Team:Bio Without Borders/Experiments


HOME
TEAM
Team Members
Collaborations
PROJECT
Description
Design
Experiments
Notebook
Contribution
Results
Attributions
PARTS
Parts Overview
SAFETY
HUMAN PRACTICES
Human Practices
Education & Engagement
JUDGING FORM ⇗

Experiments

We improved the empty cassette of TecCEM 2017 team to include BamHI and HincII sites and spacer DNA for the siRNA. We worked on cloning the empty cassette, BBa_K3277000 with a linearized chloramphenicol backbone from the iGEM Distribution kit, and an ampicillin backbone from a J04450-pSB1A3 plasmid. The empty cassette is a vector for our siRNAs targeting the chitin synthase and superoxide dismutase genes. Then, we ligated the BBa_3277000 + pSB1C3 plasmid with the oligos of the siRNAs. We also attempted to improve upon the WNT siRNA of the previous team.

Digestion of Linearized Backbones · Benchling

Digestion of Linearized Backbones

Introduction

The linearized backbones from the iGEM Distribution Kits have to digestion with EcoRI and PstI and demethylated with DpnI before ligating with the BBa_K3277000 part.

Materials

  • BSA
    • EcoRI-HF
      • PstI
        • DpnI
          • dH2O or Nuclease Free Water
            • Linearized backbone

            Procedure

            • Enzyme Mastermix for Digestions and Demethylation
            1. 5μL NEB Buffer 2
            1. 0.5μL of BSA (or nuclease free water).
            1. 0.5μL of EcoRI-HF
            1. 0.5μL of PstI
            1. 0.5μL of DpnI
            1. up to 25μL with dH2O or nuclease free water
            • Digest of Linearized Backbone
            1. 4μL of linearized backbone (25ng/μL for 100ng)
            1. 4μL of Enzyme mastermix
            1. Digest 37℃ for 30 minutes and heat inactivate at 80℃ for 20 minutes.
            Ligation with T4 DNA Ligase · Benchling

            Ligation with T4 DNA Ligase

            Introduction

            This protocol ligates an insert fragment of DNA to a vector backbone. We ligated our BBa_K3277000 g-bloxk with linearized pSB1C3 backbone and the pSB1A3 backbone from the J04450-pSB1A3 plasmid (in two separate reactions). The same procedure was followed to ligate the annealed siRNA oligos to the BBa_K3277000 + pSB1C3 plasmid.

            Materials

            • T4 Ligase
              • T4 Ligase buffer
                • Backbone DNA
                  • Insert DNA
                    • Nuclease Free Water
                      • Thermocycler
                        • Micropipetter
                          • PCR Tubes

                          Procedure

                          • Ligation with T4 Ligase
                          1. Add 2μL of T4 ligase buffer to pcr tube.
                          1. Add 1μL of backbone DNA
                          1. Add 7μL insert DNA
                          1. Add 1μL T4 Ligase
                          1. Fill up to 20μL with nuclease free water.
                          1. Inucbate overnight at 16℃ or at room temperature (25℃) for 10 minutes
                          Restriction Digest using EcoR1 and Pst1 · Benchling

                          Restriction Digest using EcoR1 and Pst1

                          Introduction

                          A restriction digest allows a plasmid to be cut at specific sites. Using EcoR1 and Pst1, the insert can removed from the backbone and can be reinserted in another backbone or vice versa. In this case, the ampicllin backbone from J04450-pSB1A3 were cut with EcoRI-HF and PstI to serve as a vector for the BBa_K3277000 g-block fragment.

                          Materials

                          • EcoRI-HF
                            • PstI
                              • Template DNA
                                • NEB Buffer 3.1
                                  • Nuclease Free Water
                                    • Thermocycler
                                      • PCR Tubes
                                        • Micropipettor

                                          Procedure

                                          • Double Digest with EcoRI-HF and PstI
                                          1. Add 2μL of NEB Buffer 3.1 to PCR tube.
                                          1. Add 1μL of EcoRI-HF
                                          1. Add 1μL of PstI
                                          1. Add 5μL of plasmid
                                          1. Fill up to 20μL with nuclease free water
                                          1. Digest at 37℃ for 60 minuts and heat inactivate at 80℃ for 20 minutes.
                                          Restriction Digest with BamHI and HincII · Benchling

                                          Restriction Digest with BamHI and HincII

                                          Introduction

                                          The ligated BBa_K3277000 + pSB1C3 was cut with BamHI and HincII to insert the annealed siRNA oligos.

                                          Materials

                                          • Mini-Prepped Plasmid
                                            • BamHI-HF
                                              • HincII
                                                • CutSmart Buffer
                                                  • Nuclease Free Water
                                                    • PCR tubes
                                                      • Thermocycler
                                                        • Micropipetter

                                                          Procedure

                                                          • Double Digest with BamHI-HF and HincII
                                                          1. Add 2μL of CutSmartbuffer to tube
                                                          1. Add 1μL of BamHI-HF
                                                          1. Add 1μL of HincII
                                                          1. Add 5μL of plasmid
                                                          1. Fill up to 20μL with nuclease free water
                                                          1. Digest at 37℃ for 60 minutes and then heat inactivate at 80℃ for 20 minutes.
                                                          Colony PCR (cPCR) with OneTaq 2X Mastermix · Benchling

                                                          Colony PCR (cPCR) with OneTaq 2X Mastermix

                                                          Introduction

                                                          Polymerase Chain Reactions on transformed bacterial colonies can be useful to determine if the desired insert DNA is present or absent in plasmid constructs.

                                                          Materials

                                                          • OneTaq 2x Mastermix
                                                            • 10μM Forward primer
                                                              • 10μM Reverse primer
                                                                • Resuspended colony (to be used as DNA template)
                                                                  • Nuclease Free Water
                                                                    • Thermocycler
                                                                      • Micropipetter
                                                                        • PCR tubes

                                                                        Procedure

                                                                        • PCR
                                                                        1. Select a transformed colony from the plate and resuspend in 20μL of nuclease free water.
                                                                        1. Add 9μL of nuclease free water to PCR tube.
                                                                        1. Add 12.5 μL of OneTaq 2x Mastermix.
                                                                        1. Add 0.5μL of forward primer.
                                                                        1. Add 0.5μL of reverse primer.
                                                                        1. Add 1μL of resuspended colony.
                                                                        1. Place in thermocycler at standard temperatures and adjust accordingly to the primer and primer concentration.
                                                                        Component25μL reaction50μL reaction[Final]
                                                                        OneTaq 2x Mastermix12.5μL25μL 1x
                                                                        Forward primer0.5μL1μL0.2μM
                                                                        Reverse primer0.5μL1μL0.2μM
                                                                        Template DNAvariablevariable<1000ng
                                                                        Nuclease Free waterup to 25μLup to 50μL<1000ng
                                                                        PCR Protocol · Benchling

                                                                        PCR Protocol

                                                                        Introduction

                                                                        Polymerase Chain Reaction is used to amplify sections of DNA using specific primers, allowing the production of many copies of that section.

                                                                        Materials

                                                                        • Q5 High-Fidelity 2X Master Mix
                                                                          • 10 µM Forawrd Primer
                                                                            • 10 µM Reverse Primer
                                                                              • Template DNA
                                                                                • Nuclease Free Water
                                                                                  • PCR Tubes
                                                                                    • Thermocylcer
                                                                                      • Micropipetter

                                                                                        Procedure

                                                                                        • PCR
                                                                                        1. Add 9 µL of water to PCR Tube
                                                                                        1. Add 12.5 µL of Master Mix to PCR tube
                                                                                        1. Add 2.5 µl of each Primer
                                                                                        1. Add 1 µL of DNA template
                                                                                        1. Place into thermocyler at standard temperatures with adjusted annealing and extension parameters. See table from NEB below.
                                                                                        Component25 µl Reaction50 µl ReactionFinal Concentration
                                                                                        Q5 High-Fidelity 2X Master Mix12.5 µl25 µl1X
                                                                                        10 µM Forward Primer1.25 µl2.5 µl0.5 µM
                                                                                        10 µM Reverse Primer1.25 µl2.5 µl0.5 µM
                                                                                        Template DNAvariablevariable< 1,000 ng
                                                                                        Nuclease-Free Waterto 25 µlto 50 µl
                                                                                        PCR Parameters
                                                                                        Annealing Oligos · Benchling

                                                                                        Annealing Oligos

                                                                                        Introduction

                                                                                        The siRNAs were ordered as separate oligos, the sense and anti-sense strands. These strands have to be annealed together.

                                                                                        Materials

                                                                                        • Resuspended sense and anti-sense oligos
                                                                                          • Heatblock
                                                                                            • Eppendorf Tubes

                                                                                              Procedure

                                                                                              • Annealing
                                                                                              1. Set the temperature on the heatblock to 98℃.
                                                                                              1. Place 10μL of each oligo into an eppendorf tube (total volume should be 20μL).
                                                                                              1. Put the Eppendorf tubes onto the heatblock and turn it off. Allow the temperature to decrease gradually to room temperature (25℃).