Team:UCLA/Notebook/Honeybee Silk

iGEM UCLA




Honeybee Silk Notebook

May 10 - May 16
May 11 Sequencing of silk biobricks

Today we sent in both our constructs to GeneWiz for sequencing.

  • For sequencing primers, I used the igem vf2 primers and vr primers that bind to the psb1c3 backbone, as well as a sequencing primer that we designed to bind to the silk sequence (p13) see https://benchling.com/s/p7bClpzQ/edit
  • The sequencing results came back and checked out for sample 1 of construct 1 (silk coding region) and samples 1 and 2 for construc 2 (regulatory elements + silk) i.e. we have our first honeybee biobricks!!!
  • For sequencing results of construct 1 see https://benchling.com/s/4XT6VRRU/edit (click on alignments on the right side of screen)
  • For sequencing results of construct 2 see https://benchling.com/s/TxD8z7Kq/edit (click on alignments on the right side of screen)
May 3 - May 9 (Cloning Honeybee Biocricks week 2)
May 4
May 5
May 6
April 26 - May 2 (Cloning honeybee biobricks week 1)
4/26 PCR off honey bee gene block
  • See Benchling for the g block sequence and primer reference code https://benchling.com/s/p7bClpzQ/edit
  • The goal of this is to clone 2 different constructs and prepare them as biobricks, one that is just the silk gene, and the other that as the promoter, rbs, etc.. required for protein expression.
  • Using primers p3, p7, and p8. (See Benchling link)

  • PCR Reaction 1 (q5 polymerase kit): primers honeybee p#7 and honeybee p#8 with gBlock, and honeybee p#3 and honeybee p#8 amplify just the honeybee coding region and the prom + coding region respectively

    Component Volume
    5X Q5 Reaction Buffer 5
    10 mM dNTPs 0.5
    10 uM Forward (primer 3/7) 1.25
    10 uM Reverse (primer 8) 1.25
    Template (diluted to 1ng/uL) 0.5
    Q5 High Fidelity DNA Polymerase 0.25
    Nuclease Free Water 16.25
  • PCR program (using benchling annealing temperatures)
    Temperature (C) Time (Min:sec)
    98 0:30
    98 x 30 cycles 0:10
    grad. 55.4/58.4/61.4 C x 30 0:20
    72 x 30 0:30
    72(diluted to 1ng/uL) 2:00
    12 hold

    • The program and pcr reaction recipe were based on the manufacturer protocol. These annealing temperatures were determined using benchling's algorithm. In the future we will use the NEB annealing temperature calculator, because it is more accurate and takes the type of polymerase into account See tomorrow's entry for the gel image of the pcr reaction

4/27 visualization and purification of honeybee gblock PCR

We visualized the gradient pcr from 4/26 on a 1% agarose gel. See image below:

  • The PCR for both constructs (silk coding region) and (regulatory elements + silk coding region) worked relatively well across all gradient temperatures.
  • We next scaled up the PCR reaction to 50 ul for each construct and gel extracted the product
  • From left to right, just silk w/ bb prefix and suffix w/ increasing annealing temp. , ladder, followed by silk+ promoter at increasing temp (see 4/26 for pcr conditions)
4/28 PCR to prepare 2 honeybee constructs for cloning We are repeating the same PCR from 4/26, but only at one temperature instead of a gradient, and we are scaling the reaction up to 50 ul.
  • PCR recipe
    Component Volume
    5X Q5 Reaction Buffer 10
    10 mM dNTPs 1.0
    10 uM Forward (primer 3/7) 2.5
    10 uM Reverse (primer 8) 2.5
    Template (diluted to 1ng/uL) 1.0
    Q5 High Fidelity DNA Polymerase 0.50
    Nuclease Free Water 32.5
  • PCR program (using benchling annealing temperatures)
    Temperature (C) Time (Min:sec)
    98 0:30
    98 x 30 cycles 0:10
    grad. 62 (silk) 58.4 (silk+prom) x 30 0:20
    72 x 30 0:30
    72(diluted to 1ng/uL) 2:00
    10 hold
  • PCR products run on 1% agarose gel.
  • There were some non specific bands, but the appropriate bands were present. However, the bands were kind of off. I think this is due to the samples running weird on the gel. B/c the first two lanes were the exact same sample, just aliquoted, and they ran fairly differently.
  • Performed a gel extraction and purified using Qiagen min elute gel extraction kit
  • Results : 115 ng/ ul for silk tube #1, 86 ng/ul tube #2 (8.8 ul each tube) 136 ng/ul promoter + silk
    • 4/30 Double digest of Honeybee PCR product and ligation into PSB1C3 plasmid backbone Vinson and Olivia already prepared ecor1 and pst1 digested psb1c3, (35 ng/ul in honeybee box) which I will be using for ligation. It is column purified as well. I am double digesting both the plain silk construct, as well as the promoter+silk construct with ecoR1 and pst1.
  • Digestion Rxn (50 ul) One for silk and One for prom+silk
    EcoR1 (C) 1 ul
    Pst1 1 ul
    NEB buffer 2.1 (10x) 5 ul
    Silk DNA / PRom+Silk DNA 8.7 ul / 7.2 ul
    H20 34.2 / 35.8 ul
  • Digestion incubation specifications: 37 degrees for 1 hour, and 80 degree heat inactivation for 15 minutes, hold 10C
  • Ligation Reaction into PSB1c3 vector
  • 20 ul ligation reaction according to protocol using T4 ligase
  • 2 reactions, (1 for silk, 1 for promoter + silk) Both are at a 3:1 molar insert:vector ratio.
    10X T4 Ligase Buffer 1 2 ul
    PSB1C3 (50 ng) 1.4 ul
    silk construct / prom + silk construct 1.4 ul / 1.06 ul
    T4 Ligase 1 ul
    H20 14.2 / 14.54
    • Reaction conditions: 16 C for 12 hours, 65 C for 15 min, hold at 10 C


    Goals

    The goal of this project is to recombinantly express honeybee silk proteins, with the intention of creating synthetic honeybee silk fibers. We hope to develop protocols to efficiently produce the raw protein, process the protein into various materials, and characterize these materials. Furthermore, by conjugating the honeybee protein with other proteins, we aim to create honeybee silk materials with a wide array of properties and functionalities.

    Achievements

    As of 5/16 we are well on our way to establishing a strong foundation for this project. We have cloned some honeybee sequences into biobrick approved format, and are on our way to expressing our first batch of honeybee silk protein. (For detailed achievements see below)

    Cloning

    We have successfully cloned and sequence verified two honeybee silk constructs into the igem psb1c3 plasmid backbone, in the correct biobrick format.

    • The first sequence is just the honeybee silk protein #3 in the psb1c3 backbone. The annotated sequence can be found here. silk #3 coding Further details on how we designed and cloned this biobrick can be found HERE
    • The second sequence is the same silk coding sequence, but with regulatory elements upstream for protein expression, including [http://parts.igem.org/Part:BBa_R0010 BBa_R0010 Promoter] and [http://parts.igem.org/Part:BBa_B0034 Promoter BBa_B0034 ]. The sequence can be found here. regulatory elements + silk#3 coding Further details on how we designed and cloned this biobrick can be found HERE

    Protein Expression

    Haven't accomplished anything yet :P

    What to accomplish next

    We are currently at the starting phases of honeybee silk protein expression. We will be expressing regulatory elements + silk#3 coding and purifying the protein with BugBuster lysozyme kit. We will then run our purified protein on an SDS PAGE gel to ascertain the purity of our purified silk protein.

    Raw lab notebook entries

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