Team:Newcastle/Labwork/9 September 2009

=Formal Lab Session - 9th September 2009= = Overview = 
 * Metal Sensor Team - 
 * Stochastic Switch Team - 
 * Sporulation Tuning/Chassis Team - 

Introduction
In the last lab session the Metal Sensing Team had shown that the version of the BBa_J33206 BioBrick submitted by Chris French (contained within the pSB1A2 plasmid) was indeed the correct one. However any further work on this BioBrick should now be put on temporary hold because of the arrival of our newly synthesized BioBricks. These synthesized BioBricks need to be transformed in E. coli bacteria as well as other processing measures.

Today's task is to tranform DH5-alpha E.coli bacteria with our newly synthesized BioBricks - one set of cells should be tranformed with the cotC-GFP-smtA BioBrick and the other group of cells should be transformed with the kinA BioBrick

Procedure
To carry out the transformations of the two sets of DH5-alpha E.coli cells with our two BioBricks, we adhered to Dr. Aldridge's transformation by heat shock protocol. The only differences to the protocol were these: Once the transformation procedure for both sets of DH5-alpha E.coli cells was completed they were plated out on some LB+kanamycin plates (seen as kanamycin is the antibiotic the plasmid pMK-RQ offers resistance to). The way in which the transformants were plated out were as follows: The remainder of the two sets of cells were then centrifuged for 1 minute. 500ul of the supernatant was then removed and pellet resuspended in the remaining solution. The resuspended transformant cells were then placed on plates as follows: The four plates were then placed in the 37C incubator overnight to be processed tomorrow.
 * Step 2 - the period in which the DH5-alpha cells were left on ice was 15 minutes instead of the indicated 30 minutes.
 * Step 4 - the amount of DNA added to the DH5-alpha cells (whether it was cotC-GFP-smtA BioBrick DNA or kinA BioBrick DNA) was 10ul.
 * Step 5 - the cells were left on ice for 15 minutes instead of the suggested 3 minutes.
 * 200ul of the cotC-GFP-smtA transformed E. coli cells were plated on an LB + kanamycin plate
 * 200ul of the kinA transformed E. coli cells were plated on an LB + kanamycin plate
 * 500ul of the resuspended cotC-GFP-smtA transformed E. coli cells were plated on an LB + kanamycin plate
 * 500ul of the resuspended kinA transformed E. coli cells were plated on an LB + kanamycin plate

Stochastic Switch Team
Today we carried out the minipreps for the E. coli cells transformed with pSB1AT3 + sspB

This time we spinned the tubes 30 minutes after adding isopropanol and we also gave a good mix to the tubes. To create some variances we centrifuges 1st and 2nd tubes for further 5 minutes before adding isopropanol and transferred the supernatant into new tubes. For 9th and 10th tubes we removed any solution after the first step. For 4t, 5th, 6th, 7th, 8th tubes we mixed wel after adding ethanol.

All the minipreps worked this time.

We then carried out restriction digest for the ten minipreps using the list below
 * H20 11ul
 * Buffer 2ul
 * DNA from minipreps 5ul
 * EcoRI 1ul
 * PstI 1ul

We had ten restriction digest for sspB + pSB1AT3 ligation. We also had another one for the control. mCherry + pSB1AT3

The tubes were centrifuged lightly and left for incubation for an hour. We then placed the 11 tubes into -20 freezer.

We then prepared some %0.8 agarose gel and 1XTAE buffer.

500ml TAE + 4 gr agarose

To prepare 1X TAE buffer, mix 980 ml of water with 20ml of 50X TAE buffer.

Today we discovered that the JM109 tramsfromations had worked and so miniprep cultures for these were set up. We did these in LB+Amp and LB +Amp +Tet to make sure that the cells still had Tet resistance.

Did digests of pGFPrrnb and Arun's ara fragment and ran these on a wide well gel to fragment prep them.

Introduction
Since we did the ligation of sleB and pSB1AT3 yestoday, we will perform transformation today. also,our new primers for cwlJ has arrived, we can PCR cwlJ fragment using new primers(clwJnoCut). New primers doen't contain restriction site for standard Biobrick. So when we get the cwlJ PCR fragment, we need to run another PCR with original primers.

DNA transformation (Phil Style)

 * Step 1:Get ready for ice box and water bath(42C-45C).
 * Step 2: take out E.coli competence cells from -80c freezer and put it into ice box immediately for 30 mins.

DH5-alpha: which were prepared by our iGEM team. JM109: which were bought form company.


 * Step3: Seperate the original cells into two tubes, one for our ligation DNA, one for control.
 * Step4: Add ligated sleB:pSB1AT3 20ul to one tube, for the control one, the pSB1AT3 backbone was added. After votex them, put on ice for 30mins.
 * Step5: Put the tubes in water bath 43C exactly 50 sec.
 * Step6: Put the tubes back to ice for 2 mins.
 * Step7: Add 0.9ml liquid LB to each tube and incubate 37C for 1 hour.
 * Step8: Pray 200ul cells on LB+Amp plate, 37C overnight.

Redo PCR for cwlJ gene using GoTaq
95C 2min -->[95C 30S --> 48C 30S --> 75C 1min] 30 runs --> 75C 5min --> store at 4C
 * PCR procedure:

Conclusion
Line 6: cwlJ fragement Line 7: control
 * New primers combined with GoTaq works well when we do the PCR for cwlJ.

Futher plan

 * Transform ligated sleB and pSB1AT3 backbone into E.coli cells