Antigen 43 Primer Design

Size: px

Start display at page:

Download "Antigen 43 Primer Design"

Delilah Norton
6 years ago
Views:

1 Antigen 43 Primer Design Background We want to amplify the flu operon off of the E. coli K12 chromosome using PCR in order to make the cell surface of E. coli and other Pseudomonas species frizzy. This frizzy cell surface will aid in the formation of biofims between bacteria of different species. Primers have already been designed by Kjaergaard et. al. for amplification of the flu operon. We are modifying their primer sequence in order to incorporate the biobrick cutsites but still using the same complementary sequence to the E. coli K12 chromosome. After this amplification we plan on placing this operon under the control of a constituative promoter. This will always have the bacteria in the biofilm to degrade NA s. This will eliminate adding sugars to the media to induce expression because ultimately we want to test biofilm formation with NA s as the sole carbon source. Reference Antigen 43 from Escherichia coli induces inter-and intraspecies cell aggregation and changes in colony morphology of Pseudomonas fluorescens. Journal of bacteriology [ ] Kjargaard yr:2000 vol:182 iss:17 pg:4789 Primer Design Reference Primer The primer sequence as reported by Kjaergaard et. al. to amplify the flu operon is as follows: 5 -CCCGCGGCCGCGATATCCTTTGTCAGTAACATGC 5 -CCCGCGGCCGCGGATCCTGTGGCGTTGAAGATCCG GC rich region EcoRV cutsite BamHI cutsite Complementary region with the E. coli K12 chromosome according to the NCBI sequence Reference Sequence We looked up the flu sequence on NCBI for E. coli K12 (NCBI ) and found it to be Ag43 flu gene to be between the 2,069,400 and 2,072,800 bp on the sequence. Our annotated sequence from NCBI is listed below

2 Upstream: ACCTGTCGTGACTGATGCCCTCCCTGACTCTGAGTCTGCTCACAAAAGCACTGTTTTCGTTACTGTCTCTCTTGTCCG TGCAATAGCTCAATAATAGAATAAAACGATCAATATCTATTTTATCGATCGTTTATATCGATCGATAAGCTAATAAT AACCTTTGTCAGTAACATGCACAGATACGTACAGAAAGACATTCAGGGAACAACAGAACCACAATTCAGAAACTC CCACAGCCGGACCTCCGGCACTGTAACCCTTTACCTGCCGGTATCCACGTTTGTGGGTACCGGCTTTTTTATTCACC CTCAATCTAAGGAAAAGCTG CODING SEQUENCE: ATGAAACGACATCTGAATACCTGCTACAGGCTGGTATGGAATCACATGACGGGCGCTTTCGTGGTTGCCTCCGAAC TGGCCCGCGCACGGGGTAAACGTGGCGGTGTGGCGGTTGCACTGTCTCTTGCCGCAGTCACGTCACTCCCGGTGC TGGCTGCTGACATCGTTGTGCACCCGGGAGAAACCGTGAACGGCGGAACACTGGCAAATCATGACAACCAGATTG TCTTCGGTACGACCAACGGAATGACCATCAGTACCGGGCTGGAGTATGGGCCGGATAACGAGGCCAATACCGGC GGGCAATGGGTACAGGATGGCGGAACAGCCAACAAAACGACTGTCACCAGTGGTGGTCTTCAGAGAGTGAACCC CGGTGGAAGTGTCTCAGACACGGTTATCAGTGCCGGAGGCGGACAGAGCCTTCAGGGACGGGCTGTGAACACCA CGCTGAATGGTGGCGAACAGTGGATGCATGAGGGGGCGATAGCCACAGGAACCGTCATTAATGATAAGGGCTGG CAGGTCGTCAAGCCCGGTACAGTGGCAACGGATACCGTTGTTAATACCGGGGCGGAAGGGGGACCGGATGCAGA AAACGGTGATACCGGGCAGTTTGTTCGCGGGGATGCCGTACGCACAACCATCAATAAAAACGGTCGCCAGATTGT GAGAGCTGAAGGAACGGCAAATACCACTGTGGTTTATGCCGGCGGCGACCAGACTGTACATGGTCACGCACTGG ATACCACGCTGAATGGGGGATACCAGTATGTGCACAACGGCGGTACAGCGTCTGACACTGTTGTGAACAGTGACG GCTGGCAGATTGTCAAAAACGGGGGTGTGGCCGGGAATACCACCGTTAATCAGAAGGGCAGACTGCAGGTGGAC GCCGGTGGTACAGCCACGAATGTCACCCTGAAGCAGGGCGGCGCACTGGTTACCAGTACGGCTGCAACCGTTACC GGCATAAACCGCCTGGGAGCATTCTCTGTTGTGGAGGGTAAAGCTGATAATGTCGTACTGGAAAATGGCGGACGC CTGGATGTGCTGACCGGACACACAGCCACTAATACCCGCGTGGATGATGGCGGAACGCTGGATGTCCGCAACGGT GGCACCGCCACCACCGTATCCATGGGAAATGGCGGTGTACTGCTGGCCGATTCCGGTGCCGCTGTCAGTGGTACC CGGAGCGACGGAAAGGCATTCAGTATCGGAGGCGGTCAGGCGGATGCCCTGATGCTGGAAAAAGGCAGTTCATT CACGCTGAACGCCGGTGATACGGCCACGGATACCACGGTAAATGGCGGACTGTTCACCGCCAGGGGCGGCACAC TGGCGGGCACCACCACGCTGAATAACGGCGCCATACTTACCCTTTCCGGGAAGACGGTGAACAACGATACCCTGA CCATCCGTGAAGGCGATGCACTCCTGCAGGGAGGCTCTCTCACCGGTAACGGCAGCGTGGAAAAATCAGGAAGT GGCACACTCACTGTCAGCAACACCACACTCACCCAGAAAGCCGTCAACCTGAATGAAGGCACGCTGACGCTGAAC GACAGTACCGTCACCACGGATGTCATTGCTCAGCGCGGTACAGCCCTGAAGCTGACCGGCAGCACTGTGCTGAAC GGTGCCATTGACCCCACGAATGTCACTCTCGCCTCCGGTGCCACCTGGAATATCCCCGATAACGCCACGGTGCAGT CGGTGGTGGATGACCTCAGCCATGCCGGACAGATTCATTTCACCTCCACCCGCACAGGGAAGTTCGTACCGGCAA CCCTGAAAGTGAAAAACCTGAACGGACAGAATGGCACCATCAGCCTGCGTGTACGCCCGGATATGGCACAGAACA ATGCTGACAGACTGGTCATTGACGGCGGCAGGGCAACCGGAAAAACCATCCTGAACCTGGTGAACGCCGGCAAC AGTGCGTCGGGGCTGGCGACCAGCGGTAAGGGTATTCAGGTGGTGGAAGCCATTAACGGTGCCACCACGGAGG AAGGGGCCTTTGTCCAGGGGAACAGGCTGCAGGCCGGTGCCTTTAACTACTCCCTCAACCGGGACAGTGATGAGA GCTGGTATCTGCGCAGTGAAAATGCTTATCGTGCAGAAGTCCCCCTGTATGCCTCCATGCTGACACAGGCAATGGA CTATGACCGGATTGTGGCAGGCTCCCGCAGCCATCAGACCGGTGTAAATGGTGAAAACAACAGCGTCCGTCTCAG CATTCAGGGCGGTCATCTCGGTCACGATAACAATGGCGGTATTGCCCGTGGGGCCACGCCGGAAAGCAGCGGCA

3 GCTATGGATTCGTCCGTCTGGAGGGTGACCTGATGAGAACAGAGGTTGCCGGTATGTCTGTGACCGCGGGGGTAT ATGGTGCTGCTGGCCATTCTTCCGTTGATGTTAAGGATGATGACGGCTCCCGTGCCGGCACGGTCCGGGATGATG CCGGCAGCCTGGGCGGATACCTGAATCTGGTACACACGTCCTCCGGCCTGTGGGCTGACATTGTGGCACAGGGAA CCCGCCACAGCATGAAAGCGTCATCGGACAATAACGACTTCCGCGCCCGGGGCTGGGGCTGGCTGGGCTCACTG GAAACCGGTCTGCCCTTCAGTATCACTGACAACCTGATGCTGGAGCCACAACTGCAGTATACCTGGCAGGGACTTT CCCTGGATGACGGTAAGGACAACGCCGGTTATGTGAAGTTCGGGCATGGCAGTGCACAACATGTGCGTGCCGGTT TCCGTCTGGGCAGCCACAACGATATGACCTTTGGCGAAGGCACCTCATCCCGTGCCCCCCTGCGTGACAGTGCAAA ACACAGTGTGAGTGAATTACCGGTGAACTGGTGGGTACAGCCTTCTGTTATCCGCACCTTCAGCTCCCGGGGAGAT ATGCGTGTGGGGACTTCCACTGCAGGCAGCGGGATGACGTTCTCTCCCTCACAGAATGGCACATCACTGGACCTG CAGGCCGGACTGGAAGCCCGTGTCCGGGAAAATATCACCCTGGGCGTTCAGGCCGGTTATGCCCACAGCGTCAGC GGCAGCAGCGCTGAAGGGTATAACGGTCAGGCCACACTGAATGTGACCTTCTGA Downstream: CAGAACCATCGCCTCTCTGTGGTCCCGGTCATCATGACCGGGACCCGGACCGGCGCAACGGATCTTCAACGCCAC ATTCGCTGGCATTAACAATAACATGATATTCATCACGGAGTGACTATGTTACAGATAGTCGGCGCGCTGATCCTGC TGATCGCAGGATTTGCCATTCTTCGCCTTTTGTTCAGAGCATTAATCAGCACGGCTTCTGCGCTGGCAGGGCTCATA TTGCTGTGTCTGTTCGGCCCGGCCTTACTGGCTGGCTATATCACCGAACGCATAACCCGGTTGTTCCATATTCGCTG GCTGGCAGGCGTATTTCTGACGATTGCCGGAATGATCATCAGCTTCATGTGGGGACTTGATGGTAAACATATCGC GCTGGAGGCTCACACCTTTGACTCTGTGAAATTTATTCTGACCACCGCTCTCGCCGGTGGTCTGCTGGCTGTTCCCC TGCAGATCAAAAACATTCAGCAGAACGGGATCACACCAGAAGATATCAGCAAGGAAATTAACGGGTATTACTGCT GTTTTTATACTGCCTTTTTCCTTATGGCGTGTTCTGCATGCGCACCATTGATCGCGTTACAGTACGATATTTCACCGT CACTGATGTGGTGGGGCGGGTTGTTGTACTGGCTGGCTGCATTAGTGACGCTGCTATGGGCGGCCAGCCAGATCC AGGCGCTGAAAAAACTGACCTGTGCCATCAGCCAGACACTGGAAGAACAACCGGTGCTCAACAGTAAATCGTGGC TGACCAGTTTGCAAAACGATTACAGCCTTCCTGACTCACTGACGGAGCGCATCTGGCTGACGCTCATTTCTCAACG GATTTCCCGGGGAGAGCTGAGGGAATTTGAACTGGCAGACGGAAACTGGTTACTGAACAATGCCTGGTATGAAA GAAACATGGCAGGGTTTAACGAACAGTTGAAA Primer sequence analogous with the Kjargaard et. al. paper Slightly different primer sequence to lower Tm Our Biobrick Primers To design our primers we added on our cutsites to the same complementary regions as reported in the paper The part will be cut if digested by PstI. This means you will only be able to insert a part in front. We only want to insert the promoter infront of this part so it is okay. We arrive upon the following primer sequences: 5 3 UPSTREAM OF THE CODE Forward flu primer

4 ATTAgaattcatatctagaCCTTTGTCAGTAACATGC Reverse flu primer 5- to 3 CCTTACTAGTCGTTGAAGATCCGTTG CGTTGAAGATCCGTTG AT rich region to lower Tm EcoRI cutsite XbaI cutsite SpeI cutsite Region analogous to E. coli K12 chromosome for original designed primers NOTE: we have modified the biobrick suffix inbetween the EcoRI and XbaI cutsites. We had to cut some of the base pairs in between the cut sites to lower the Tm of the primer. Melting Temp, Length and Product length The Tm s were calculated according to the finnenzyme website ( with a normal 50mM salt concentration as suggested Primer melting temps must be less than 68C otherwise it is too close to the temperature DNA polymerase replicates at (72C) The Tm was calculated for the parts only homologous to the E. coli K12 chromosome for the first 5 cycles and then the Tm for the entire primer with the biobrick cutsites for the last 25 cycles Forward primer homologous to E. coli K12 chromosome Tm=54.4 C Length=18 Reverse primer homologous to E. coli K12 chromosome Tm=56.4 C Length=16 Forward primer with added biobrick cutsites Tm=68.4 C

5 Length=37 Reverse primer with added biobrick cutsites Tm=66.7 C Length=26 NOTE: 3C may have to be added to the Tm because the primers are over 20nt long Blast Sequence The sequence was blastn against the entire E. coli K12 chromosome to see if there would be any unwanted amplification Forward Primer Score = 35.6 bits (38), Expect = Identities = 19/19 (100%), Gaps = 0/19 (0%) Query ACCTTTGTCAGTAACATGC Sbjct 19 ACCTTTGTCAGTAACATGC 37 Score = 24.7 bits (26), Expect = 2.8 Identities = 13/13 (100%), Gaps = 0/13 (0%) Query TACTGACAAAGGT Sbjct 31 TACTGACAAAGGT 19 Score = 24.7 bits (26), Expect = 2.8 Identities = 15/16 (93%), Gaps = 0/16 (0%) Query GCATTTTACTGACAAA Sbjct 37 GCATGTTACTGACAAA 22 Score = 24.7 bits (26), Expect = 2.8 Identities = 13/13 (100%), Gaps = 0/13 (0%) Query GTCAGTAACATGC Sbjct 25 GTCAGTAACATGC 37

6 Query GATATGAATTCT Sbjct 15 GATATGAATTCT 4 Query AATTCATATCTA Sbjct 6 AATTCATATCTA 17 Query TGACAAAGGTCT Sbjct 28 TGACAAAGGTCT 17 Query TGACAAAGGTCT Sbjct 28 TGACAAAGGTCT 17 Query CTGACAAAGGTC Sbjct 29 CTGACAAAGGTC 18 Query CTTTGTCAGTAA Sbjct 21 CTTTGTCAGTAA 32

7 Query TTACTGACAAAG Sbjct 32 TTACTGACAAAG 21 Query TGTCAGTAACAT Sbjct 24 TGTCAGTAACAT 35 Reverse Primer Score = 30.1 bits (32), Expect = Identities = 16/16 (100%), Gaps = 0/16 (0%) Query CAACGGATCTTCAACG Sbjct 26 CAACGGATCTTCAACG 11 Score = 24.7 bits (26), Expect = 1.3 Identities = 13/13 (100%), Gaps = 0/13 (0%) Query TCTTCAACGACTA Sbjct 19 TCTTCAACGACTA 7 Identities = 17/19 (89%), Gaps = 1/19 (5%) Query CTGACTA-TCGTTGAAGAT Sbjct 2 CTTACTAGTCGTTGAAGAT 20 Query TAGTCGTTGAAG Sbjct 7 TAGTCGTTGAAG 18

8 Query TCTTCAACGACT Sbjct 19 TCTTCAACGACT 8 Identities = 15/17 (88%), Gaps = 0/17 (0%) Query AACGGATCTTCTACCAC Sbjct 25 AACGGATCTTCAACGAC 9 Identities = 14/15 (93%), Gaps = 0/15 (0%) Query CGGTTCTTCAACGAC Sbjct 23 CGGATCTTCAACGAC 9 Query TCGTTGAAGATC Sbjct 10 TCGTTGAAGATC 21 Query TCGTTGAAGATC Sbjct 10 TCGTTGAAGATC 21 Query GATCTTCAACGA Sbjct 21 GATCTTCAACGA 10 Query TGAAGATCCGTT

9 Sbjct 14 TGAAGATCCGTT 25 Query AACGGATCTTCA Sbjct 25 AACGGATCTTCA 14 Primer Dimers/Hairpins checked using IDT (Checked using IDT) Primer Hairpins Forward None Reverse None Primer self dimmers Forward Delta G kcal/mole Base Pairs 6 :: :: Delta G kcal/mole Base Pairs 6 : : : : : : Delta G kcal/mole Delta G kcal/mole : : ::::

10 Delta G kcal/mole : : : : : : Delta G kcal/mole : : : : ::: Delta G kcal/mole ::: Delta G -3.3 kcal/mole : : ::: Delta G kcal/mole Base Pairs 2 Delta G kcal/mole : ::: : Delta G kcal/mole : : ::: Reverse Delta G kcal/mole Base Pairs 6 3' GTTGCCTAGAAGTTGCTGATCATTCC Delta G kcal/mole

11 3' GTTGCCTAGAAGTTGCTGATCATTCC Delta G kcal/mole Base Pairs 2 : : : : 3' GTTGCCTAGAAGTTGCTGATCATTCC Delta G kcal/mole Base Pairs 2 :: 3' GTTGCCTAGAAGTTGCTGATCATTCC Delta G kcal/mole Base Pairs 2 3' GTTGCCTAGAAGTTGCTGATCATTCC Delta G kcal/mole : : ::: 3' GTTGCCTAGAAGTTGCTGATCATTCC Primer hetero dimmers Delta G kcal/mole Base Pairs 5 : : : Delta G kcal/mole : Delta G kcal/mole : : : Delta G kcal/mole : : :

12 Delta G kcal/mole : : :: Delta G kcal/mole : :: : : Delta G kcal/mole :: Delta G kcal/mole Delta G kcal/mole : : : Delta G kcal/mole : : Results This PCR will give us a product length of 3392 bp. We plan on doing the PCR with the Phusion polymerase (not hot start because the annealing temperature of just the portion homologous to the E. coli chromosome is less than 60 C)

Design. Construction. Characterization

Design. Construction. Characterization Design Construction Characterization DNA mrna (messenger) A C C transcription translation C A C protein His A T G C T A C G Plasmids replicon copy number incompatibility selection marker origin of replication