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| - | __TOC__
| + | #redirect [[Team:Edinburgh/Assembly]] |
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| - | It may be necessary to plan in detail how stuff is going to be assembled into the final DNA products...
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| - | The setup for our two systems are as follows:
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| - | * '''Phage display''': Promoter-RBS-Leader-Enzyme-pVIII
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| - | * '''Cell display''': Promoter-RBS-INP-Enzyme
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| - | Some notes from Chris:
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| - | # 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).
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| - | # Check preexisting BioBricks for BglII sites.
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| - | # We will have RFC10 reverse primers for the cellulases.
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| - | # 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.
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| - | # It may not be necessary to have reverse primers with and without stop codons, as the stop codon can be supplied by the spacer. | + | |
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| - | ==Primers actually made==
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| - | ===malS (amylase)===
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| - | ''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.
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| - | Using this as a test system would involve comparing the starch-degrading activity of the gene by itself, as compared to the relevant fusions.
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| - | The actual primers ordered are as follows:
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| - | * '''EcMalSf1''': ACT AGATCT gaa atc gca gca ata agg act c
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| - | ** 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]).
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| - | * '''EcMalSf2''': ACT AGATCT atg aaa ctc gcc gcc tgt ttt c
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| - | ** Forward; adds BglII site; starts at start codon.
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| - | * '''EcMalSr1''': ACT ACTAGT A TTA tta ctg ttg ccc tgc cca g
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| - | ** Reverse; adds SpeI site; RFC10 compliant; has double stop codon.
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| - | * '''EcMalSr2''': ACT ACTAGT ACC ctg ttg ccc tgc cca gac g
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| - | ** Reverse; adds SpeI site; RFC23 and BioSandwich compliant; no stop codons, adds a glycine.
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| - | ===bglX (B-glucosidase)===
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| - | ''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.
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| - | 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...
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| - | The actual primers ordered are as follows:
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| - | * '''EcBglXf1''': ACT AGATCT gcc acg tcg ggc aac
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| - | ** Forward; adds BglII site; starts upstream of gene to catch RBS.
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| - | * '''EcBglXf2''': ACT AGATCT atg aaa tgg cta tgt tca gta gg
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| - | ** Forward; adds BglII site; starts at start codon.
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| - | * '''EcBglXr1''': ACT ACTAGT A TTA tta cag caa ctc aaa ctc gcc
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| - | ** Reverse; RFC10 compliant; has double stop codon.
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| - | * '''EcBglXr2''': ACT ACTAGT ACC cag caa ctc aaa ctc gcc ttt c
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| - | ** Reverse; adds SpeI site; RFC23 and BioSandwich compliant; no stop codons, adds a glycine.
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| - | ===pVIII leader sequence===
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| - | The pVIII sequence is [[Team:Edinburgh/Sequences#pVIII|here]].
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| - | This part is intended to have '''stuff fused at its 3' (C-terminal) end'''.
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| - | * '''p8f_cfus''': ACT AGATCT atg aaa aag tct tta gtc c
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| - | ** Forward, adds BglII site, start codon present.
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| - | * '''p8r_cfus''': AAA ACTAGT ACC agc gaa aga cag cat cgg
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| - | ** Reverse, adds a glycine, adds SpeI.
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| - | ===pVIII mature protein===
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| - | The pVIII sequence is [[Team:Edinburgh/Sequences#pVIII|here]].
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| - | This part is intended to have '''stuff fused at its 5' (N-terminal) end'''.
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| - | * '''p8f_nfus''': ACT AGATCT gct gag ggt gac gat ccc gc
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| - | ** Forward, adds BglII site.
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| - | * '''p8r_nfus''': AAA ACTAGT A tca gct tgc ttt cga ggt g
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| - | ** Reverse, has stop codon, adds standard RFC10 suffix.
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| - | ===BBa_K118023 / BBa_K392006 (Endoglucanase)===
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| - | '''This part contains a BglII site, so that will first have to be dealt with, using MABEL:'''
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| - | * '''cenAmutf1''': atc tcg cag cgg ctg gg
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| - | ** Forward, perfect correspondance to sequence
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| - | * '''cenAmutr1''': ttg ctg gcc gta cgc ctt g
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| - | ** Reverse, replaces CAG (glutamine) with CAA (glutamine).
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| - | We can then PCR the part itself. SignalP predicts a cleavage site between amino acids 31 and 32. The following primers fix that problem:
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| - | * '''cenAf_nosig''': ACT AGATCT ATG GCA CCA GGA tgc cgc gtc gac tac gcc gtc '''[Warning: strong secondary structure]'''
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| - | ** Forward, adds BglII site, adds start codon, starts at a.a. 32. but changes first three codons to avoid ''very strong'' primer secondary structure.
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| - | * '''cenAr_nostop''': AAA ACTAGT ACC cca cct ggc gtt gcg cg
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| - | ** Reverse, no stop codon; adds a glycine, adds SpeI.
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| - | ===BBa_K118022 / BBa_K392007 (Exoglucanase)===
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| - | We already have reverse primers that ought to be suitable for cell display, though not phage display.
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| - | This primer deletes the signal peptide.
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| - | * '''cexf_nosig''': ACT AGATCT ATG gcg acc acg ctc aag gag gc
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| - | ** Forward, adds BglII site, adds start codon, starts at a.a. 43.
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| - | ===BBa_K392008 (B-glucosidase)===
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| - | We already have reverse primers that ought to be suitable for cell display, though not phage display.
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| - | This primer is slightly misnamed as there is no signal peptide predicted for this; the primer just starts at the CDS start.
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| - | * '''Cfbgluf_nosig''': ACT AGATCT ATG ggc gac cgg ttc cag cag gc
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| - | ** Forward, adds BglII site. Start codon.
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| - | ===Xylose isomerase===
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| - | 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.
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| - | * '''EcxylAf1''': ACT AGATCT atg caa gcc tat ttt gac c
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| - | ** Forward, adds BglII site, has start codon.
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| - | * '''EcxylAr1''': AAA ACTAGT A TTA tta ttt gtc gaa cag ata atg g
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| - | ** Reverse, adds 2nd stop codon, adds standard suffix.
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| - | ==Primers in planning (amylase, mature peptide only)==
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| - | ''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.
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| - | * ACT AGATCT gcc agc tgg act tct ccg gg
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| - | ** Forward, adds SglII site.
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| - | ==Primers in planning (bglX)==
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| - | ===MABEL primers to destroy PstI site===
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| - | The BioBrick we made of ''bglX'' has a PstI site in it, rendering it illegal under RFC10. We can fix with MABEL:
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| - | * a cca gca ctg gcg gat g
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| - | ** Forward, first base is a synonymous change (original was G)
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| - | * tg cag ggc cag act cac
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| - | ** Reverse, perfect match to sequence
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| - | ===Mature peptide only===
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| - | ''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.
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| - | * ACT AGATCT gat gat tta ttc ggc aac c
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| - | ** Forward, adds BglII site.
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| - | ==Primers in planning (cellulases)==
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| - | 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?
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| - | 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)'''.
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| - | ===BBa_K118022 / BBa_K392007 (Exoglucanase)===
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| - | * AAA ACTAGT AGA gcc gac cgt gca ggg cgt gc
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| - | ** Reverse, no stop codon; adds a serine, adds SpeI.
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| - | ** (I was unable to add the usual glycine due to very strong secondary structure arising.)
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| - | ===BBa_K392008 (B-glucosidase)===
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| - | * AAA ACTAGT ACC ggg ctg gta ggt cgc ggc g
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| - | ** Reverse, no stop codon; adds a glycine, adds SpeI.
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| - | ==Primers in planning (cell display)==
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| - | ===BBa_K265008 (INP)===
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| - | 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.
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| - | This part will have '''stuff fused at its 3' (C-terminal) end'''.
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| - | * ACT AGATCT atg acc ctg gat aaa gcg c
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| - | ** Forward, adds BglII site. Start codon is present.
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| - | * AAA ACTAGT ACC ttt cac ttc gat cca atc
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| - | ** Reverse, no stop codon; adds a glycine, adds SpeI.
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| - | ==Primers in planning (biorefinery)==
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| - | 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.
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| - | ===Aldose reductase===
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| - | 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:
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| - | * [http://www.ncbi.nlm.nih.gov/nuccore/159162017?from=1026949&to=1027815&report=gbwithparts ''Lactobacillus acidophilus'' NCFM]
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| - | * [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)
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| - | * [http://www.ncbi.nlm.nih.gov/nuccore/224798838?from=118139&to=119005&report=gbwithparts ''Lactobacillus acidophilus'' ATCC 4796 == LMG 11470]
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| - | Primers for DSM 4680:
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| - | * ACT AGATCT gtg aat aat ctt aaa gac
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| - | ** Forward, adds BglII site, has start codon (gtg).
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| - | * AAA ACTAGT A TTA tta aat ttt ttt gtt aaa ta
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| - | ** Reverse, adds 2nd stop codon, adds standard suffix.
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| - | ==Primers in planning (zippers)==
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| - | 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.
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| - | ===BBa_K415124 (GR1)===
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| - | We can PCR this out of <partinfo>BBa_K415151</partinfo>, which is in the latest DNA distribution. Note though that sequencing is claimed to be inconsistent.
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| - | * ACT AGATCT gag gag aag tcc cgg ctg
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| - | ** Forward, adds BglII site.
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| - | * AAA ACTAGT ACC aca acc tcc tac aga ctg g
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| - | ** Reverse, adds a glycine, adds SpeI.
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| - | ===BBa_K415125 (GR2)===
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| - | We can PCR this out of <partinfo>BBa_K415152</partinfo>.
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| - | * ACT AGATCT aca tcc cgc ctg gag ggc c
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| - | ** Forward, adds BglII site
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| - | * AAA ACTAGT ACC gca acc tcc gac gtc ttg c
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| - | ** Reverse, adds a glycine, adds SpeI.
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