Team:Groningen/Notebook/2 September 2009

GVP Cluster

 * → Choose colonies from plates for growth of E.coli TOP10 with GVP behind LAC/pBAD (+RBS) promoter in pSB1AC3
 * → ✅ Choose colonies from plates for growth of E.coli TOP10 with pSB2K3 plasmid from the registry (plate 1, 7C with RFP Coding Device BBa_J04450


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


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


 * → Order synthetic DNA for GVP
 * → Order primer for PstI site removal


 * → Test promoter strenght compared to BBa_J23101 promoter (Sven)
 * → 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 pSB2K3-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 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:


 * → 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

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 VR/VF primers expected size is 994 bp. However, sequencing results of the SmtA (without pLac) already showed that it was incorrect.