Lab Session 01/09/2009

Promoter Library Sub-Project

Introduction

Up until now, this sub-project has seen the pGFP-rrnB plasmid backbone digested and linearised with restriction enzymes EcoRI and NheI. The resulting 8kb fragment (which is the plasmid backbone) has been successfully excised from agarose gel and purified; it is into this plasmid backbone in which we will place our sigA promoters.

Today's work involves the amplification of the promoter library (a collection of sigA promoters specified by degenerate sequences) using PCR.

Preparing the Primers

The first task carried out was the rehydration of the primers involved with this sub-project. A final concentration of 200uM is the desired value and to get this for each primer, the following calculation was observed:

Amount of water to be added (ul) = amount of primer (nMoles) x 5

The table below shows the oligos involved in this practical along with the primer amounts (in nMoles) and the amount of PCR water needed (in ul) to make the primers at a concentration of 200uM

A table showing the oligos with the amount of water needed
Oligo	Purpose	Amount (nMoles)	PCR Water added (ul)
IDT 60396420	Forward Primer	19.6	98.0
IDT 60396419	Reverse Primer	21.9	109.5
alta D68423AW	Control (C0)	0.06	300
alta D68425AW	Variant 2 (V2)	0.06	300
alta D68424AW	Variant 3 (V3)	0.06	300
alta D68426AW	Variant 1 (V1)	0.06	300
alta D68427AW	19mer Primer	0.08	400

All of these oligos were then aliquoted and further diluted 1:100 with PCR water and stored in the -20C freezer. The primer stocks are kept in Neil's yellow box whereas the dilutions in enzymes are kept in the a separate box.

Preparing the PCR

The table below shows the amounts of substances added to each PCR reaction:

PCR Reaction Table
Sample	No.	Water (ul)	PCR Mix (ul)	Forward Primer (ul)	Reverse Primer (ul)	Template (ul)	Polymerase used (ul)	Total Volume (ul)
C0	1	31	15	1	1	200uM (1ul)	Taq (1ul)	50
V1	2	31	15	1	1	200uM (1ul)	Taq (1ul)	50
V2	3	31	15	1	1	200uM (1ul)	Taq (1ul)	50
V3	4	31	15	1	1	200uM (1ul)	Taq (1ul)	50

The PCR Mix was composed of the following substances:

30ul dNTP's (6 x 5ul)
30ul Buffer (6 x 5ul)
30ul DMSO (6 x 5ul)

The Taq polymerase used needed to be prepared too. 6ul of pure Taq enzyme was taken and 1ul was diluted in 5ul of PCR water. 1ul of this diluted Taq was used per tube.

Carrying out the PCR

The cycle for the PCR was as follows:

One cycle involving the reverse primer pGFGrrnBrev only with the large degenerate oligo as a template - this is to convert the single-stranded oligo into double stranded DNA. To denature the oligo 90C was needed, to anneal the oligo 60C was needed and to extend the oligo 72C was needed.

The forward primer should then be added and the mixture given 30 cycles (each cycle lasting 1 minute) of 90C denaturing, 54C annealing and 72C extending.

Stochastic Switch Team

Today we run the gel for pSB1AT3 plasmid backbone purified last week from the gel. It was cut with EcoRI and SpeI sites. The length of the band confirmed that we have the right product.

We also used PCR clean-up kit to extract sspB from last week's PCR. We used sigma-d's PCR clean-up protocol. The final volume was measured as 48ul. We measured the concentration and run it on the gel using 5ul of sspb + 4ul of water + 1ul of the loading buffer. The band confirmed that we had the right product after the clean-up process.

The concentration of sspB was 37.5ng/ul

Today we also made ON cultures of the following strains in order to freeze them down into the TPA collection. First the TPA book had to be filled out to record the strains:

2489- BBa_CO178
2490- BBa_ROO62
2491- BBa_CO161
2492- BBa_CO179
2493- BBa_ROO79
2494- BBa_J44000

These will be frozen down in DMSO tomorrow.

Metal Sensing Team

Today we are going to prepare genomic DNA from B.subtilis 168 using the Genomic DNA kit by Sigma-Aldrich as we need some more to be able to PCR up required genes for all 3 teams. Also, we will carry out PCR in order to ampplify czrA gene for our team and sac, ara and sspb genes for the stochastic switch team.

B.subtilis 168 Genomic DNA preparation

We fellowed the instructions in the kit and got our genomic DNA.

Results from the NanoDrop reading for our 6 preparations:

We then stored the genomic DNA in the -20C freezer in the midiprep box.

PCR

We carried out PCR reactions in the same way as described on 21/08/2009.

Exp.No.3 PCR experiment plan
No.	Water (ul)	PCR Mix (ul)	Forward Primer (ul)	Reverse Primer (ul)	Template (ul)	Polymerase used (ul)	Total Volume (ul)
1	24	15	pMA1 2.5ul	pMA2 2.5ul	168 DNA 5ul	MolTaq (1ul)	50
2	29	15	pMA1 2.5ul	pMA2 2.5ul	0	MolTaq (1ul)	50
3	24	15	sac_forward 2.5ul	sac_reverse 2.5ul	168 DNA 5ul	MolTaq (1ul)	50
4	29	15	sac_forward 2.5ul	sac_reverse 2.5ul	0	MolTaq (1ul)	50
5	24	15	araR_forward 2.5ul	araR_reverse 2.5ul	168 DNA 5ul	MolTaq (1ul)	50
6	29	15	araR_forward 2.5ul	araR_reverse 2.5ul	0	MolTaq (1ul)	50
7	24	15	sspb_forward 2.5ul	sspb_reverse 2.5ul	168 DNA 5ul	MolTaq (1ul)	50
8	29	15	sspb_forward2.5	sspb_reverse 2.5	0	MolTaq (1ul)	50