From 2012.igem.org

Revision as of 08:22, 25 September 2012

• Home
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  • The Team
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• Project
  • 1. Introduction
  • 2. PnA System
  • 3. Virus-Like Particles
    • CCMV
    • HepB
    • Polerovirus Natural BioBrick
  • 4. Outside Modification
  • 5. Inside Modification
  • 6. Detection of VLPs
  • 7. Applications
  • 8. The Constructor
  • 9. Final Overview
• Human Practices
  • Human Practices Home
  • 1. Mini Symposium
  • 2. Visiting Secondary Schools
  • 3. Discovery Festival
    • Communication Science
  • 4. Stakeholders
  • 5. Munich CAS Conference
• Safety
  • Introduction
  • 1. General safety
  • 2. Virus-related safety
  • 3. Regulations
  • 4. Safety suggestions
  • 5. Safety of applications
• Modeling
  • Human Body Model
  • Tertiary folding prediction
  • VLP assembly model
• Attributions
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Last year's project: Synchronized Oscillatory System

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week 12: 16 july - 22 july

Office work

PLRV

This week we designed and ordered all the primers we needed.

Lab work

General

BBa_J04450 (RFP coding device) transformation, digestion check

• BBa_J04450 was solubilized from the Standard registry parts plate I with 10µl MQ water
• Transformation of electrocompetent E.coli with the plasmid
• Plated on LB-agar with corresponding antibiotic (chloramphenicol)
• 6 red-fluorescing colony were picked after overnight growth in 37°C
• Subsequent miniprep

• Digestion check with EcoRI and SpeI

Digestion of BBa_K1970- 22/38

From the transformation plate of week 9 a new colony was grown and subsequently a miniprep was done. The yield was about 160 ng/µl. To check if the transformed E.coli contains the right plasmid a digestion check with the restriction enzymes BamHI and BgEII was done.

CCMV

18th July (Hugo)

First PCR on CCMV-Delta26 to create the CCMV_NEG construct. Phusion Mastermix for 4 + 1 = 5 samples:

Substance	Amount (µl)
H2O	176.25 uL
5x Phusion Buffer	50 uL
dNTP's	5 uL
Phusion enzyme	1.25 uL
Total	232.5 uL

Preparation samples:




Substance	Amount (µl)
Mastermix	44 uL
DNA template	1 uL
Forward Primer (10x diluted)	2.5 uL
Reverse Primer (10x diluted)	2.5 uL
Total	50 uL

PCR reaction:

Step	Time	Temperature (°C)
Step 1	10 minutes	98 °C
Step 2	30 seconds	98 °C
Step 3	30 seconds	58 °C
Step 4	30 seconds	72 °C	Return to step 2 (35 times)
Step 5	10 minutes	72 °C
Step 6	As long as it takes	4 °C

Gel:

Lane	Sample
Lane 1	Marker
Lane 2	Negative control: No template, same primers as duplo’s
Lane 3	Positive control: Delta26-Histag CCMV with Forward + Reverse (suffix) primer.
Lane 4	duplo 1: CCMV-Delta26 with Forward (Part 2) + Reverse (suffix) primer.
Lane 5	duplo 2: CCMV-Delta26 with Forward (Part 2) + Reverse (suffix) primer.

File:Gel 18-07.tif

19th July (Hugo)
Second PCR on CCMV-Delta26. Before PCR was started, fragments of the day before were purified using a PCR purification kit.

20th July (Hugo)
PCR repeat of 18-07.




HepB

• Digestion of the HepB core protein PCR product with pre- and suffix (from 11.July) and ligation into BBa_J04500 (an IPTG inducible promoter with RBS)as well as BBa_PSB1K3.ml (linearized plasmid backbone)
• Electro transformation of these constructs with DH5α
• Colony PCR (20 samples of the transformants containing HepB core protein + BBa_J04500; 10 samples of the transformants containing HepB core protein + BBa_PSB1K3.ml

-> The ligation and transformation of the HepB core protein into the linear backbone (BBa_PSB1K3.ml) was not successful -> The colony PCR shows that we have 6 out of 20 colonies with the expected insert size (around 600bp)








Figure 1: Colony PCR 20 July - HepB in BBa_J04500





TuYV

• Mach1 electrocompetent E. coli cells were transformed with last week's ligation mixtures (TuYV coat protein and TuYV coat protein with his-tag). Results were unsatisfying. No colonies were showed on the sample plates.
• 'Coat Protein + readthrough' gene amplicons were digested, ligated into pSB1K3 backbone and transformed into our electrocompetent Mach1 E. coli cells. After two attempts and a colony PCR, results were still unsatisfying.




PLRV

After a two week quest for PLRV infected plant material, we obtained infected potato leafs from the Dutch General Inspection Service for Agricultural seed and seed potatoes.

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DLS boundary experiment


19th July (Mark)

• DLS measurment: 20 runs, 45 seconds
• Changed 4 times to different vials to see if the vials are important to the measurment




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