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From 2011.igem.org

Contents 1 Primers actually made 1.1 malS (amylase) 1.2 bglX (B-glucosidase) 1.3 pVIII leader sequence 1.4 pVIII mature protein 1.5 BBa_K118023 / BBa_K392006 (Endoglucanase) 1.6 BBa_K118022 / BBa_K392007 (Exoglucanase) 1.7 BBa_K392008 (B-glucosidase) 1.8 Xylose isomerase 2 Primers in planning (amylase, mature peptide only) 3 Primers in planning (bglX) 3.1 MABEL primers to destroy PstI site 3.2 Mature peptide only 4 Primers in planning (cellulases) 5 Primers in planning (cell display) 5.1 BBa_K265008 (INP) 6 Primers in planning (biorefinery) 6.1 Aldose reductase 7 Primers in planning (GFP: green fluorescent protein) 8 Primers in planning (zippers) 8.1 BBa_K415124 (GR1) 8.2 BBa_K415125 (GR2)

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:

1. 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).
2. Check preexisting BioBricks for BglII sites.
3. We will have RFC10 reverse primers for the cellulases.
4. 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.
5. 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]).
  • Complementary Tm = 63.7 C.
• EcMalSf2: ACT AGATCT atg aaa ctc gcc gcc tgt ttt c
  • Forward; adds BglII site; starts at start codon.
  • Complementary Tm = 70.1 C.
• EcMalSr1: ACT ACTAGT A TTA tta ctg ttg ccc tgc cca g
  • Reverse; adds SpeI site; RFC10 compliant; has double stop codon.
  • Complementary Tm = 66.2 C.
• 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.
  • Complementary Tm = 71.6 C.

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.
  • Complementary Tm = 65.6 C.
• EcBglXf2: ACT AGATCT atg aaa tgg cta tgt tca gta gg
  • Forward; adds BglII site; starts at start codon.
  • Complementary Tm = 60.8 C.
• EcBglXr1: ACT ACTAGT A TTA tta cag caa ctc aaa ctc gcc
  • Reverse; RFC10 compliant; has double stop codon.
  • Complementary Tm = 64.2 C.
• 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.
  • Complementary Tm = 67.0 C.

pVIII leader sequence

The pVIII sequence is 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.
  • Complementary Tm = 49.7 C.

• p8r_cfus: AAA ACTAGT ACC agc gaa aga cag cat cgg
  • Reverse, adds a glycine, adds SpeI.
  • Complementary Tm = 64.2 C.

pVIII mature protein

The pVIII sequence is 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.
  • Complementary Tm = 73.6 C.

• p8r_nfus: AAA ACTAGT A tca gct tgc ttt cga ggt g
  • Reverse, has stop codon, adds standard RFC10 suffix.
  • Complementary Tm = 64.4 C.

BBa_K118023 / BBa_K392006 (Endoglucanase)

This part contains a BglII site, so that will first have to be dealt with, using MABEL:

• cenAmutf1: atc tcg cag cgg ctg gg
  • Forward, perfect correspondance to sequence
  • Tm = 70.3 C.

• cenAmutr1: ttg ctg gcc gta cgc ctt g
  • Reverse, replaces CAG (glutamine) with CAA (glutamine).
  • Tm = 71.5 C.

We can then PCR the part itself. SignalP predicts a cleavage site between amino acids 31 and 32. The following primers fix that problem:

• cenAf_nosig: 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.
  • Complementary Tm = 78.4 C.

• cenAr_nostop: AAA ACTAGT ACC cca cct ggc gtt gcg cg
  • Reverse, no stop codon; adds a glycine, adds SpeI.
  • Complementary Tm = 75.1 C.

BBa_K118022 / BBa_K392007 (Exoglucanase)

The forward 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.

• cexr_nostop: 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)

The forward 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.
  • Complementary Tm = 77.2 C.

• cfbglur_nostop: AAA ACTAGT ACC ggg ctg gta ggt cgc ggc g
  • Reverse, no stop codon; adds a glycine, adds SpeI.
  • Complementary Tm = 76.2 C.

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.
  • Complementary Tm = 59.2 C.

• EcxylAr1: AAA ACTAGT A TTA tta ttt gtc gaa cag ata atg g
  • Reverse, adds 2nd stop codon, adds standard suffix.
  • Complementary Tm = 58.1 C.

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.
  • Complementary Tm = 72.5 C.

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)
  • Tm = 66.4 C.

• tg cag ggc cag act cac
  • Reverse, perfect match to sequence
  • Tm = 64.1 C.

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.
  • Complementary Tm = 59.4 C.

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).

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.
  • Complementary Tm = 63.9 C.

• AAA ACTAGT ACC ttt cac ttc gat cca atc atc
  • Reverse, no stop codon; adds a glycine, adds SpeI.
  • Complementary Tm = 61.5 C.
  • An earlier version of this primer left off the last anticodon and had Tm = 55.9 (was this ordered?)

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 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).
  • Complementary Tm = 42.3 C.

• AAA ACTAGT A TTA tta aat ttt ttt gtt aaa ta
  • Reverse, adds 2nd stop codon, adds standard suffix.
  • Complementary Tm = 46.6 C.

Primers in planning (GFP: green fluorescent protein)

This can be PCR'd from <partinfo>BBa_E0040</partinfo> which is has been in the distribution forever. I assume we'll have some floating around the lab somewhere?

• ACT AGATCT atg cgt aaa gga gaa gaa ctt ttc
  • Forward, adds BglII site, has start codon.
  • Complementary region Tm = 62.6 C.

• AAA ACTAGT ACC ttt gta tag ttc atc cat gcc atg
  • Reverse, no stop codon; adds a glycine, adds SpeI.
  • Complementary region Tm = 64.4 C.

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)

It ought to be possible to PCR this out of <partinfo>BBa_K415151</partinfo>, which is in the latest DNA distribution. Note though that sequencing is claimed to be inconsistent.

• 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)

We can PCR this out of <partinfo>BBa_K415152</partinfo>.

• ACT AGATCT aca tcc cgc ctg gag ggc c
  • Forward, adds BglII site

• AAA ACTAGT ACC gca acc tcc gac gtc ttg c
  • Reverse, adds a glycine, adds SpeI.