Formal Lab Session - 31st July 2009

Team Newcastle 2009 iGEM 31-07-09 IMG 0231.JPG

Introduction

Over the past week we have attempted to transform five BioBricks from the Spring Distributions. Of these BioBricks, E. coli plus cinR (BBa_C0077) and E. coli + cinI (BBa_C0076) have not seemed to grow on LB + kan plates suggesting problems with transforming in JM109 cells. With this scenario, we attempted one final transformation but this time using DH5-alpha E. coli cells.

However JM109 E. coli transformations with the CinR sensitive promoter(BBa_R0077), cI coding sequence(BBa_C0056) and the double terminator(BBa_B1002) have all worked and grown on LB + amp plates. Three colonies from each set of BioBrick transformants were then used to inoculate 9 x 5ml LB tubes and allowed to grow overnight.

Today's lab session will see us make a decision on BioBricks BBa_C0077 and BBa_C0076 based on whether our final transformation attempt has worked. Today's lab session will also see the team carry out midi-preps from the inoculated LB tubes (one set of three tubes inoculated with BBa_C0056, another set of three tubes inoculated with BBa_B1002 and the final set of three tubes inoculated with BBa_R0077).

Practical Outline

Team Newcastle 2009 iGEM 31-07-09 IMG 0227.JPG

Here are the objectives for today:

Carry out midi-preps on E. coli cultures containing BBa_C0056, BBa_B1002 and BBa_R0077
Check LB and LB + kan plates for any DH5-alpha cells transformed with BBa_C0077 and BBa_C0076
1. If unsuccessful, abandon,
2. If successful, prepare 5ml LB inoculations

Procedures

1) Midi-preps of BBa_C0056, BBa_B1002, and BBa_R0077

Protocol

Jess tipping off the supernatant after spinning the overnight cultures in the centrifuge

We did a midi prep for the overnight cultures. We followed the protocol from GenElute (NA0200S_NA0200). The list of plasmid kits can be accessed from Gen Elute's plasmid kits page.

Procedure

To transfer the binding column, we used two eppendorf tubes for each culture. The volume of the eppendorfs were 450ul and we used 45ul of sodium acetate buffer and 315ul of isopropanol for "DNA concentration" step. We centrifuged the tubes at 18000rpm for 30 minutes before adding ethanol to rinse the DNAs. We then centriguged the tubes again for 20 minutes at 13000rpm. If we had used the faster machine we would centrifuge the tubes at 18000rpm for 10 minutes. Since it was a slower machine, we used 20 minutes instead.

At the end we air-dried the pellets until the ethanol evaporated. We then added 50ul of PCR water to each eppendorf tube and mixed the solution well. Two eppendorf tubes from the same colony were combined.

Results

Goksel adding one of the agents needed for the plasmid midiprep

Finally we measured the concentration of the DNA in the tubes using spectrometer. We used a sensitive pipet to wash the machine wth 2ul of water twice. We then loaded and measured the concentration for each tube. Between the steps we cleaned the machine with tissues. At the end we loaded with water again for a final cleaning procedure.

Tubes with plasmid DNAs were then placed to the -20C freezer.

Results for each tube are as below.

Results for BBa_C0056

We had high DNA concentration for the first tube.

The concentration of DNA: 124.6 ng/uL
The ratio of DNA concentration to protein concentration(260/280): 1.84
The ratio of protein concentration to RNA concentration(260/230): 1.73

Results for BBa_B1002

We had low DNA concentration for the second tube.

The concentration of DNA: 28.3 ng/uL
The ratio of DNA concentration to protein concentration(260/280): 1.66
The ratio of protein concentration to RNA concentration(260/230): 1.13

Results for BBa_R0077

The concentration of DNA: 53.8 ng/uL
The ratio of DNA concentration to protein concentration(260/280): 1.75
The ratio of protein concentration to RNA concentration(260/230): 1.39

2) BBa_C0077 and BBa_C0076 Transformants

Team Newcastle 2009 iGEM 31-07-09 IMG 0225.JPG

At last, colonies were found on the plates which contained DH5-alpha transformed with BBa_C0077 and BBa_c0076! The 2 LB-alone plates for both BBa_C0077 and BBa_C0076 transformants was completely covered in a lawn of bacteria. The LB + kan plate with the DH5-alpha cells transformed with BBa_C0077 yielded about 5 colonies whereas the LB + kan plate with the BBa_C0076 transformants yielded 100+ colonies.

However the plates will be stored in the fridge, over the weekend, for LB inoculations and future mini-preps to take place later (hopefully in Monday's lab session).