Lab Work

From 2006.igem.org

Revision as of 13:21, 14 July 2006 by Jenwilson (Talk | contribs)
Jump to: navigation, search

13th July 2006

We performed ligations with the DNA purified from the colonies transformed on the 6th, 7K (promoter) to 30 (RBS) and 9E (lacZ) to 1I (terminator). The recombinant plasmids were transformed (hopefully) into competent cells and plated on medium containing Xgal and IPTG, with a pBluescript colony also plated as a control. We also suspended more original colonies in liquid culture as a backup.

The primers ordered for a better lacZ gene, and the arsR and ars promoter arrived and we did PCR with cells of two different E. coli strains.

In order to determine the ideal conditions to achieve a pH response, three different growth mediums were established:

Parameter: 50% culture 25% culture 12.5% culture
Ampicillin: 50 microlitres 50 microlitres 50 microlitres
IPTG: 50 microlitres 50 microlitres 50 microlitres
Culture: 25 ml 12.5 ml 6.25 ml
Sterile water: 25 ml 37.5 ml 43.75 ml


From these cultures we achieved the following results:

Parameter: 50% culture (pH) 25% culture (pH) 12.5% culture (pH)
Time (in min)
0 8.48 8.47 8.43
30 8.28 8.17 7.97
60 8.18 8.05 7.85
90 7.99 7.83 7.62
120 7.67 7.61 7.61
150 7.53 7.49 7.54
180 7.48 7.47 7.47
210 7.38 7.34 7.38
240 7.33 7.33 7.46


At the end of the experiment the pH meter was tested using pH 7.0 and pH 4.0 buffers. The 4.0 buffer was measured at 3.42 and the 7.0 buffer measured 7.21. Although there is a degree of inaccuracy in the measurements, the electrode was still functioning correctly after the experiments.

12th July 2006

The pH response over time was again measured but this time, we used liquid cultures which were already saturated (i.e. in the stationary phase). The results will be put up soon.

Parameter: Blue saturated culture white saturated culture
Ampicillin: 50 μlitres 50 microlitres 50 microlitres
IPTG: 50 microlitres 50 microlitres 50 microlitres
Culture: 25 ml 12.5 ml 6.25 ml
Sterile water: 25 ml 37.5 ml 43.75 ml


11th July 2006

Today we tested the timed pH response in 2 cell cultures: 1 with the LacZ gene, which was activated by IPTG, and one that had this gene absent.

We cut the isolated plasmid DNA with restriction enzymes to remove the inserts from the promoter and lacZ part, and open the vectors for the RBS and terminator. We ran gels, and these showed that the restriction had succeeded, but had not yielded much DNA. The correct bands were cut out of the gel to purify the inserts and vectors, ready for ligation.

10th July 2006

The colonies transformed on the 6th made it this time, and we isolated the plasmid DNA from three individual colonies for each biobrick. We also transformed some E. coli with

23E pSB1A3 Plasmid Plate 1 AmpR

to serve as an empty plasmid for the new LacZ and arsenic promoter/repressor parts which we will create.

7th July 2006

Acid Production with LacZ and Lactose

When bacteria with the lacZ gene inserted are present in a medium containing lactose, the pH does drop significantly.








6th July 2006

Unfortunately the colonies we plated on the 4th did not survive due to a problem with the competent cells we used, so today we repeated transforming and plating colonies containing the following parts:

9E BBa_E0033 LacZ alpha Plate 2 KanR
1I BBa_B0015 Terminator Plate 1 AmpR
7K BBa_R0010 IPTG responsive promoter Plate 1 AmpR
3O BBa_B0034 RBS Plate 1 AmpR

4th July 2006

We plated colonies containing plasmids with the following parts:

9E BBa_E0033 LacZ alpha Plate 2 KanR
3P BBa_0010 Terminator Plate 2 AmpR
7K BBa_R0010 IPTG responsive promoter Plate 1 AmpR


[http://2006.igem.org/Standard_Protocols Standard Protocols]


[http://2006.igem.org/University_of_Edinburgh_2006 Main page]

Personal tools
Past/present/future years