Team:UCLA/Notebook/Honeybee Silk
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.| 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 |
| 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 |
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.| 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 |
| 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 |
Contents
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
Protein Expression
What to accomplish next
Raw lab notebook entries
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