Team:Groningen/Notebook/2 September 2009

From 2009.igem.org

Wet

GVP Cluster

→ TODO Choose colonies from plates for growth of E.coli TOP10 with GVP behind LAC/pBAD (+RBS) promoter in pSB1AC3

→ DONE Choose colonies from plates for growth of E.coli TOP10 with [http://partsregistry.org/Part:pSB2K3 pSB2K3] plasmid from the registry (plate 1, 7C with RFP Coding Device [http://partsregistry.org/Part:BBa_J04450 BBa_J04450]

→ TODO Make glycerol stocks of pBad/araC, pBad/araC+RBS+GVP, and pLacI+RBS+GVP (both in pSB1A2 and pSB1AC3 plasmid)

→ TODO Test control of bouyancy in Saline solution (grow plates with GVP constructs)

→ TODO Order synthetic DNA for GVP

→ TODO Order primer for PstI site removal

→ TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)

→ TODO Enter sequences of constructs to Sandbox

Colonies on Plates

→ The plates showed very low colony growth, and can be caused by the fact it is a low copy plasmid which can be induced by iptg to increase the copy number. Additional growth with IPTG should increase the amount of colonies and available plasmid.

→ The positive control failed due to growth of ampicillin resistance plasmids on kanamycin plates.

→ From the plate with four colonies 5mL LB-amp₁₀₀ medium was inoculated, and additional plates were swiped with the used colonies to see if single colony growth on plate can occur.

New o.n. precultures

The colonies on the [http://partsregistry.org/Part:pSB2K3 pSB2K3]-[http://partsregistry.org/Part:BBa_P1010 BBa_P1010] plate were used to inocculate 5mL LB-kan₅₀-IPTG medium to see if growth takes place.

→ The stock solution of IPTG was 0.5M (mol/L), and the concentration needed for maximum plasmid copy (~150) of [http://partsregistry.org/Part:pSB2K3 pSB2K3] is 100 μmol/L. This is 0.1 μmol/mL, which accounts for 0.2 μL/mL of medium used.

New o.n. plates

The plates of E.coli TOP10 with GVP behind LAC/pBAD (+RBS) promoter in pSB1AC3 were used to grow single colonies on new LB-amp₁₀₀ plates, to test this method of getting pure colonies. If it is not succesfull the old approach of first growing precultures, plating, growing single colonies, and growing new precultures has to be repeated.

Ligation

A total amount of vector of 100ng was used (GVP) in a 1:3 ratio with insert.

(1:3)

• 3 uL Ligase buffer
• 1 ul T4 Ligase
• 13 uL plasmid pSB1AC3 pBad/araC digested with SpeI and PstI
• 3 uL GVP restricted with XbaI and PstI

(1:3)

• 3 uL Ligase buffer
• 1 ul T4 Ligase
• 5 uL plasmid pSB1A2 pLacI digested with SpeI and PstI
• 3 uL GVP restricted with XbaI and PstI
• 8 uL MQ

Incubate:

• 25°C 50min.
• kept on ice for 10min.

Tranformation

• add 10uL of the ligation product 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 plates

→ Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ.

Observations

Here are the allignments of the expected sequence with sequencing results, the part is underlined with a blue line and restriction sites XbaI and SpeI with a red line:

BioBrick BBa_K190015

BioBrick BBa_K190016

BioBrick BBa_K190017

→ The pZntR has two insersions and one mutation in the -35/-10 region, during out testing of the biobricks we will see if it is still Zn induced and compareable to literture values.

Transporters

HmtA

Touchdown PCR 1 pcr2 + pcr 742 + rw + mut1 2 pcr1.2 + pcr 742 + F1 Mut2 3 pcr1.2 + pcr 742 + F2 Mut2

Metal Accumulation

MBP-ArsR

• Check sequence
• Put promotors in front
• Put transporters in back

fMT

• fMT#4 was transformed and plated out in preparation for biobrick mailing
• fMT+promotors were put in ON culture from -80 C

SmtA

Colony PCR of colonies obtained from a pure culture of ON cultures of pLac-SmtA #4 & #5 (31august2009). pLac+SmtA should be about 886 bp. PCR was performed with V_R/V_F primers expected size is 994 bp. However, sequencing results of the SmtA (without pLac) already showed that it was incorrect.

Vectors

Dry