29

    1. Purify OVA sequence by gel extraction.
    2. Ligate pX63HYG and OVA.

    7/30 Transform pX63HYG-OVA into DH5α.





August 2016

  • Week 1 (8/1~8/7)

    8/1 Colony PCR of pX63-HYG-OVA
    8/2 Design primers for 2.3 intron sequence.
    8/3 Purify leishmania genomic DNA.
    8/4 Perform PCR with 2.3 intron primers and leishmania genomic DNA as template. Fail to proliferate the sequence.
    8/5 Receive the p6.5 plasmid from Dr. KPC. Elute the DNA.

    Week 2 (8/8~8/14)

    8/8

    1. Ligate pX63HYG and HA.
    2. Transform pX63HYG and HA ligation product.
    3. Transform p6.5 plasmid into DH5α. No colony forms.

    8/9

    1. Transform pX63HYG-OVA.
    2. Transform p6.5 into DH5α.

    8/10

    1. Transform pX63HYG-OVA.
    2. Transform p6.5 into DH5α.

    8/11

    1. Purify pX63HYG-OVA by miniprep.
    2. Digest pX63HYG-OVA by EcoRI and PstI.
    3. Sequencing of pX63HYG-OVA. The sequence of OVA is incorrect.


    Week 3 (8/15~8/21)

    8/15 KOD PCR of 2.3kb intron region from p6.5 plasmid.
    8/16 Re-KOD PCR of 2.3kb intron region with gradient from p6.5 plasmid.
    8/17

    1. Enzyme digestion of 2.3 kb intron.
    2. Purify the digested 2.3 kb intron by gel extraction.
    3. Ligation of pSB1C3 and 2.3 intron.

    8/18 Transform pSB1C3 and 2.3 intron ligation product. No colony forms.

    Week 4 (8/22~8/28)

    8/23

    1. Re-KOD PCR of 2.3kb intron region from p6.5 plasmid.
    2. Still fail to get the 2.3kb intron region by KOD PCR.

    8/24
    Using NEB cloner to divide the 2.3kb intron region into 3 parts, and each digested by double restriction enzyme to
    form sticky end for ligation. Double restriction enzyme(EcoRIHF and KpnIHF) of part I; double RE(KpnIHF and NdeI) of part II;
    double RE(NdeI and PstI) of part III; double RE(EcoRIHF and PstI) of pSB1C3.
    8/25 Ligate pSB1C3 with part I, II, III to form pSB1C3-2.3kb intron region.
    8/26

    1. Transform the ligation sample(pSB1C3-2.3kb from 160825) into DH5α.
    2. Due to wrong sequencing of OVA, re-ligate linear pSB1C3 with OVA1 and OVA2.

    Week 5 (8/29~9/4)

    8/29

    1. Re-ligate pSB1C3 with part I, II, III to form pSB1C3-2.3kb intron region.
    2. Colony PCR of pSB1C3-2.3 kb.

    8/30

    1. Colony PCR of pSB1C3-2.3 kb and pSB1C3-OVA.
    2. Still fail to get the correct pSB1C3-2.3 kb plasmid.

    8/31

    1. Double restriction enzyme(EcoRIHF and KpnIHF) of part I; double RE(KpnIHF and NdeI) of part II;
    double RE(NdeI and PstI) of part III; double RE(EcoRIHF and PstI) of pSB1C3.
    2. Before ligate the 2.3kb with pSB1C3, conduct ligation and PCR to get the intact 2.3kb insert then cut by RE.
    3. Also ligate pSB1C3 with part I, II, III to form pSB1C3-2.3kb.

    9/1

    1. Test the ligation of three parts of 2.3kb by RE.
    2. Transform ligation sample(pSB1C3-2.3 from 160831) into DH5α.

    9/2

    1. Ligate 2.3kb which has KOD PCR with pSB1C3.
    2. Ligate three parts to form 2.3kb and ligate pSB1C3 with 2.3kb.
    3. Colony PCR of pSB1C3-2.3.
    4. KOD+ PCR of 2.3kb intron region from p6.5 plasmid.
    5. Enzyme digestion(EcoRIHF and PstI) of 2.3kb which has ligated and KOD+ PCR.
    6. Transform ligation sample(pSB1C3-2.3 from 160901) into DH5α.

    9/3

    1. Colony PCR of pSB1C3-2.3.
    2. Re-ligate 2.3kb which has KOD PCR with pSB1C3.

    9/4

    1. Transform ligation sample(pSB1C3-2.3 from 160903).
    2.Successfully get the correct pSB1C3-OVA.

  • Week 1 (8/1~8/7)

    8/5
    1.pretest B6 mouse for first immunization
    2.Animal trails (trail1 saline, trail2 saline+OVA,trail3 high,Alum ,trail4 106 leishmania+OVA,
    trail4 107 leishmania+OVA,trail5 108 leishmania+OVA

    Week 2 (8/8~8/14)

    8/15
    collect D10 serum from mouse facial vein

    Week 3 (8/15~8/21)

    8/20
    collect D15 serum from mouse facial vein

    Week 4 (8/22~8/28)

    8/25
    collect D20 serum from mouse facial vein

    Week 5 (8/19~9/4)

    8/30
    collect D25 serum from mouse facial vein





September 2016

  • Week 1 (9/5~9/11)

    9/5

    1. Restriction enzyme digestion(EcoRIHF and SpeI) of pSB1C3-HA/OVA; Restriction enzyme digestion
    (EcoRIHF and XbaI) of pSB1C3-3’UTR; Restriction enzyme digestion(BamHI) of pX63HYG and pSB1C3-HA/OVA.
    2. Ligate pSB1C3-OVA/HA with 3’UTR and ligate pX63HYG with HA/OVA.
    3. Re-ligate 2.3kb which has KOD PCR with pSB1C3.

    9/6

    1. Colony PCR of pSB1C3-OVA and pSB1C3-3’UTR and pSB1C3-2.3kb.
    2. Restriction enzyme digestion(EcoRIHF and PstI) of pSB1C3; Restriction enzyme digestion
    (XbaI and PstI) of pSB1C3-3’UTR; Restriction enzyme digestion(EcoRIHF and SpeI) of
    pSB1C3-5’HYG/OVA; Restriction enzyme digestion(EcoRIHF and PstI) of pSB1C3-3’UTR;
    Restriction enzyme digestion(BamHIHF) of pSB1C3-OVA; Restriction enzyme digestion(PstI and XbaI)
    of pSB1C3-3’UTR.
    3. Ligate pX63HYG with OVA/HA; Ligate pSB1C3 with 2.3kb; Ligate pSB1C3 with 3’UTR.

    9/7

    1. Restriction enzyme digestion(EcoRIHF and SpeI) of pSB1C3-OVA; Restriction enzyme digestion(BamHI)
    of pSB1C3-OVA; Restriction enzyme digestion(XbaI and PstI) of pSB1C3-2.3kb and pSB1C3-3’UTR.
    2. Ligate three parts to form 2.3kb and ligate pSB1C3 with 3’UTR.
    3. Transform ligation samples from 160906 and 160907.

    9/8

    1. KOD+ PCR of 2.3kb intron region from p6.5 plasmid.
    2. Colony PCR of the colonies.
    3. Double restriction enzyme digestion(EcoRIHF and KpnIHF) of 2.3 kb part I; double RE digestion
    (KpnIHF and NdeI) of part II; double RE digestion (NdeI and PstI) of part III; double RE digestion
    (EcoRIHF and PstI) of pSB1C3.

    9/9

    1. Ligate pSB1C3 with 2.3kb.
    2. Ligate three parts to form 2.3kb.
    3. Restriction enzyme digestion(PvuII) of pX63HYG; double restriction enzyme(PstI and XbaI) of pSB1C3-3’UTR.
    4. Transform ligation samples from 160908.

    9/10

    1. Double restriction enzyme digestion (EcoRIHF and XbaI) of pSB1C3-3’UTR; restriction enzyme digestion
    (EcoRIHF and SpeI) of pSB1C3-OVA.
    2. KOD+ PCR of 2.3kb intron region from p6.5 plasmid.
    3. Ligate three parts to form 2.3kb.
    4. Transform ligation samples from 160909.
    5.Successfully get the correct pX63Hyg-OVA/HA.

    Week 2 (9/12~9/18)

    9/11

    1. Ligate pSB1C3-3’UTR with OVA/HA.
    2. KOD+ PCR of 2.3kb intron region from p6.5 plasmid.
    3. Double restriction enzyme digestion (EcoRIHF and XbaI) of pSB1C3-3’UTR; double restriction enzyme digestion
    (EcoRIHF and SpeI) of pSB1C3-HA/OVA.
    4. Only ligate two parts(I, II) of 2.3kb and perform PCR to amplify them.

    9/13

    1. Colony PCR of pSB1C3-HA-3’UTR.
    2. Ligate pSB1C3-3’UTR with HA/OVA.
    3. Only ligate two parts(I, II) of 2.3kb and perform PCR to amplify them.
    4. Ligate part(I-II) of 2.3kb with part(III) of 2.3kb.
    5. Transform ligation sample from 160912.

    9/14

    1. KOD+ PCR of 2.3kb intron region from individually ligation of three parts of 2.3kb.
    2. Double restriction enzyme digestion (EcoRIHF and XbaI) of pSB1C3-HA-3’UTR.
    3. Ligate part(I-II) of 2.3kb and part(III) of 2.3kb with pSB1C3.
    4. Transform ligation sample from 160913.

    9/15

    1. Ligate part(I-II) of 2.3kb and part(III) of 2.3kb with pSB1C3.
    2. KOD+ PCR of 2.3kb intron region from individually ligation of three parts of 2.3kb.
    3. Transform ligation sample from 160914.

    9/16 Do the same thing on 160915.

    Week 3 (9/19~9/25)

    9/19

    1. KOD+ PCR of 2.3kb intron region from individually ligation of three parts of 2.3kb.
    2. Double restriction enzyme digestion (SpeI and PstI) of pSB1C3-HA/OVA.
    3. Colony PCR of pSB1C3-5’HYG-HA-3’.
    4. RE digestion (EcoRIHF and NdeI) of part I+II of 2.3kb; RE digestion (PstI and NdeI) of part I+II of 2.3kb.
    5. Ligate pSB1C3-5’HYG with OVA-3’; ligate part I+II of 2.3kb, part III of 2.3kb with pSB1C3.

    9/20

    1. KOD+ PCR of 2.3kb intron region from individually ligation of three parts of 2.3kb.
    2. Transform ligation sample from 160919.

    9/21

    1. Double restriction enzyme digestion (SpeI and PstI) of pSB1C3-HA/OVA.
    2. KOD+ PCR of 2.3kb intron region from individually ligation of three parts of 2.3kb.
    3. Ligate pSB1C3-5’HYG with OVA-3’; ligate pSB1C3 with part I+II+III of 2.3kb.
    4. Transform ligation sample from 160920.
    5. Double restriction enzyme digestion (EcoRIHFand PstI) of pSB1C3/2.3kb; double restriction enzyme digestion
    (SpeI and EcoRIHF) of pSB1C3-5’HYG.

    9/22

    1. Colony PCR of pSB1C3-2.3kb.
    2. Still fail to get the correct pSB1C3-2.3kb.

    9/23

    1. Re-ligate pSB1C3 with 2.3kb.
    2. Transform ligation sample from 160921.
    3.Successfully get the pSB1C3-5’HYG-HA-3’UTR.

    Week 4 (9/26~10/2)

    9/26

    1. Double restriction enzyme digestion (EcoRIHF and XbaI) of pSB1C3-3’UTR; double RE digestion
    (EcoRIHF and SpeI) of pSB1C3-OVA/HA.
    2. Ligate pSB1C3-3’UTR with OVA/HA.

    9/27 Transform the ligation sample from 160926.
    9/28

    1. Colony PCR of pSB1C3-OVA/HA-3’UTR.
    2. Double restriction enzyme digestion (EcoRIHF and SpeI) of pSB1C3-5’HYG.
    3. Inoculate correct colonies of pSB1C3-OVA/HA-3’UTR into 4ml LB broth.

    9/29

    1. Mini preparation of pSB1C3-OVA/HA-3’UTR.
    2. Double restriction enzyme digestion (EcoRIHF and XbaI) of pSB1C3-OVA/HA-3’UTR.
    3. Ligate pSB1C3- OVA/HA-3’UTR with 5’HYG.

    9/30 Transform ligation sample from 160929. Successfully get the pSB1C3-5’HYG-OVA-3’UTR.

    Week 5 (10/3~10/10)

    10/3

    1. Colony PCR of pSB1C3-5’HYG-OVA/HA-3’UTR.
    2. Inoculate correct colonies of pSB1C3-5’HYG-OVA/HA-3’UTR into 4ml LB broth.

    10/4 Get the correct plasmid of pSB1C3-5’HYG-OVA/HA-3’UTR.
    10/5

    1. Double restriction enzyme digestion (EcoRIHF and SpeI) of pSB1A2-GFP; double RE digestion
    (EcoRIHF and XbaI) of pSB1C3-3’UTR.
    2. Ligate pSB1C3-3’UTR with GFP.

    10/6 Transform the ligation sample from 161005.
    10/7

    1. Colony PCR of pSB1C3-GFP-3’UTR.
    2. Double restriction enzyme digestion (EcoRIHF and SpeI) of pSB1C3-5’HYG.
    3. Inoculate correct colonies of pSB1A2-GFP-3’UTR into 4ml LB broth.

    10/8

    1. Mini preparation of pSB1C3-5’HYG-GFP.
    2. Double restriction enzyme digestion (EcoRIHF and XbaI) of pSB1C3-GFP-3’UTR.
    3. Ligate pSB1C3-GFP-3’UTR with 5’HYG.

    10/9 Transform ligation sample from 160929. Successfully get pSB1C3-5’HYG-OVA/HA-3’UTR.
    10/10

    1. Colony PCR of pSB1C3-5’HYG-GFP-3’UTR.
    2. Inoculate correct colonies of pSB1A2-5’HYG-GFP-3’UTR into 4ml LB broth.

    10/11 Mini preparation of pSB1C3-5’HYG-GFP-3’UTR. Successfully get pSB1C3-5’HYG-GFP-3’UTR.

  • Week 1 (9/5~9/11)

    9/5
    collect D31 serum from mouse facial vein
    9/8
    Detection antibody IgG1 and IgG2a titer from mouse serum via ELISA

    Week 2 (9/12~9/18)

    9/15
    collect preimmune serum, B6 mouse(trail1-5) for first immunization,Animal trails (injection way: s.c, site:
    both site of shoulder)(trail1 saline, trail2 saline+OVA, trail3 high Alum,
    trail4 107 leishmania+OVA, trail5 108 leishmania+OVA)

    Week 3 (9/19~9/25)

    9/20
    B6 mouse(trail6 108 leishmania-OVA+OVA) get first immunization
    9/25
    collect serum from mouse facial vein

    Week 4 (9/26~10/2)

    9/30
    B6 mouse(trail1-5) boost, collect serum from mouse facial vein

    Week 5 (10/3~10/7)

    10/4
    spleen dissection and culture of pretest trails
    10/5
    B6 mouse(trail6) boost, collect serum from mouse facial vein
    10/7
    collect serum from mouse facial vein

    Week 6 (10/8~10/13)

    10/10
    collect splenocyte supernatant, collect serum from mouse facial vein
    10/11
    spleen dissection and culture(from trails 1-6)
    10/13
    collect serum from mouse facial vein

    Week 7 (10/14~10/18)

    10/15
    collect serum from mouse facial vein
    10/17
    Detection antibody IgG1 and IgG2a titer from mouse serum via ELISA (so much samples and we eager for
    triple cheese sandwiches as many as samples.)

    10/18
    Detection cytokines, IFN gamma and IL10, from splenocyte supernatant.

  • Week 1

    10/8

    1. Double restriction enzyme digestion (EcoRIHF and XbaI) of pSB1C3-HA; double restriction enzyme digestion
    (EcoRIHF and SpeI) of pSB1C3-J04500.
    2. Ligate pSB1C3-HA with J04500.

    10/9 Transform ligation sample from 161008.

    Week 2

    10/10

    1. Colony PCR of pSB1C3-J04500-HA.
    2. Inoculate correct colonies of pSB1C3-J04500-HA into 4ml LB broth.
    3. Double restriction enzyme digestion (EcoRIHF and XbaI) of pSB1A2-GFP; double restriction enzyme digestion
    (EcoRIHF and SpeI) of pSB1C3-J04500.
    4. Ligate pSB1A2-GFP with J04500.

    10/11

    1. Mini preparation of pSB1C3- J04500-HA.
    2. Transform ligation sample from 161010.

    10/12

    1. Colony PCR of pSB1A2-J04500-GFP.
    2. Mini preparation of pSB1A2- J04500-GFP.
    3.Successfully get pSB1C3-J04500-HA and pSB1A2- J04500-GFP.






Protocol
  1. Affinity purification of MHC class I and II molecules from the infected DC2.4 cells
  2. Cryopreservation of DC2.4 cells
  3. Cryopreservation of leishmania
  4. Double photo inactivation and endocytosis of photo inactivated leishmania in DC2.4 cells
  5. Drug selection of Leishmania transfectants
  6. Leishmania and DC2.4 cells culture
  7. Ligation protocol (follows iGEM official protocol)
  8. Plasmid DNA ethanol precipitation for electroporation
  9. Transfection of Leishmania promastigotes by electroporation
  10. Transformation protocol
  11. Prepare Leishmania transfectants lysate / Western blot analysis

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