Team:Newcastle/Labwork/18 September 2009

=Formal Lab Session - 18th September 2009=

= Overview = 
 * Stochastic Switch Team - 
 * Metal Sensor Team - 
 * Sporulation Tuning/Chassis Team - 

Stochastic Switch Team
Today we did midipreps of the ara transformation and digested 10ul to run on a gel (EcoRI + PstI) Two bands could be seen one for the backbone and one correctly sied for the insert (~200bp) so this midiprep can be taken forward in order to do the ara-sspb double clone.

Today we also made up some fresh DNA ladder as we have had problems with degradation.

=400ul which was aliquoted into 4 tubes; 3 put in the freezer 1 in the fridge.
 * 40ul of 500ug/ml promega lambda HindIII digest (20ug in total)
 * 280ul H2O
 * 80ul loading dye

Practical Outline
This is the initial list of tasks which need to be carried out by the end of the day:
 * 1) Start the Bacillus subtilis transformation process ("Day of transformation" steps of protocol) in the morning.
 * 2) Design primers for checking B. subtilis cotC integration
 * 3) Plate out some TPA2 cells for production of competent DH5-alpha E. coli cells
 * 4) Run digests from yesterday (midi-prep digests) on gel
 * 5) If successful transform B. subtilis with midi-prep DNA
 * 6) If unsuccessful prepare another ligation attempt
 * 7) Carry out a second attempt at digesting the 5 midi-prep samples from the 11/09/09 Lab Session, i.e. cotC-GFP-smtA, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3

Starting Bacillus subtilis transformations
The team started the first stage of the Bacillus subtilis transformation process by carrying out the beginning steps of the "Day of transformation" section shown in the protocol. If the midi-prep test digest analysis shows that the ligations and the transformation of cotC-GFP-smtA in the pMUTIN4 plasmid have worked then some of the remaining midi-prep DNA can be used to transform B. subtilis when the time is right.

Designing primers for cotC BioBrick integration
It is key that if the team manage to transform Bacillus subtilis with the cotC-GFP-smtA BioBrick in pMUTIN4 plasmid, they can determine that the BioBrick has successfully integrated into the B. subtilis genome. We have designed the BioBrick so that it integrates within the cotC gene of the B. subtilis genome so any primers designed should flank this region. Unfortunately, due to time restrictions, we didn't manage to carry this step out; it will be carried out another day.

Plating out TPA2 cells
It has been noted that the stock of competent DH5-alpha E. coli cells stored in the -80C freezer has been greatly reduced. To generate some more competent E. coli cells, some TPA2 cells (DH5-alpha E. coli) from the stocks freezer were taken and spread on an LB + agar plate. This is to fulfil the first step of the "Pre-culture" stage in the "Preparing Competent Cells" protocol. The plate will then be grown overnight in the 37C incubator.

Analysing midi-prep digests
The midi-prep digest, which we are analysing today, was carried out in yesterday's lab session. The team hopes that the midi-prep DNA is that of cotC-GFP-smtA ligated into pMUTIN4 plasmid and only an analytical digest will determine this.

Just as a recap: we had attempted to ligate the cotC BioBrick into pMUTIN4 and had attempted to transform DH5-alpha E. coli with this product. Several colonies were found on the LB + chloramphenicol plate the trasnformants had been plated onto which suggested both the ligations and the transformations had worked well. Mini-preps were then carried out for 12 colonies in the 16/09/09 Lab Session and digested. When ran on gel, the 2 bands created did not match the expected sizes but there was consistency for most of the samples. This led us to think that it was the restriction enzymes to blame and ruled out contamination.

Based on this decision, the Metal Sensing team conducted a midi-prep on colony 8 LB culture (seen as colony 8 had the clearest bands) and had digested the DNA (see the 17/09/09 Lab Session). The team then attempted to run 10ul of the digest on agrose gel through DNA gel electrophoresis but the DNA ladder was faulty and the bands seemed faint. Today's midi-prep analysis is a second attempt of this procedure.

It can be seen that the gel photograph shows some erroneous results; there is only one band in sight when there should be two (pMUTIN4 and cotC-GFP-smtA bands) and the band formed lies just underneath the 4,361 band from the HindIII DNA ladder. This size neither matches pMUTIN4 or cotC-GFP-smtA indicating a huge error.

Conclusion
We will have to abandon the potential Bacillus subtilis transformations and consider carrying out a second attempt at ligating together pMUTIN4 and cotC-GFP-smtA.

Analysing cotC, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3 midi-preps
Today, we only got as far as digesting the five midi-prep samples with restriction enzymes (this should linearise the plasmids) - tomorrow the team will run products on gel. The restriction digests were allowed to run for 1 hour at 37C (in waterbath). The contents of the digest reactions were as follows: Once the digests were completed, the samples were placed in the -20C freezer for storage.

New Tasks
In light of the ligation/transformation not working, the team needs to start again with the cotC-GFP-smtA work. This involves carrying out a PCR amplification of the cotC BioBrick, digesting both cotC and pMUTIN4, ligating the two fragments together and then using this DNA to transform E. coli cells. Today, we will do the following:
 * Carry out a PCR amplification of the cotC-GFP-smtA BioBrick (which is found within the pMK-RQ plasmid) using primers cotC_forward and cotC_reverse

Introduction

 * We cultured two sets of transformation cells yesterday, two Mini prep need to be processed today.
 * After Mini Prep, we digested those plasmid to find the strain which contains right clone.
 * Continued with yesterday's PCR result, a PCR clean-up process was carried out today.

Mini Prep for L1 and L2

 * Followed the Phil's Mini Prep protocal.

Digest the Mini Prep result
dd H2O                    7ul 10X fast digest buffer    2ul Fast enzyme 1           0.5ul Fast enzyme 2           0.5ul Mini Prep Plasmid        10ul --                           20ul
 * Prepare the digest reaction.
 * Incubate 1 hour at 37 degree.

enzyme 1 EcoRI enzyme 2 SpeI enzyme 1 XbaI enzyme 2 SpeI Control plasmid : pSB1AT3:sleB
 * For digestion of L1 Mini Prep result:
 * For digestion of L2 Mini Prep result:

Run the digested result on 0.8% agarose gel

 * 2ul stop buffer added before loading sample

Conclusion
L1: all stains worked well except No.10. L2: Strain No.4,5,6,11 were not the right clone.
 * From the gel picture.





Further plan

 * Culture the right strain for Midi prep of plasmid.
 * PCR sleB+cwlJ part and ligate to pMutin4 plasmid.
 * Midi Prep L1 and L2 strain.