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From 2012.igem.org

week 11: 9 july - 15 july

Lab work

General

cloning of BBa_I13522 (pTet GFP)

• BBa_I13522 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
• a green-fluorescing colony was picked after 3 days of growth in 37°C
• subsequent miniprep

TuYV

• Two (non-readthrough) gene variants 1(TuYV coat protein) and 1H(TuYV coat protein with his-tag) were digested and ligated into pSB1A3 linearized backbone.

Written by: Han Yue

PLRV

The start of the lab work on PLRV

We went out to the potato fields near Wageningen to look for PLRV infected potatoes. We thought we found some infected plants, based on the primary symptoms namely smaller sizes of the plants and curled leafs (figure 1).

We contacted Kees Bus and Chris Cuperus, who are experts at the Wageningen University on potato diseases. Judging the pictures, they concluded that these plants were probably not infected by PLRV.

This week we started trial experiments for RNA isolation from potato plants. We used a RNA isolation protocols using Trizol (see protocol page). The RNA product concentration was very high (upto 3500 ng/µL).

RNA isolate purity checked. RNA is of very good quality. Note that smaller rRNA bands visible in the leaf sample are derived from plant organelles such as plastids and chloroplasts

HepB

• Production of a HepB PCR fragment with pre- and suffix by means of a PCR using primers annealing to a plasmid containing the Hepatitis B core protein encoding gene. These primers also have an overhang that encode for pre- and suffix

CCMV

colony PCR pJET constructs CCMV biobrick

6 transformation plates in total with different CCMV constructs:

• A CCMV wt His-tag I
• B CCMV wt His-tag II
• C Δ26 CCMV MachI
• D Δ26 CCMV DH5α
• E Δ26 CCMV His DH5α
• F Δ26 CCMV His MachI

3 colonies were picked from each plate (18 samples in total) and stipped in the PCR rack and following growth in 5ml liquid LB medium.

Grown cultures were miniprepped according to Fermentas GeneJet protocol

sample	concentration (ng/µl)
A1	130.1
C1	131.3
C3	141.2
D2	207.7
D3	77.2
E2	113.9
E3	223.3
F1	177.7

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DLS boundary experiment

11th - 12th July

• Ultracentrifuge 2 times
• Followed protocol of purification CCMV
• Total end volume is 6 ml

13th July

• FPLC with the S400 column
• 1 run with 4 ml sample
• A very good separation also high concentration

14th July

• DLS Measurement: 20 runs, 45 seconds

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