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-	__TOC__	+	#redirect [[Team:Edinburgh/Assembly]]
-		+
-	It may be necessary to plan in detail how stuff is going to be assembled into the final DNA products...	+
-		+
-	The setup for our two systems are as follows:	+
-		+
-	* '''Phage display''': Promoter-RBS-Leader-Enzyme-pVIII	+
-	* '''Cell display''': Promoter-RBS-INP-Enzyme	+
-		+
-	Some notes from Chris:	+
-		+
-	# Check the annealing temperature of the portion without the non-complementary tail (as this is the only part that will anneal during the first round).	+
-	# Check preexisting BioBricks for BglII sites.	+
-	# We will have RFC10 reverse primers for the cellulases.	+
-	# It may not be necessary to have two different forward primers with and without start codon, since the start codon will just be interpreted as a methionine.	+
-	# It may not be necessary to have reverse primers with and without stop codons, as the stop codon can be supplied by the spacer.	+
-		+
-		+
-	==Primers actually made==	+
-		+
-	===malS (amylase)===	+
-		+
-	''malS'' ([http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=3735520&to=3737550&report=gbwithparts sequence]) is an amylase gene from ''E. coli''. It seems to be periplasmic, and for whatever reason does not cause noticable starch degradation, whether because of its location or its expression levels.	+
-		+
-	Using this as a test system would involve comparing the starch-degrading activity of the gene by itself, as compared to the relevant fusions.	+
-		+
-	The actual primers ordered are as follows:	+
-		+
-	* '''EcMalSf1''': ACT AGATCT gaa atc gca gca ata agg act c	+
-	** Forward; adds BglII site; starts upstream of gene to catch RBS ([http://heptamer.tamu.edu/cgi-bin/gb2/gbrowse/MG1655/?plugin=FastaDumper;q=NC_000913%3A3735491..3737550;plugin_action=Go see here]).	+
-	* '''EcMalSf2''': ACT AGATCT atg aaa ctc gcc gcc tgt ttt c	+
-	** Forward; adds BglII site; starts at start codon.	+
-	* '''EcMalSr1''': ACT ACTAGT A TTA tta ctg ttg ccc tgc cca g	+
-	** Reverse; adds SpeI site; RFC10 compliant; has double stop codon.	+
-	* '''EcMalSr2''': ACT ACTAGT ACC ctg ttg ccc tgc cca gac g	+
-	** Reverse; adds SpeI site; RFC23 and BioSandwich compliant; no stop codons, adds a glycine.	+
-		+
-	===bglX (B-glucosidase)===	+
-		+
-	''bglX'' ([http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=2217714&to=2220011&report=gbwithparts sequence, but gene is on reverse strand]) is a periplasmic Β-glucosidase from ''E. coli''. Again, it seems to not be expressed very strongly.	+
-		+
-	Note presence of PstI site in sequence; we could insert via BglII and SpeI. It would then be necessary to use [http://www.openwetware.org/wiki/Cfrench:MABEL MABEL] to remove the PstI site. Still, all of this may be unnecessary if <partinfo>BBa_K392008</partinfo> or something similar works...	+
-		+
-	The actual primers ordered are as follows:	+
-		+
-	* '''EcBglXf1''': ACT AGATCT gcc acg tcg ggc aac	+
-	** Forward; adds BglII site; starts upstream of gene to catch RBS.	+
-	* '''EcBglXf2''': ACT AGATCT atg aaa tgg cta tgt tca gta gg	+
-	** Forward; adds BglII site; starts at start codon.	+
-	* '''EcBglXr1''': ACT ACTAGT A TTA tta cag caa ctc aaa ctc gcc	+
-	** Reverse; RFC10 compliant; has double stop codon.	+
-	* '''EcBglXr2''': ACT ACTAGT ACC cag caa ctc aaa ctc gcc ttt c	+
-	** Reverse; adds SpeI site; RFC23 and BioSandwich compliant; no stop codons, adds a glycine.	+
-		+
-	===pVIII leader sequence===	+
-		+
-	The pVIII sequence is [[Team:Edinburgh/Sequences#pVIII|here]].	+
-		+
-	This part is intended to have '''stuff fused at its 3' (C-terminal) end'''.	+
-		+
-	* '''p8f_cfus''': ACT AGATCT atg aaa aag tct tta gtc c	+
-	** Forward, adds BglII site, start codon present.	+
-		+
-	* '''p8r_cfus''': AAA ACTAGT ACC agc gaa aga cag cat cgg	+
-	** Reverse, adds a glycine, adds SpeI.	+
-		+
-	===pVIII mature protein===	+
-		+
-	The pVIII sequence is [[Team:Edinburgh/Sequences#pVIII|here]].	+
-		+
-	This part is intended to have '''stuff fused at its 5' (N-terminal) end'''.	+
-		+
-	* '''p8f_nfus''': ACT AGATCT gct gag ggt gac gat ccc gc	+
-	** Forward, adds BglII site.	+
-		+
-	* '''p8r_nfus''': AAA ACTAGT A tca gct tgc ttt cga ggt g	+
-	** Reverse, has stop codon, adds standard RFC10 suffix.	+
-		+
-	===BBa_K118022 / BBa_K392007 (Exoglucanase)===	+
-		+
-	We already have reverse primers that ought to be suitable for cell display, though not phage display.	+
-		+
-	This primer deletes the signal peptide.	+
-		+
-	* '''cexf_nosig''': ACT AGATCT ATG gcg acc acg ctc aag gag gc	+
-	** Forward, adds BglII site, adds start codon, starts at a.a. 43.	+
-		+
-	===BBa_K392008 (B-glucosidase)===	+
-		+
-	We already have reverse primers that ought to be suitable for cell display, though not phage display.	+
-		+
-	This primer is slightly misnamed as there is no signal peptide predicted for this; the primer just starts at the CDS start.	+
-		+
-	* '''Cfbgluf_nosig''': ACT AGATCT ATG ggc gac cgg ttc cag cag gc	+
-	** Forward, adds BglII site. Start codon.	+
-		+
-	===Xylose isomerase===	+
-		+
-	From the ''[http://www.ncbi.nlm.nih.gov/nuccore/148278?from=213&to=1535&report=gbwithparts xylA]'' gene of ''E. coli''. Designed for fusion to INP or expression on its own. We probably won't try for phage.	+
-		+
-	* '''EcxylAf1''': ACT AGATCT atg caa gcc tat ttt gac c	+
-	** Forward, adds BglII site, has start codon.	+
-		+
-	* '''EcxylAr1''': AAA ACTAGT A TTA tta ttt gtc gaa cag ata atg g	+
-	** Reverse, adds 2nd stop codon, adds standard suffix.	+
-		+
-	==Primers in planning (amylase, mature peptide only)==	+
-		+
-	''malS'' is a periplasmic gene which has a signal peptide. In order to fuse it to INP, it may be necessary to isolate the mature protein sequence, which is [http://www.ncbi.nlm.nih.gov/protein/CAA41740.1 claimed] to be amino acids 18-676; i.e. we would need to delete the first 51 bases in the DNA.	+
-		+
-	* ACT AGATCT gcc agc tgg act tct ccg gg	+
-	** Forward, adds SglII site.	+
-		+
-	==Primers in planning (bglX)==	+
-		+
-	===MABEL primers to destroy PstI site===	+
-		+
-	The BioBrick we made of ''bglX'' has a PstI site in it, rendering it illegal under RFC10. We can fix with MABEL:	+
-		+
-	* a cca gca ctg gcg gat g	+
-	** Forward, first base is a synonymous change (original was G)	+
-		+
-	* tg cag ggc cag act cac	+
-	** Reverse, perfect match to sequence	+
-		+
-	===Mature peptide only===	+
-		+
-	''bglX'' is a periplasmic gene which has a signal peptide. In order to fuse it to INP, it may be necessary to isolate the mature protein sequence, which SignalP predicts to be amino acids 21 onwards; i.e. we would need to delete the first 60 coding bases in the DNA.	+
-		+
-	* ACT AGATCT gat gat tta ttc ggc aac c	+
-	** Forward, adds BglII site.	+
-		+
-	==Primers in planning (cellulases)==	+
-		+
-	The enzymes of interest will need start codons so they can be used as controls for assays, where we ask: do they work without the carrier?	+
-		+
-	When part of a display system, the different products will '''either be fused only at their 5' end (cell display), or at both ends (phage display)'''.	+
-		+
-	===BBa_K118023 / BBa_K392006 (Endoglucanase)===	+
-		+
-	'''This part contains a BglII site, so that will first have to be dealt with, before we can use these primers.'''	+
-		+
-	Note: SignalP predicts a cleavage site between amino acids 31 and 32. The following primers fix that problem:	+
-		+
-	* ACT AGATCT ATG GCA CCA GGA tgc cgc gtc gac tac gcc gtc '''[Warning: strong secondary structure]'''	+
-	** Forward, adds BglII site, adds start codon, starts at a.a. 32. but changes first three codons to avoid ''very strong'' primer secondary structure.	+
-		+
-	* AAA ACTAGT ACC cca cct ggc gtt gcg cg	+
-	** Reverse, no stop codon; adds a glycine, adds SpeI.	+
-		+
-	===MABEL primers to destroy BglII site, above===	+
-		+
-	* atc tcg cag cgg ctg gg	+
-	** Forward, perfect correspondance to sequence	+
-	* ttg ctg gcc gta cgc ctt g	+
-	** Reverse, replaces CAG (glutamine) with CAA (glutamine).	+
-		+
-	===BBa_K118022 / BBa_K392007 (Exoglucanase)===	+
-		+
-	* AAA ACTAGT AGA gcc gac cgt gca ggg cgt gc	+
-	** Reverse, no stop codon; adds a serine, adds SpeI.	+
-	** (I was unable to add the usual glycine due to very strong secondary structure arising.)	+
-		+
-	===BBa_K392008 (B-glucosidase)===	+
-		+
-	* AAA ACTAGT ACC ggg ctg gta ggt cgc ggc g	+
-	** Reverse, no stop codon; adds a glycine, adds SpeI.	+
-		+
-	==Primers in planning (cell display)==	+
-		+
-	===BBa_K265008 (INP)===	+
-		+
-	Continuing on the assumption of '''BioSandwich''' assembly. Ice Nucleation Protein will be present at the start of the coding fusion. A promoter and RBS will have to be added upstream.	+
-		+
-	This part will have '''stuff fused at its 3' (C-terminal) end'''.	+
-		+
-	* ACT AGATCT atg acc ctg gat aaa gcg c	+
-	** Forward, adds BglII site. Start codon is present.	+
-		+
-	* AAA ACTAGT ACC ttt cac ttc gat cca atc	+
-	** Reverse, no stop codon; adds a glycine, adds SpeI.	+
-		+
-	==Primers in planning (biorefinery)==	+
-		+
-	These things may be produced on their own (as a control) or fused to INP. We probably won't bother with fusion to phage? In any case, the unfused versions used as controls will need start codons.	+
-		+
-	===Aldose reductase===	+
-		+
-	From the [[Team:Edinburgh/Biorefineries#Sorbitol|reverse complement]] of [http://www.ncbi.nlm.nih.gov/nuccore/15594346?from=538328&to=539275&report=gbwithparts this sequence] which we can get from [http://www.straininfo.net/strains/149324/browser DSM 4680]. Might be problems with low GC content of gene. Some alternative strains that might be used instead:	+
-		+
-	* [http://www.ncbi.nlm.nih.gov/nuccore/159162017?from=1026949&to=1027815&report=gbwithparts ''Lactobacillus acidophilus'' NCFM]	+
-	* [http://www.ncbi.nlm.nih.gov/nuccore/209953764?from=197281&to=198111&report=gbwithparts ''Providencia alcalifaciens'' DSM 30120] (but BLAST says this is probably a 2,5-diketo-D-gluconate reductase)	+
-	* [http://www.ncbi.nlm.nih.gov/nuccore/224798838?from=118139&to=119005&report=gbwithparts ''Lactobacillus acidophilus'' ATCC 4796 == LMG 11470]	+
-		+
-	Primers for DSM 4680:	+
-		+
-	* ACT AGATCT gtg aat aat ctt aaa gac	+
-	** Forward, adds BglII site, has start codon (gtg).	+
-		+
-	* AAA ACTAGT A TTA tta aat ttt ttt gtt aaa ta	+
-	** Reverse, adds 2nd stop codon, adds standard suffix.	+
-		+
-	==Primers in planning (zippers)==	+
-		+
-	The GR1 and GR2 zippers may have applications in phage display. In both cases, stuff will be fused to both N and C terminals (see [[Team:Edinburgh/Phage_Reactors]]) so neither start nor stop codons are required.	+
-		+
-	===BBa_K415124 (GR1)===	+
-		+
-	* ACT AGATCT gag gag aag tcc cgg ctg	+
-	** Forward, adds BglII site.	+
-		+
-	* AAA ACTAGT ACC aca acc tcc tac aga ctg g	+
-	** Reverse, adds a glycine, adds SpeI.	+
-		+
-	===BBa_K415125 (GR2)===	+
-		+
-	* ACT AGATCT aca tcc cgc ctg gag ggc c	+
-	** Forward, adds BglII site	+

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Latest revision as of 16:01, 28 July 2011

1. redirect Team:Edinburgh/Assembly