Team:Melbourne/Notebook


Melbourne iGEM 2016

Melbourne iGEM 2016

Melbourne iGEM 2016

Notebook

  • Week 1 (27/06/16 - 01/07/16)

    • Streak plated empty DH5α and BL21(DE3) cells on LB agar from glycerol stocks.
    • Inoculated DH5α and BL21 colonies in LB media, grew to OD 0.6 and chemically treated to make competent cells.

    June-27-2016

  • Week 2 (04/07/16 – 08/07/16)

    • Tested DH5α and BL21(DE3) competent cell for cell competency. Cells worked very well with DNA >10 ng during transformation.
    • Transformed DH5α with empty pSB1C3 and pET23a plasmids individually and plated them on LB-CAP and LB-AMP agar respectively. These will be used in midiprep to create stock solutions of empty plasmid DNA.
    • Inoculated DH5α colonies with empty pSB1C3 and pET23a plasmids in LB-CAP and LB-AMP respectively.
    • Performed a miniprep on the two DH5α cultures and a restriction digest on the plasmids with EcoRI-HF and XhoI, and then ran agarose gel electrophoresis to confirm the identity of the plasmids with predicted banding pattern.
    • Made glycerol stock of DH5α cell + pSB1C3 (empty) and DH5α cell + pET23a (empty) for future use.
    • Performed a midiprep on DH5α cell + pSB1C3 (empty) and DH5α cell + pET23a (empty) culture.

    July-04-2016

  • Week 3 (11/07/16 – 15/07/16)

    • Performed a restriction digest on the midiprep plasmids with EcoRI-HF and XhoI, and then ran agarose gel electrophoresis to confirm the identity of the plasmids with predicted banding pattern.
    • Performed PCR optimisation using the BioBrick prefix and suffix primers (BBa_G10004 and BBa_G10005), Taq polymerase and No Helix StarScaffold DNA (IDT synthesised) with a gradient of annealing temperature between 50-60 °C. The predicted annealing temperature was predicted to be within this range. Gel electrophoresis showed no band.
    • Performed another PCR optimisation with the same condition with No Helix StarScaffold DNA (IDT synthesised) and with a plasmid as a positive control. Gel electrophoresis showed no band.

    July-11-2016

  • Week 4 (18/07/16 – 22/07/16)

    • To troubleshoot the PCR, a positive control was set up using Taq polymerase, a plasmid which has been showed to be capable of being amplified and VF2, VR primers. Gel electrophoresis showed very strong bands, indicating that there is no fault with PCR reagents. The problem may come from the template, primers or PCR program.
    • More PCRs were performed to investigate the problem. Primer concentration, Mg2+ concentration and lower annealing temperature range were adjusted. It is found that the best annealing temperature for the BioBrick prefix and suffix primers was 40.0 °C when amplifying StarScaffold DNA (IDT synthesised).
    • Performed PCR optimisation using the BioBrick prefix and suffix primers, Q5 polymerase and No Helix StarScaffold DNA (IDT synthesised) with a gradient of annealing temperature between 40-60 °C. Gel showed that the best annealing temperature was 51.6 °C but all samples at different annealing temperature had non-specific amplicons.
    • Performed a touch down PCR in an attempt to reduce non-specific, but it did not succeed.

    July-18-2016

  • Week 5 (25/07/16 – 29/07/16)

    • Performed PCR with Q5 polymerase on all StarScaffold and non-StarScaffold DNA (IDT synthesised). Gel showed that most PCR samples did not have non-specific amplicons.
    • All PCR-amplified samples were cleaned with PCR purification.
    • All PCR-amplified-purified DNA (StarScaffold and non-StarScaffold) were digested with EcoRI-HF and PstI-HF and cleaned with PCR purification.
    • Digested pSB1C3 with EcoRI-HF and PstI-HF and cleaned with PCR purification.
    • Digested and PCR-purified StarScaffold DNA inserts were ligated into digested and PCR-purified pSB1C3 plasmid.

    July-25-2016

  • Week 6 (01/08/16 – 05/08/16)

    • Transformed the ligated products (StarScaffold DNA in pSB1C3) into DH5a and plated on LB-AMP agar.
    • Performed colony PCR with VF2 and VR primers on the transformed colonies. Most of the colonies did not have an insert.
    • Performed more colonies PCR to identify successful colonies. Again, most of the colonies did not have an insert.
    • Performed a restriction digest of the purified PCR products of non-StarScaffold DNA with EcoRI-HF and PstI-HF, and the PCR-purified.

    August-01-2016

  • Week 7 (08/08/16 – 12/08/16)

    • Inoculate the successful colonies (Prototype 1, Prototype 2, Non-Seq 1, Non-Seq 2 and No Helix in pSB1C3) in LB-CAP, miniprep and sequence with VF2 and VR primers. Sequencing showed that the plasmid did not contain the right insert. BLAST was performed and found that the insert could be from genomic contamination which happened to be of the right size.
    • Ligation of StarScaffold DNA into pSB1C3 was aborted because ligation of StarScaffold DNA into pET23a and subsequently the protein expression have a higher priority.
    • Thinking that genomic contamination could come from the midiprep of pET23a, more pET23a was digested with NotI and NdeI and treated with shrimp alkaline phosphatase (SAP). This may prevent plasmid religation and reduced the change of having colonies without the insert, and also prevent digested genomic contamination from being ligated into the plasmid.
    • Ligated the digested and SAP-treated pET23a with StarScarffold DNA.

    August-08-2016

  • Week 8 (15/08/16 – 19/08/16)

    • PCR optimisation using VF2 and VR primers, as well as T7 promoter and T7 terminator primers with known plasmids. The optimal annealing temperature were 53 °C and 51 °C respectively.
    • Transformed the new ligated products (StarScaffold DNA in pET23a) into DH5a and plated on LB-AMP agar.
    • Performed colony PCR of the transformed cells. Gel electrophoresis of the samples showed a much high percentage of successful colonies.

    August-15-2016

  • Week 9 (22/08/16 - 26/08/16)

    • Inoculated successful colonies (Prototype 1, Prototype 2, Non-Seq 1 and Double Helix in pET23a) in LB-AMP
    • Performed miniprep of the grown culture and sequenced with T7 promoter and terminator.
    • Perform ligation of digested Non-StarScaffold DNA into digested and SAP-treated pSB1C3.
    • Transformed the ligated products (Non-StarScaffold DNA in pSB1C3) into DH5α and plated on LB-CAP agar.
    • Performed colony PCR with VF2 and VR primers on the transformed DH5α colonies (Non-StarScaffold DNA in pSB1C3). Most of the colonies did not have an insert.

    August-22-2016

  • Week 10 (29/08/16 - 02/09/16)

    • Some sequences of the pET23a with StarScaffold DNA (Prototype 1, Prototype 2, Non-Seq 1 and Double Helix) were verified.
    • Ligation and transformation of Non-StarScaffold DNA into pSB were aborted as the protein expression with ligated pET23a plasmids had a high priority.

    August-29-2016

  • Week 11 (05/09/16 - 09/09/16)

    • Performed PCR with Q5 polymerase on StarScaffold DNA again.
    • Digested the PCR-amplified StarScaffold DNA with EcoRI-HF and PstI-HF and then gel purified.
    • Digested pSB1C3 with EcoRI-HF and PstI-HF, gel purified and treated with SAP.
    • Prepared pET23a with StarScaffold DNA (Prototype 1, Prototype 2, Non-Seq 1 and Double Helix) for transformation in BL21(DE3) next week.

    September-05-2016

  • Week 12 (12/09/16 - 16/09/16)

    • Transformed pET23a plasmid with StarScaffold DNA (Prototype 1, Prototype 2, Non-Seq 1 and Double Helix) into BL21(DE3) and plated on LB-AMP agar.
    • BL21(DE3) colonies were inoculated and grown to the appropriate OD for induction.
    • Protein expression of BL21(DE3) were induced by IPTG. A small amount of culture was pelleted at different points of time during the overnight expression at 16 °C.

    September-12-2016

  • Week 13 (19/09/16 - 23/09/16)

    • Pelleted cells were prepared and ran on SDS-PAGE but there were problems with running the gel.
    • SDS-PAGE was run again. Although there were issues with staining and destaining procedures, protein bands were observed with poor resolution.
    • Digested pSB1C3 and StarScaffold DNA from Week 11 were ligated and transformed into DH5α cells. They were then plated on LB-CAP plates.
    • Performed colony PCR with VF2 and VR primers on the transformed DH5α colonies (StarScaffold DNA in pSB1C3).

    September-19-2016

  • Week 14 (26/09/16 - 30/09/16)

    • Gel electrophoresis of the colony PCR indicated that there were some successful DH5α colonies (StarScaffold DNA in pSB1C3). They were inoculated into LB-CAP media and then a miniprep was performed.
    • Streak plated empty NEB Shuffle T7 cells on LB agar which will be used for making more competent cells.
    • Inoculated Shuffle T7 cells in LB media, grew to OD 0.6 and chemically treated to make ultra-competent cells.
    • New BL21(DE3) cultures were grown and induced for protein expression at 37 °C. A small amount of culture was pelleted at different points of time during the protein expression.

    September-29-2016

  • Week 15 (03/10/16 - 07/10/16)

    • Pelleted cells were prepared and ran on SDS-PAGE. There were issues with staining and destaining procedures again.

    October-03-2016

  • Week 16 (10/10/16 - 14/10/16)

    • Performed PCR on the StarScaffold DNA in pSB1C3 with VF2 and VR primers and PCR purified before sending for sequencing.
    • pET23a with StarScaffold DNA was transformed into BL21(DE3) again and plated on LB-AMP agar plate. Colonies were inoculated in LB-AMP media.
    • The BL21(DE3) cultures were grown and induced by IPTG at 37 °C. A small amount of culture was pelleted at different points of time during the protein expression.
    • Pelleted cells were prepared and ran on SDS-PAGE. Successful results were observed on SDS-PAGE.

    October-10-2016