IGEM:MIT/2005/Inventory

File:PlasmidInventory IGEM2005.xls

Strains

E.coli Strain Genotypes

1. NB115 (pCT-4-4-20) -- 7.02 -80°C
2. NB116 (pCT302 S101A) -- 7.02 -80°C
3. NB118 (pCT-4M5.3) -- 7.02 -80°C
4. MC4100
   • Genotype: F- araD139 delta(argF-lac)U169 prsL150 relA1 deoC1 rbsR fthD5301 fruA25 lambda- [s]

Plasmids

1. File:PCT302.pdf
2. [[../PCT302 (aka pCT-4-4-20)/]]-- 7.02 4°C
3. [[../PCT302 S101A/]] -- 7.02 4°C
4. [[../PCT-4M5.3/]] -- 7.02 4°C

PCR Amplification

1. Spin PCR purification kit ()
   
2. Platinum, TAQ polymerase (DNA polymerase for PCR)
   
3. DNTP mix (25mm ea. dntp) (nucleotides for PCR)
   

Agarose gels

1. Agarose 100g (pouring gels)
   
2. 10X TAE buffer (pouring/running gels)
   
3. 1 kb DNA ladder 250µg (molecular weight standard)
   
4. Ethidium Bromide Solution (10mg/mL) (staining DNA)
   

Cutting/joining DNA fragments

1. EcoR1 10,000 u (Restriction Enzyme)
   
2. PstI 10,000 u (Restriction Enzyme)
   
3. SpeI 2500 u (Restriction Enzyme)
   
4. Xba1 3,000 u (Restriction Enzyme)
   
5. T4 DNA Ligase 100,000un (Ligating DNA fragments)
   

Purifying DNA

1. Spin Plasmid Kit, Mini(for minipreps)
   
2. Gel Extraction Kit 50 (purifying from gel slices)
   
3. Spin PCR Purification Kit (purifying PCR products)
   

Microbiology supplies

1. LB amp -- Cold Room
2. Amp stock -- -20 Fridge
3. VWR Petri Dish (100x15mm, case of 500) (for pouring plates)
   
4. Test Tube, Culture (Polystyrene, 12x75, case of 1000) (special tubes for FACS)
   

Bio Bricks

1. R0053 -- Amp -- KBlab -- 4°C
2. R0040 -- Amp -- KBlab -- 4°C
3. R0011 -- Amp -- KBlab -- 4°C
4. Q04121 -- Kan -- KBlab -- 4°C
5. Q04510 -- Kan -- KBlab -- 4°C
6. Q04400 -- Kan -- KBlab -- 4°C
7. E0420 -- Amp -- KBlab -- 4°C
8. E0430 -- Amp -- KBlab -- 4°C
9. Z0251 -- still in planning stage
10. I13504 -- still locating

Planning Bio Bricks

1. BBa_J07005 [msr] E.coli JM83 alkaline phosphatase gene (PhoA) 1416 bp
2. BBa_J07006 [tmp] malE 1191 bp
3. BBa_J07007 [reg] ctx promoter 145 bp
4. BBa_J07008 [tmp] ToxR w.t. (from Vibrio Cholarae, aa's 1-294) 1160 bp
5. BBa_J07009 [reg] ToxR' (aa's 1-210) 630 bp
6. BBa_J07010 [reg] ToxR inner (aa's 1-198; cytoplasm + TM) 594 bp
7. BBa_J07011 [rpt] ctx promoter :: gfp reporter 871 bp
8. BBa_J07012 [sig] scFv anti-fluorescein [4-4-20][1/3] 762 bp
9. BBa_J07013 [sig] scFv anti-fluorescein [4M5.3][2/3]
10. BBa_J07014 [sig] scFv anti-fluorescein [S101A][3/3]

Primers

• scFv (pCT-4-4-20, pCT302 S101A, pCT-4M5.3 anti-fluorescein plasmids)

FWD: 5' CATCAT GAATTC GCGGCCGC T TCTAGA GACGTCGTTATGACTCAAACACC 3' T_m,i=71.8 T_m,f=88.5
         rand   EcoRI   NotI      XbaI       Primer

REV: 5' GCTCAT  ACTAGT TTATTA CTTATCATCATCATCCTTATAATC GGAGACGGTGACTGAGGTTCC 3' T_m,i=74.7 T_m,f=87.1
         rand    SpeI   Stop          FLAG Tag                 Primer

• FecA

fecA-Fwd  5’CATCAT  GAATTC  GCGGCCGC  TCTAGA TGACGCCGTTACGCGTTTTTCG 3’ T_m,i=73.2 T_m,f=89.6
             rand    EcoRI    NotI    XbaI     primer
fecA-Rev  5’GCCATA  ACTAGT  TTATTA  TCAGAACTTCAACGACCCCT 3’ T_m,i=68.2 T_m,f=79.9
             rand    SpeI     stop    primer

Middle Fwd- 5'GGACGGCAGCGGACTGCAAGTAAAACCGCTGGGAAATAAC 3' T_m=84.7
Middle Rev- 5’GTTATTTCCCAGCGGTTTTACTTGCAGTCCGCTGCCGTCC 3' T_m=84.7

fecA Promoter Fwd- 5’CATCAT  GAATTC  GCGGCCGC T TCTAGA  TGGATAAACATTTCACCACTG 3' T_m,i=65.0 T_m,f=86.2
                     rand    EcoRI    NotI      XbaI     primer
fecA Promoter Rev- 5'GCCATA  CTGCAG  CGGCCGC  T ACTAGT  CACAAGTCGATACTCAGCTTG 3' T_m,i=68.9 T_m,f=89.8
                     rand    PstI     NotI      SpeI     primer

• FecR

fecR-Fwd 5' CATCAT  GAATTC  GCGGCCGC TCTAGA tgaatcctttgttaaccgattccc 3'T_m 66 
             rand   EcoRI     NotI     XbaI   primer
fecR-Rev 5' GCCATA  ACTAGT  TTATTA ttacagtggtgaaatgtttatccag 3' T_m 68
             rand   SpeI     stop   primer

Middle Fwd 5'gtgaaagcctacagttcagcgcctcag3'  T_m 84
Middle Rev 5'ctgaggcgctgaactgtaggctttcac3'  T_m 84

• FecI

fecI-Fwd 5'CATCAT GAATTC GCGGCCGC T TCTAGA tgtctgaccgcgccactacc3' T_m 68
           rand   EcoRI    NotI     XbaI  primer
fecI-Rev 5'GCCATA CTGCAG CGGCCGC T ACTAGT A TTATTA tcataacccatactccagacggaa3' T_m 70
            rand  PstI    NotI      SpeI     stop  primer

• MalE (updated/fixed)

fwd: 5' CCGTTA  GAATTC GCGGCCGC T TCTAGA atgaaaataaaaacaggtgcacgc3' T_m,i=68.8 T_m,f=85.5
         junk    EcoRI    NotI     XbaI       Primer
*
rev: 5' ACTGAG  CTGCAG GCGGCCGC T ACTAGT TTATTA  TTACTTGGTGATACGAGTCTGC 3' T_m,i=69.5 T_m,f=89.4
         junk    PstI    NotI      SpeI   Stop        Primer

• PhoA

fwd: 5'     CATGAG  GAATTC GCGGCCGC T TCTAGA ATGAAACAGAGCACCATTGCG 3' T_m,i=68.9 T_m,f=88.8
             junk    EcoRI    NotI     XbaI       Primer
rev: 5'     AAGCTA  CTGCAG GCGGCCGC T ACTAGT TTATTA TTATTTCAGGCCCAGCGCC 3' T_m,i=69.7 T_m,f=89.8
             junk    PstI    NotI      SpeI   Stop        Primer

Middle(1)
 fwd: 5' caccgcggaactgcaagatgcgaccccggcg 3' T_m=88.8
                        *
 rev: 5' CGCCGGGGTCGCATCTTGCAGTTCCGCGGTG 3' T_m=88.8
                        * 

Middle(2)
 fwd: 5' aggcttttttctgcaagtggaaggcgcgagc 3' T_m=82.2
                        *
 rev: 5' GCTCGCGCCTTCCACTTGCAGAAAAAAGCCT 3' T_m=82.2
                        *