Team:Groningen/Notebook/8 September 2009

From 2009.igem.org

Wet

GVP Cluster

→ DONE Check transfer of pArsR-GVP, pZntR-GVP, pCueO-GVP, and pLacI-GVP from J61035 and pSB1A2 to pSB2K3. (preculture growth, isolation, and restriction (the colonies all grew on kanamycin plates with IPTG, this is the first selection, because the parts come from an ampicillin plasmid))

→ DONE Cut out GVP cluster from biobrick vector (can be used for fragment formation)

→ TODO Create fragments for 400bp repeat removal from the gvpL gene. (restriction, gel purification)

→ TODO Perform ligation of all fragments into pSB1AC3 vector. (the pSB1AC3 vector has been the most succesfull vector to use in assemblies)

→ DONE Grow L-GVP, M-GVP, and pLacI-GVP (with IPTG) on plates for test of buoancy in Saline solution. (as control use pNL29 with IPTG, and J61002-pCueO)(precultures, plates)

→ TODO Check Saline solutions and take pictures!

O.n. precultures

→ All tubes for plasmid isolation (pArsR-GVP, pZntR-GVP, pCueO-GVP, and pLacI-GVP in pSB2K3, and pSB2K3 (2x)) showed growth. The pArsR-GVP in pSB2K3 had less growth (and smaller pellet) than the other tubes, and pSB2K3 no.1 was red coloured.

→ All tubes for Saline Test (for plating on LB-amp₁₀₀ and LB-amp₁₀₀-IPTG) showed growth and 300uL was plated.

Plasmid isolation

Restriction for Control

The plasmids from the o.n. precultures were cut with PstI and EcoRI to cut out the entire part between the pre- and suffix.

→ From left to right: 1kB ladder, pArsR-GVP, pZntR-GVP, pCueO-GVP, pLacI-GVP, Empty Slot, pSB2K3 no.1 (2x), Empty Slot, pSB2K3 no.2

→ The fragments were not as expected, but the fragments of GVP with promoter seem correct.

→ The pSB2K3 vector should be ~4425bp in size, instead the fragments correspond to ~2000bp and ~3000bp. Close inspection of the registry gave a possible answer on this [http://partsregistry.org/assembly/plates.cgi?id=681 page]. The sequencing result was bad, and only the resistance was checked, no gel restriction was performed. The resistance to kanamycin was also observed by us, but restriction shows incorrect fragment sizes.

→ Sequencing results of the pSB2K3 vector with RFP reporter system are expected any day now, and may or may not show a differnce in vector part.

Because of the result, all previously thought pSB2K3 products were trown away (to be sure the wrong plasmid does not show up again).

→ From the registry, two parts were chosen which are stored in pSB2K3. The idea is to have the correct plasmid by cutting out the parts: [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0071 BBa_C0071] plate 1, 12J and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0072 BBa_C0072] plate 1, 12L. The dry plasmid was dissolved in 15uL MQ and stored in -20C box of Michael.

→ Also new E.coli DB3 competent cells will be made to transform pSB2K3 with death gene (plate 1, 7K). Problem is that the sequencing results were inconsistent for this part as well as for the RFP containing one. Growth of E.coli TOP10 cells with this gene resulted in no colonies, demonstrating the work of the death gene, because on the possitive control ~2000 colonies were observed.

Tranformation

• add 4uL of the dissolved registry plasmid to 50uL competent E.coli TOP10 cells and 1uL of HmtA (for SJ) to 50uL competent E.coli TOP10 cells.

Incubate:

• 30 min @ ice
• 90 sec 42°C
• 2 min @ ice
• add 800uL LB-medium
• incubate for 1 h at 37°C
• plate on LB-amp₁₀₀ (HmtA) and LB-kan₅₀-IPTG (pSB2K3) plates
• plates were stored at 37°C o.n.

New o.n. plates

Plates of LB-agar-amp₁₀₀ (IPTG if required) were used grow L-GVP, M-GVP, and pLacI-GVP (with IPTG) on plates for test of buoancy in Saline solution (as control pNL29 with IPTG, and J61002-pCueO).

• 300uL of preculture was plated and incubated at room temperature for 1 hour to let medium absorp the liquid, the plated were stored at 37°C o.n.
• Remaining preculture was stored in fridge for future use.

New o.n. precultures

Four cultures of E.coli DB3 with pSB3K3 and death gene were made in LB-kan₅₀ medium. The plasmids will be used to transform the pArsR-GVP, pZntR-GVP, pCueO-GVP, and pLacI-GVP into. Transformation to E.coli TOP10 will eliminate the selfligation product due to the death gene.

Transporters

Metal Accumulation

SmtA
Part made in registry and on which the primers were based was false. Smta was canceled due to time issues. It did explain all the issues with PCR.

1% agarose (1xTBE) gel of SmtA PCR using different templates.

Vectors

Dry