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From 2012.igem.org

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

sample	concentration (ng/µl)	260/280
A1	115.5	1.92
C1	96.0	1.98
C3	100.1	1.92

• 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)

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 measurement

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