Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk"

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)

• 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

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)