Team:UCLA/Notebook/Spider Silk Genetics/25 June 2015

• Home
• Team
  • Meet the Team
  • Attributions
  • Collaborations
• Projects
  • Programming Spider Silk
  • Functionalizing Silk
  • Honeybee Silk
  • Materials Processing
  • Protein Cages
  • Human Practices
  • Interlab Study
• Parts
  • Team Parts
  • Basic Parts
  • Composite Parts
  • Part Collection
• Notebook
  • Programming Spider Silk
  • Functionalizing Silk
  • Honeybee Silk
  • Materials Processing
  • Protein Cages
  • Interlab Study
• Safety
• Judging

6/25/2015

To-Do Plan

• set up IABCT, IT and verify on gel (maybe e-gel)
• amplify using post-elution primers.
  • verify this on gel too,
• If both of these work, then bind the first IABCT to beads. Wash, then elute, then attempt amplification.
• This will test whether the beads can bind to the biotin or not.

Making IABCT and IT

• General scheme: Assemble IAB and CT, then combine both together.
• Here is the concentrations of all ICA pieces used in this experiment.

	Concentration	Volume Used (uL)
AB	41.5 ng/uL	1.2
BC	54.3 ng/uL	0.93
CA	15.1 ng/uL	3.3
Initiator	5 uM	1
Terminator	5 uM	1

• Here is the scheme for assembling the pieces.

	IAB	CT	IT
I	1 uL	1 uL	1 uL
A	1.2 uL	n/a	n/a
B	0.93 uL	n/a	n/a
C	n/a	3.3 uL	n/a
2x T7 Ligase Buffer	5 uL	5 uL	5 uL
T7 Ligase	0.5 uL	0.5 uL	0.5 uL
ddH2O	1.39 uL	0.2 uL	2.5 uL
Total	10 uL	10 uL	10 uL

• Incubated 25 C for 10 min.
• Combined IAB and CT, incubated 25 C for 10 min.

Making IABCT results

• Cast 1.5% TAE gel; 2 uL of 100 bp ladder.
• Ran 10 uL of IABCT.
• Ran 5 uL of CT, diluted to 10 uL.

Fig. 1 Result of IABCT generated without using beads. The band at ~406 bp corresponds to the IABCT construct. Compare to the ~100 bp band corresponding to the IT construct only. The DNA runs slightly larger due to presence of biotinylation.

• The IABCT construct can be successfully generate without using beads. We will try incubating the leftover IABCT solution onto the streptavidin beads.
• In the IABCT sample, there is also IT present due to leftover IT that was subsequently ligated. This IT may bind onto the beads as well.

PCR Amplification with Post-Elution Primers

• Testing to see if the post-elution primers can be used to amplify IABCT, IT, prior to testing on beads.
• Set up one 25 uL reaction each for IABCT, IT from earlier.

	IABCT	IT
5x Q5 Buffer	5 uL	5 uL
10 mM dNTPs	0.5 uL	0.5 uL
10 uM For	1.25 uL	1.25 uL
10 uM Rev	1.25 uL	1.25 uL
Template	1 uL	1 uL
Q5 Polymerase	0.25 uL	0.25 uL
5x GC enhancer	5 uL	5 uL
ddH2O	10.75 uL	10.75 uL
Total	25 uL	25 uL

98 C	30 sec
98 C	10 sec
66 C	15 sec
72 C	20 sec
repeat from step 2	20x
72 C	2 min
12 C	hold

IABCT, IT can be amplified with Post-elution primers

• Cast 1.5 % TAE gel. 2 uL of 100 bp ladder.

Fig. 2 Amplification of IABCT, IT with post-elution primers. The ~400 bp product corresponds to the IABCT construct. The 100 bp band corresponds to the IT construct.

• The amplification of IABCT is not very strong due to competition from the IT that is also present in the mixture.
• BUT we show that post-elution amplification is possible. At this point, we will bind the IABCT and IT onto the streptavidin beads.
• If amplification does not work, then it is most likely a problem with bead binding.

IABCT, IT amplification with beads

1. 10 uL of magnabind beads.
   • washed 2x using 100 uL 2x BW buffer.
2. Added 5 uL of 2x BW buffer, and 5 uL of relevant DNA (IABCT or IT).
3. Incubate 45 min at RT (~23 C) with rotation.
4. Removed and saved supernatant.
5. Washed 2x in 0.5x BW buffer.
6. Elute at 95 C for 5 min uisng 0.01% Tween-20, with occasional mixing.
7. PCR amplify bead eluate and post-incubation supernatant in 25 uL reactions.
   • use 2 uL of each sample as template.
   • same temperature cycle as above section, but with 25x cycles.
8. Visualize on 1.5% TAE gel, with 2 uL 100 bp ladder.

Fig. 3 Amplification of IABCT, IT after binding and eleution from beads. The expected product for IABCT amplification is ~406 bp. The expected product for IT amplification is ~100 bp.

• The results show that the beads bind all of the present biotinylated DNA. Subsequent calculation show that the beads are in excess.
• IT is amplified from both bead elutions, but IABCT is not amplified.
  • This result is confusing. May be due to selective amplification of IT.

BsaI Digestion of MaSp2 CA

• We digested more CA monomers.
• Digested 5 ug of 2CA plasmid in 50 uL reaction using 4 uL BsaI.
• Incubated 50 C for 2 hours, then heat killed at 65 C for 20 min.
• Ran on 1.5% TAE gel to visualize; used 2 uL NEB 100 bp ladder.

Fig. 4 BsaI digestion of MaSp2 CA. The band at 102 bp corresponds to the digested desired product.

• We excised and gel extracted the 102 bp band using qiagen's kit.
  • 23.66 ng/uL for the final concentration.