Glasgow/Wetlab

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== Week 1 ==
=== 03/07 ===
#Maija and Christine prepared LB broth and LB agar with [[Glasgow/Wetlab/Protocols# Protocol 1: LB Broth and Agar|Protocol 1]].  Made 4 batches of plates, 2 with kanamycin and 2 with carbenicillin.

=== 04/07 ===
#All wetlab researched BioBricks.
#Reporter constructs and mini-Tn5 stocks looked out.
#Streaked the following:
#*''P. putida'' PAW 340 pJAK14 (Carb. plate)
#*Tn5 lux AB (Carb. plate)
#*Mini-Tn5 lux AB (Carb. plate)
#*''P. fluorescens'' NCIMB 9815 (Carb. plate)
#*''P. putida'' KT2440 (LB)
#*JM109 pBluescript 5k+ (Carb. plate)
#*Mini-Tn5 Tc (Carb. Plate)
#*pQF52 (Carb. plate)
#*''P. fluorescens'' 9815 (LB)
#*''E. coli'' pJAK14 (Km plate)
#*Il DntR in pOF52 (Carb. plate)
#*pUCINR in Ω strain C (Carb. plate)
#*pGLTUR (Carb. plate)
#*Mini-Tn5 Kan (Carb. plate)
#*Mini-Tn5 Sm/Sp (Carb. plate)
#*Mini-Tn5 1cc2 (Carb. plate)
#*''E. coli'' Sa1 (LB)
#*DmpR #24 (Carb. plate)
#*Mini-Tn5 lac 32 in ''E. coli'' 517 (Carb. plate)
#*Mini-Tn5 Tc (Carb. Plate)
#*Mini-Tn5 Cm (Carb. plate)
#*DmpR WT (Carb. plate)
#*''E.coli'' sm 10 pESD15 Tn5 GFP (Carb. plate)
#*pUJ8 (Carb. Plate)

=== 05/07 ===
#BioBricks – Maija and Scott transformed using [[Glasgow/Wetlab/Protocols | Protocol 2]].
#*BBa_p1010 (DB3.1) (death gene plasmid) Plate 4: 7A – p5B1A10, 11E – p5B1A10 and 11C – p5B3K3
#*BBa_IS2001 (Top10) (high copy number plasmid) Plate 4: 5I – p5B4A5, 5D – p5B4K5, 6B – p5B3K5, 6D – p5B4K5 and 6E – p5B4A5 (Also 5K, 5M, 4J, 4L, 4N, 4P)
#*BBa_J23119 (Top 10) (strong constitutive promoter) Plate 3: 19A – pB1A2 (V1013)
#*BBa_R0062 (Top 10) (HSL and luxR inducible) Plate 1: 9G – pSB1A2 (V1004)
#*BBa_J04500 (Top 10) (IPTG inducible prom + RBS) Plate 1: 16P – p5B1AK3 (V1009)
#*BBa_E0040 (Top 10) (GFP mutant no promoter (3b)) Plate 1: 5H – p5B1A2 (V1001)
#Maia and Christine researched restriction enzymes for the reporter constructs and Mini-Tn5s streaked on 4/7/07 and digested some.  Had planned to make biobricks using Mini-Tn5s etc but discovered that a previous attempt to use transposable elements to make biobricks had been unsuccessful due to scarring at the restriction site. http://partsregistry.org/Part:BBa_J61206

=== 06/07 ===
#Maija and Christine made 10x stocks for M9 (see [[Glasgow/Wetlab/Protocols|Protocol 3.2]]).
#Tutorial in [http://www.mbio.ncsu.edu/BioEdit/bioedit.html BioEdit] and [http://biotools.umassmed.edu/bioapps/primer3_www.cgi Primer3].
#Started designing primers for amplification and site directed mutagenesis of DmpR and DmpR #24 (see [[Glasgow/Wetlab/Protocols|Protocol 4]]).

== Week 2 ==
=== 09/07 ===
#Christine and Maija designed primers for site directed mutagenesis in DmpR, DmpR #24, ****, **** and ****, and amplification of DmpR and DmpR #24.  Used [[Glasgow/Wetlab/Protocols|Protocol 4]], and to check Tm http://www.itt-biotech.de/itt-cgi/oligo-tm.pl
#Mai carried out restriction digests of DmpR and DmpR #24.  Results were poor and gel gave poor visibility.
#Scott retransformed any of the transformations that did not work from the transformations from 5/7/07.
#*4/11C	BBa_p1010	pSB3K3	death gene
#*4/5I	BBa_I522001	pSB4A5	hi-copy
#*4/5D	BBa_I522001	pSB4K5	hi-copy
#*4/6B	BBa_I522001	pSB3K3	hi-copy
#*4/6D	BBa_I522001	pSB4K	hi-copy
#*1/5H	BBa_E0040	pSB1A2	GFP non promoter
#Used transformations that did work and set them up tubes of LB for minipreps tomorrow.
#*BBa_I52001	death gene plasmid (hi copy number)
#*BBa_J23119	strong constitutive promoter
#*BBa_R0062	HSL and luxR inducible
#*BBa_306500	IPTG inducible and RBS
Plan is to use DmpR and DmpR #24 to detect phenol and produce lacZ.  Grown on Xgal the better the bacteria detect phenol, the more blue they will be in a spectrophotometer.

=== 10/07 ===
#Mai did minipreps, according to Qiagen prepkit manual (see [[Glasgow/Wetlab/Protocols|Protocol 5]]), of the transformations grown in LB last night (9/7/07).
#Wetlab and Drylab gave presentations to the team to explain key terms used in the lab (see [[Wet to Dry|Tutorials]]).
#Christine made tetracycline stock – 250 mg tetracycline in 50 ml 100% ethanol to make 5 mg/ml stock.
#Maija made thiamine stock (0.8 g thiamine in 20 ml dH2O and filter sterilized).  Kept in freezer in foil (light sensitive).
#Christine grew ''E. coli'' pJAK14 in LB overnight at 37°C following protocol from Wise et al, 2000).
#Scott and Lynsey grew DmpR and DmpR #24 overnight in LB with Tc and Carb.  This is because the plates streaked on 4/7/07  for DmpR and DmpR #24 did not grow, and restriction digests on 9/7/07 used up most of the DNA.  What is grown will be mini-prepped and restriction digested tomorrow.
#*2x  5ml LB containing carb (50 µg/ml) with DmpR
#*2x  5ml LB containing carb (50 µg/ml) with DmpR #24
#*2x  5ml LB containing Tc (50 µg/ml) with DmpR
#*2x  5ml LB containing Tc (50 µg/ml) with DmpR #24
#*2x  5ml LB containing Tc (10 µg/ml) with DmpR
#*2x  5ml LB containing Tc (10 µg/ml) with DmpR #24
#Scott's retransformations (9/7/07) all worked (esp Top 10s, not so much DB3.1).  To be mini-prepped tomorrow.

=== 11/07 ===
#Lynsey mini-prepped Scott's retransformed biobricks (9/7/07) according to Qiagen Prepkit Manual.
#Maija prepared glycerol for freezing the transformations in LB.
#Can not mini-prep DmpR or DmpR #24 in Tc because it did not grow well overnight.  Time for Plan B.
#*'''Plan B'''<br>DmpR is not working – not growing or digesting as we would expect it to.  Instead of DmpR we will try XylR which detects BETX compounds (benzene, toluene, and xylene) and DntR which detects salicylate and could be modified to detect TNT and DNT.  For this we will be using pGLTUR and pQF52.
#We all began to design primers for site directed mutagenesis (SDM) and amplification of XylR, Pr, Pu and DntR.

=== 12/07 ===
<u>XylR, Pr, and Pu</u>
#Searched [http://www.ncbi.nlm.nih.gov/Genbank GenBank] for pWW0 which contains XylR, Pr and Pu.  Saved in BioEdit.
#Using the XylR sequence (Inouye et al, 1988) we were able to design primers for the amplification of XylR.
#Using previously designed primers (Willardson et al, 1998) we were able to locate the beginning of Pr and designed primers to amplify the sequence between the beginning of Pr and the beginning of XylR.
#From previously designed primers (Willardson et al, 1998) we were able to locate a sequence we believed to be Pu and designed primers. To be sure we also searched pWW0 sequence with [http://biotools.umassmed.edu/bioapps/primer3_www.cgi Primer3] to locate on the plasmid where the Willardson Pu primers would attach. Using BioEdit we located another sequence we also suspect to be Pu. We now have primers designed for both suspected sequences.
#Also designed primers for site directed mutagenesis of the PstI site in XylR.
(We were unable to use the Willardson primers for our purposes because they were designed to contain restriction sites, instead we used them to locate the genes of interest).

<u>DntR</u>
#Scott found an article containing the sequence for DntA and some of DntR, then we used BlastX to find the sequence of DntR.  From this we were able to design primers to amplify the sequence.
#Also designed primers so we can sequence pQF52 because the lacZ gene is not complete in the plasmid and we need to know its sequence.

=== 13/07 ===
#Began typing up protocols for the Wiki.
#Ordered primers, made changes to DntR_suffix_1 which will arrive later. (See [[Glasgow/Wetlab/Orders|Orders]])
#Wiki meeting.  Maija, Christine H, Majeik, Toby, Christine M, Mai, Scott and Lynsey.

== Week 3 ==
=== 17/07 ===
#Restriction Digests:
#*BBa_J23119 (strong constitutive promoter) pSB1A2.  1 x NheI, 1 x PvuI.  680bp, 1430bp
#*BBa_R0062 (HSL and luxR) pBB1A2.  1 x EcoRI, 1 x PvuI. 1460bp, 660bp
#*BBa_J04500 (IPTG inducer and RBS) pSB1AK3.  1 x PvuII, 2 x PvuI. 2250bp, 1030bp, 730bp
#*BBa_p1010 (death gene) pSB3K3. 1 x BamHI, 2 x XhoI. 190bp, 2390bp,840bp. 
#*BBa_E0040 (GFP no promoter) pSB1A2. 1 x Hime II, 1 x PvuI. 1630bp, 1170bp.
#*BBa_I52001 (6D) p5B4K5. 2 x AvaI, 1 x PvuI. 920bp, 1470bp, 2120bp.
#*See [[Glasgow/Wetlab/Protocols|Protocol 7]] for Restriction Digests.

=== 18/07 ===
#Last night we diluted primers to 100 pmol/ul by adding the amount of dH2O given on the order sheet, and made working solutions to 10 pmol/ul (2 x 200 ul each - 20 ul stock and 180 ul dH2O).
#Maija ran a gel of the restriction digests to check the sizes of the biobricks.  These were the maps we made yesterday.
#Mai is extracting DNA from 3 samples of ****, which were plated yesterday and grown in liquid media, using MoBio PowersoilTM DNA purification kit (see [[Glasgow/Wetlab/Protocols|Prorocol 8]]), only change was to shorten "shaking" time from 10 to 2 minutes.
#PCR trial run using Reddymix and Touch 2 done on:
#*pGLTUR: XylR_prefix / XylR_suffix, Pr_prefix / Pr_suffix, Pr_prefix / XylR_suffix, Pu_prefix_EM / Pu_suffix_EM.
#*pQF52 plasmid: DntR_prefix / DntR_suffix
#*DmpR WT/24: DntR_prefix / DntR_suffix
#*Biobricks: R0062, E0040, J04500, J23119, p1010, I52001.
#*See [[Glasgow/Wetlab/Protocols|Prorocol 9]] for PCR.

=== 19/07 ===
#Mai redid PCR on pGLTUR and pQF52 using Reddymix and a gradient program (See [[Glasgow/Wetlab/Protocols|Prorocol 9.1]]) to minimise the non-specific amplification evident in previous gels. New DntR suffix primer has arrived. Used the following primer sets.
#*XylR prefix and XylR suffix
#*Pr prefix and Pr suffix
#*Pr prefix and XylR suffix
#*Pu prefix and Pu suffix
#*DntR prefix and DntR suffix 2

=== 20/07 ===

== Week 4 ==
=== 23/07 ===
#PCR Program "Touch 2" with Emma's primers (see [[Glasgow/Wetlab/Orders|Orders 1]]) using Reddy Mix (See [[Glasgow/Wetlab/Protocols|Protocol 9.1]]) and Pseudomonas as our template DNA. The primer pairs used:
##Methyl_1 and Methyl_2
##Oxy_1 and Oxy_2
##Bbp_Methyl_1 and Bbp_Methyl_2
##Bbp_Oxy_1 and Bbp_Oxy_2
##(***)_S_for_1 and (***)_S_rev_1
##Bbp_(***)_S_1 and Bbp_(***)_S_1
##(***)_M_for_1 and (***)_M_rev_1
##Bbp_(***)_M_for_1 and Bbp_(***)_M_rev_1
#PCR of 7 gene operon using Program "Touch 2" with extension time of 8 mins with Emma's primers (see [[Glasgow/Wetlab/Orders|Orders 1]]) and site directed mutagenesis primers (see [[Glasgow/Wetlab/Orders|Orders 2]]) using Reddy Mix (See [[Glasgow/Wetlab/Protocols|Protocol 9.1]]) and Pseudomonas as our template DNA. The primer pairs used:
##(***)Up and (***)Low
##bbp(***)Up and bbs(***)Low
##bbp(***)Up and (***)E_SDM_EcoRI_rev
##bbs(***)Low and (***)B_SDM_EcoRI_for
#KOD polymerase PCR on functioning DntR primers using KOD polymerase [[Glasgow/Wetlab/Protocols|Protocol 9]])and KOD Program [[Glasgow/Wetlab/Protocols|Protocol 9]]).  
#Redid PCR for XylR, Pr and Pu as from 19/07/07 from glycerol stocks, DNA from plates and colony PCR.
#Set up overnights of pGLTUR and pQF52 to do lux and lacZ assays.

=== 24/07 ===
#Redid 7 gene operon PCR (23/07/07 (2)) with Reddymix and Touch 2, and then redid again with KOD polymerase and KOD program with extension time of 2 mins, 55ºC annealing temperature, and 40 cycles [[Glasgow/Wetlab/Protocols|Protocol 9]]).  Added the extra primers combinations for both reactions:
#*bbp(***)Up and (***)B_SDM_EcoRI_rev
#*(***)B_SDM_EcoRI_for and (***)E_SDM_EcoRI_rev
#**And for the KOD polymerase reaction also:
#*bbs(***)_Low and (***)B_SDM_EcoRI_for
#Repeated PCR reaction to amplify (*m*) and (*s*) using KOD polymerase and KOD program (see [[Glasgow/Wetlab/Protocols|Protocol 9]]). Results show that bbs(*s*)_rev_1 is faulty, will redesign. Primer combinations also included:
#*(*s*)_for_1 and bbs(*s*)_rev_1
#*bbp_(*s*)_for_1 and (*s*)_rev_1
#Gel-extracted from the PCR to amplify (*M*) and (*s*), and purified in order to transform ([[Glasgow/Wetlab/Protocols | Protocol 2]]) into Top10 cells. Transformations did not work, will do again tomorrow.
#Colony PCR for XylR, Pr and Pu (23/07/07 (4.)) showed no unique bands in any of the primer pairs.  Will redesign primers for pGLTUR.

=== 25/07 ===
#Overnights of pQF52 did not grow, so done again.
#Overnights also set up for the Bba_p1010 death gene biobrick to see if cloning could be made faster by inserting our biobrick constructs into the biobrick plasmid.
#Transformations for (*m*) and (*S*) done again (24/07/07 (3.)) and overnights set up.
#The rest of the KOD PCR (24/07/07 (2.)) products were run on gel in order to PCR amplify the (*M*) and (*S*) genes (not gel extraction because A overhangs are required for 7A cloning).  From the results, another PCR is required for B7 and C4 over a gradient.  Primer pairs used:
#*A2: Methyl_1 and Methyl_2
#*A7: bbp_Oxy_1 and bbp_Oxy_2
#*B7: bbp_(*m*)_for_1 and bbs_(*m*)_rev_1
#*C4: bbp_(*s*)_for_1 and (*s*)_rev_1
#Restriction digests of Bba_p1010 pSB3K3 were redone using Roche recipe ([[Glasgow/Wetlab/Protocols|Protocol 7]]) as previous attempts were unsuccessful.
##AraI, predicted fragment sizes 274bp, 604bp, 569bp, 1978bp.
##EcoRI and SpeI, predicted fragment sizes 698bp, 2727bp.
##BamHI, XhoI, predicted fragment sizes 188bp, 843bp, 2394bp.
#An alternative source of the XylR gene, P. Putida mt-2 strain, which contains the TOL plasmid plated out and overnights made up (28ºC).
#PCR of 7 gene operon does not show bands of interest, will redesign primers (see [[Glasgow/Wetlab/Orders|Orders 2]]) for site directed mutagnesis on (*d*), and forward and reverse primers for (*a*) and (*g*) respectively.

=== 26/07 ===
#Minipreps of DntR transformant and death gene plasmid overnights done (see [[Glasgow/Wetlab/Protocols|Protocol 5]]).
#Digests of death gene plasmid show the wrong sizes of bands (see 25/07/07 (2.)).
#Overnights of pQF52 with the DntR gene for the Miller assay have still not grown, new overnights set up.
#mt-2s have not grown overnight either, possibly because they were done from glycerol stocks. New colonies set up with colonies from LB plates.
#PCR of 7 gene operon does not show bands of interest, will redesign primers (see [[Glasgow/Wetlab/Orders|Orders 2]]) for site directed mutagnesis on (*d*), and forward and reverse primers for (*a*) and (*g*) respectively.
#Redesigned XylR, Pr and Pu primers using sequence for XylR (Iouye et al, 1988) and located Pr and Pu sequences using predesigned primer sequences (Willardson et al, 1998) ([[Glasgow/Wetlab/Orders|Orders 2]]).
#New primers also designd for the death gene plasmid ([[Glasgow/Wetlab/Orders|Orders 2]]).
#(*d*) site directed mutagenesis primers, and (*a*) forward and (*g*) reverse primers designed.
#PCR done on DntR transformants (2 colonies) using Reddymix and Touch 2 with extension time of 1min 30secs (see [[Glasgow/Wetlab/Protocols|Protocol 9]]).   PCR transformants give the right sizes of bands (M13 rev starts a little outside the mcs of the TOPO vector).
#PCR with KOD on various combinations of the (*m*) and (*s*) gene primers with different PCR programs ([[Glasgow/Wetlab/Protocols|Protocol 9]]) with the intention of cloning them into TOPO TA vectors and BB construction vectors.  A1 and A7 gave good bands but thair KOD equivalents C1 and C7 gave a closer match.  Again and combinations with Bbs_(*s*)_rev_1 fail. Primer combinations as follows:
#*A1 Bbp_(*m*)_for_1 and bbs_(*m*)_rev_1, Gradient program 58-65ºC
#*A3 Bbp_(*s*)_for_1 and Bbs(*s*)_rev_1, Gradient program 58-65ºC
#*A5 Bbp_(*s*)_for_1 and (*s*)_rev_1, Gradient program 58-65ºC
#*A7 Bbp_Methyl_1 and Bbs_Methyl_2, Touch 2
#*B1 Bbp_(*m*)_for_1 and Bbs_Methyl_2, KOD
#*B3 Bbp_(*s*)_for_1 and Bbs_Oxy_2, KOD
#*B5 Bbs_Oxy_1 and Bbs_Oxy_2, KOD
#*B7 Bbp_Oxy_1 and Bbs(*s*)_rev_1, KOD
#*C1 Bbp_Methyl_1 and bbs_(*m*)_rev_1, KOD
#*C3 Bbp_(*s*)_for_1 and Bbs(*s*)_rev_1, KOD
#*C5 Bbp_(*m*)_for_1 and bbs_(*m*)_rev_1, KOD
#*C7 Bbp_Methyl_1 and Bbs_Methyl_2, KOD
#B1, B3, B5, C5 and A7 were gel-etracted and PCR-purified into TOPO vectors in Top10 cells.
#*'Loose' M, A7: Bbp_Methyl_1 and Bbs_Methyl_2 
#*'Close' M, B1: Bbp_(*m*)_for_1 and Bbs_Methyl_2
#*'Close' S, B3: Bbp_(*s*)_for_1 and Bbs_Oxy_2
#*'Loose' S, B5: Bbs_Oxy_1 and Bbs_Oxy_2
#*'Tight' M, C5: Bbp_(*m*)_for_1 and bbs_(*m*)_rev_1

=== 27/07 ===
#Order placed for primers (see 26/07/07 (5., 6., and 7.)).
#Miniprep done of the mt-2 overnights (1 and 2)according to Qiagen prepkit manual (see [[Glasgow/Wetlab/Protocols|Protocol 5]]).  
#Further PCR done from mt-2 with the existing primers for the XylR system using Reddymix and Touch 2 (see [[Glasgow/Wetlab/Protocols|Protocol 9]]). Used the following primer pairs:
#*XylR prefix and XylR suffix
#*Pr prefix and Pr suffix
#*Pr prefix and XylR suffix
#*Pu prefix EMMA and Pu suffix EMMA
#*Pu prefix AFTER XylR and Pu suffix AFTER XylR
#Restriction Digest of DntR in TOPO vector successful, DntR DNA to be sent for sequencing.
#*PvuI - expected fragments 878bp, 2175bp, 1851bp, - observed fragments 878bp, 2175bp, 1851bp.
#*BamHI and NcoI - expected fragments - 2280bp, 2624 bp, -  observed fragments 2280bp, 2624bp.
#DntR overnights for the Miller assays finally grew.  To be subcultured on Sunday for use on Monday.
#Construction vectors results (26/07/07 (11.))
#*4/11E Bba_p1010 pSB1A10 - no growth
#*4/11C Bba_p1010 pSB3K3 - 1 colony - inoculated overnight
#*4/7A Bba_p1010 pSB1A10 - 2 colonies - inoculated overnight
#*B1: Bbp_(*m*)_for_1 and Bbs_Methyl_2 - 2 colonies - inoculated overnight
#*B3: Bbp_(*s*)_for_1 and Bbs_Oxy_2 - 3 colonies - inoculated overnight
#*B5: Bbs_Oxy_1 and Bbs_Oxy_2 - no growth, will try cloning direct from gel extraction into biobrick vector
#*C5: Bbp_(*m*)_for_1 and bbs_(*m*)_rev_1 - no growth, will try cloning direct from gel extraction into biobrick vector
#Observation: TOPO pCR4 vector uses disruption of the ccdB gene as a selection method and the PCR transformants were plated on carbenicillin, perhaps this has killed the transformants.  Colony PCR will be done on the successful ones. Overnights were then grown from 3 or more colonies from each (*m*) and (*s*) transformation plate on carbenicillin again.  Also leftovers from the gel extractions and purifications(26/07/07) were plated on LB only for later analysis.
#More constuction vectors (constructors) were selected for their multiple resistances from the registry to transform into DB3.1.  These were then diluted from the kit plates and transformed into DB3.1 using [[Glasgow/Wetlab/Protocols | Protocol 2]] and plated overnight.  They are as follows:
#*2/23N Bba_p1010 pSB1AK3 V1005
#*2/23P Bba_p1010 pSB1AT3 V1005
#*2/2B Bba_p1010 pSB1AC3 V1005
#*3/20G Bba_p1010 pSB1A2 V1015

== Week 5 ==
=== 30/07 ===
#Minipreps done:
#*(*m*): B1 – colonies 1, 2, 
#*(*s*): B3 – colonies 1, 2, 3
#*Constructor 4/7A – colonies 1, 2, 3, 4, 5, 6, 7, 8, 9
#*Constructor 4/11C – colony 1
#*Constructor 4/11C colonies 10 – 13 did not grow when inoculated overnight.
#*(*s*): Bbp_(*s*)_for_1 and Bbs_Oxy_2 – colonies 20, 21, 22
#*(*m*): Bbp_(*m*)_for_1 and Bbs_(*m*)_rev_1 – colonies 23, 24, 25
#*Bbp_Oxy_1 and Bbs_Oxy_2 – colonies 14, 15, 16
#*Bbp_Methyl1 and Bbs_Methyl_2 – colonies 17, 18, 19
#*Constructor 2/23N – colonies 26, 27, 28, 32
#*Constructor 3/20G – colonies 29, 30, 31, 33, 34, 35
#PCR to check if the BioBrick transformants contain the expected size inserts (used VF2 and VR primers) with Reddymix and Touch 2 program.  Tested:
#*4/11C – colonies 10, 11, 12
#*4/7A – colonies 5, 6, 7
#*4/5I – colonies 1, 2, 3
#*4/5O – colonies 1, 2
#*4/6B –  colonies1, 2, 3
#*3/19A – colonies1, 2, 3, 4
#*1/9G – colonies 1, 2, 3
#*1/16P – colonies 1, 2, 3
#*1/5H – colonies 1, 2, 3
#*B1 – colonies 17, 18, 19, 25, 26, 27
#*B3 – colonies 20, 21, 22, 23, 24
#*B5 – colonies 14, 15, 16, 28, 29
#Diluted new primers which arrived according to the labels.
#Ran new PCR testing the XylR, Pr and Pu primers (original and new which arrived 30/7/07) on mt-2 with Reddymix and Touch2. Both sets of primers successful.  The pGLTUR stock was unreliable.
#PCR of the 7 gene operon with Reddymix, Touch 2 with an extension time of 8 mins and ***** DNA as template.  No desired bands seen.  Primer combinations as follows:
#*Bbp_(*a*)_for and Bbs_(*g*)_rev
#*Bbp_(a*)_for and Bbs_(*d*)_rev
#*Bbp_(*d*)_for and Bbs_(*g*)_rev
#Restriction digest of 3/20G and 2/23N. Used Promega's AvaI for both digests and buffer B, 1hr at 37°C.  Ran 10ul of the reaction with 2ul of loading dye on a 1% agarose gel.  No bands showed up on the gel picture.  Expected band sizes as follows:
#*3/20G: Colonies 29, 30, 31, 33, 34, 35: Bba_p1010 in pSB1AC3 V1005: 1462bp, 1376bp, 892bp
#*2/23N: Colonies 26, 27, 28, 32: Bba_p1010 in pSB1AK3 V1005: 1462bp, 1376bp, 569bp, 457bp
#To understand why restriction digests did not appear, ran gel of miniprepped DNA.  No bands present.  Minipreps ran:
#*2/23N: Colonies 26, 27, 28
#*3/20G: Colonies 30, 31
#Cleaned out cells and reassembled according to NCBE protocol, using one as a negative control replacing yeast with water.


=== 31/07 ===
#Cells reassembled because they had leaked over night.  
#PCR of the 7 gene operon repeated using KOD polymerase and KOD program.  Amplified A-D 3.5kb and D-G 3.5kb. Primer pair as follows:
#*Bbp_(*a*)_for and Bbs_(*g*)_rev
#*Bbp_(a*)_for and Bbs_(*d*)_rev
#*Bbp_(*d*)_for and Bbs_(*g*)_rev
#PCR of the 7 gene operon repeated using KOD XL polymerase and KOD XL program.  Amplified A-G 7kb. Primer pair as follows:
#*Bbp_(*a*)_for and Bbs_(*g*)_rev
#*Bbp_(a*)_for and Bbs_(*d*)_rev
#*Bbp_(*d*)_for and Bbs_(*g*)_rev
#Gel ran of yesterday's BioBrick confirmation (VF2 and VR) PCR.  Expected sizes and results as follows:
#*4/11C: 981bp - Correct
#*4/7A: 4096bp - All wrong
#*4/5I: 1396bp - Correct
#*4/5O: 1396bp - Correct
#*4/6B: 1396bp - 2 and 3 correct
#*3/19A: 273bp - Correct
#*1/9G: 293bp - Correct
#*1/16P: 535bp - Correct
#*1/5H: 958bp - Correct
#*B1: 1072bp - 17, 18, 19, 26 correct
#*B3: 1262bp - 23 correct
#*B5: 1260bp - All wrong
#Restriction digests of constructors 2/23N and 3/20G carried out again using Roche enzymes and protocol, Buffer B and incubation for at least 1hr at 37°C. All bands on gel are between 2 and 2.5 kb.  Digests as follows:
#*2/23N in pSB1AK3, colonies 26, 27, 32:  BamHI, XhoI – 1046bp, 1026bp, 1792bp
#*3/20G in pSB1AC3, colonies 29, 30, 31, 33, 34, 35: BamHI, XhoI – 1046bp, 892bp,1792bp
#Overnights set up to test resistances of the 2/23N and 3/20G constructors.  All colonies 26-35 grown on carbenicillin LB, and colonies 31, 33 and 34 also grown in chloramphenicol LB.

=== 01/08 ===
#Meeting with modellers about biobricks.
#Of yesterday's overnights, the 3 with chloramphenicol did not grow.  This confirms that 3/20G is 3/20G and not 2/2B as previously believed as a result of mislabelling.  All overnights then miniprepped according to Qiagen protocol.
#New restriction digest for new miniprepped 3/20G and 2/23N.  3/20G using Roche enzymes, Buffer B and incubating for at least 1hr at 37°C, digestion worked.  2/23N using Promega's AvaI restriction enzyme as on 30/7/07, AvaI cut the plasmid but not the biobrick. 
#*3/20G in pSB1AC3, colonies 30 and 31: BamHI, PvuI – 1411bp, 1343bp (Xho does not cut).
#*Overnights set up: 2/23N – 26, 27, 28 and 3/20G – 33 (all carb).
#Gel run of 7 gene operon PCR from 31/7/07 and DNA extracted according to Qiagen Gel Extraction Microcentrifuge Protocol.
#PCR repeated for XylR, Pr and Pu because the previous Pu primer combinations did not work.  Primer combinations as follows:
#*XylR preffix and XylR suffix
#*Pr prefix and Pr suffix
#*Pr_for_2 and XylR_rev_2
#*Pu_Pre_EM and Pu_Suf_AFT
#*Pu_Pre_AFT and Pu_Suf_EM
#Researched restriction maps for plasmids with death genes.
#Overnights set up with the intention of digesting constructors of (*m*) and (*s*) after miniprepping in the morning, hopefully getting ligations that will transform TOP10 cells to grow overnight.  Colonies were chosen that gave a positive result of the right predicted size (see 31/7/07) on a gel of PCR with VF2 and VR primers.
#*4/11C constructor – 10, 11, 12 (Kan)
#*4/5I constructor – 1, 2, 3 (Carb)
#*4/5O constructor – 1, 2, 3 (Kan)
#*4/6B constructor – 2, 3 (Kan)
#*B1 (*m*) - 17, 18, 19, 26 (Carb)
#*B3 (*s*) - 20 (Carb)
#*C5 (*m*) - 24, 25 (Carb)
#Miller Assay was done on E.coli cell culture aliquots containing the DntR system grown up in LB according to the Miller assay [[Glasgow/Wetlab/Protocols | Protocol 10]]. A range of salicylate concentrations (0 mM, 20 uM, 50 uM, 100 uM and 200 uM)were added to aliqouts and then allowed to grow for a further 3 hours. As this was a trial run, measurements at only one time point (58 minutes)were taken.  
The very high level of LacZ expression throughout the range of concentrations of salicylate could be due to the content of tryptophan in the LB media which could act upon the DntR responsive promoter. In order to try and minimize the background LacZ expression due to the presence of aromatic compounds present in the LB media, cell cultures will be grown up in minimal media instead.

=== 02/08 ===
#PCR done with death gene primers for the miniprepped DNA to make sure Bba_p1010 is in the plasmid (esp 23N as it does not correc on the gel, using Reddymix and Touch 2 with Tm of 60C and 28 cycles, saved as PCR60DEG.
#Minimal media prepared for the Miller Assay because it does not interfere with enzyme activity (see Protocol).
#Minipreps done of yesterday's overnights for (*m*) and (*s*).
#Digestions done of these (*m*) and (*s*) minipreps
{| border="1" cellspacing="0" cellpadding="5" align="center"
!Gene
!Label
!Colonies
!Primers
!Enzyme
!Orientation 1
!Orientation 2
|-
|(*m*)
|B1
|17, 18, 19, 26
|(*m*)_for_1 + Methyl_2
|EcoRI
|4243bp, 443bp, 275bp
|3683bp, 835bp, 443bp 
|-
|(*m*)
|C5
|24, 25
|(*m*)_for_1 + (*m*)_rev_1
|EcoRI
|4243bp, 443bp, 275bp
|3683bp, 835bp, 443bp 
|-
|(*s*)
|B3
|20
|(*s*)_for_1 + Oxy_2
|PstI
|4012bp, 534bp, 344bp, 275bp
|3683bp, 604bp, 534bp, 344bp
|-
|(*s*)
|B3
|20
|(*s*)_for_1 + Oxy_2
|EcoI
|3656bp, 1491bp, 18bp
|4682bp, 285bp, 18bp
|}

=== 03/08 ===
#Miller assay repeated on E.coli cultures grown up in minimal media. Measurements were taken at three time points (10, 30 and 60 minutes) and the range of salicylate concentrations used was expanded (0mM, 10 uM, 100 uM, 1mM and 10 mM)
#A 1% gel was run for the p1010 PCR products from 02/08.
#*23N (pSB1AK3) plasmid size ~3800bp - AvaI did not cut the biobrick.
#*20G (pSB1A2) plasmid size ~ 2700bp - digest worked!