From 2013.igem.org

Breakfast

• socializing =)

Catching Up on Lab Work (Hanna)

• news on the lab work
• present protocol

Gibson Cloning Primer Design (Nils)

presentation:

• Backbone pSB1C3, lacI, RBS, mRFP1, Cm-R, pMB! ori
• finding data for introduction of RBS-DelE (PacI and KpnI)
  • design of four primers
    • 2 for backbone
    • 2 for insert
  • go to parts registry, search pSB1C3
    • shows sequence and features --> get selected sequence
  • look at sequence in Serial Cloner
    • graphic map should show sequence and particular RE sites (bold names are unique sites, italic names are not existing sites)
    • look for cutting site that is not present in backbone nor insert/cassette
  • find cassette BBa_503495 sequence in parts registry (not standardized, not best example)
  • add cassette sequence to backbone sequence
    • show in graphic map
  • get DelE sequence (has a lot of PstI cutting sites, but EcoRI only a single site --> can be mutated)
  • Primer for adding RBS and cuttingssites between backbone and insert
  • If primer gets too large one can make two PCR steps
  • methyl specific cutting enzymes can digest backbone to separate the PCR amplified
  • Primer nomenclatur: Kürzel_Nummer:Protein_CuttingSite_fw/rev
  • Buffer of Restriction enzymes should be compatible
  • Forward primer protein: Overhang - cutting site e.g. PacI - RBS - 6bp (can be used to enhance specifity when taken from gDNA) - atg-Start of Protein - additional 22-27 bp for specificity (or smaller for big primers) - last one should be G/C, no repetitive G/C if possible
  • Reverse primer protein: Overhang - cutting site e.g. KpnI (reverse complementary in case of non palindromic enzymes) - reverse complementary end of protein
  • Primers should be same melting temperature / length
  • Watch out for first and second melting temperature and other reactions run in parallel!
  • Forward primer backbone: Overhang - cutting site e.g. KpnI - backbone specific sequence starting with RBS
  • Reverse primer backbone: Overhang - cutting site e.g. PacI - reverse complementary sequence of backbone until RBS
• Fold DNA on mfold:
  • 3' end should be accessible
  • 5' end can be varied (overhang)
  • wobble base pairs can be replaced
  • change length of primer to reduce folding
  • target would be a folding free energy > -2
• Run in silico PCR in serial cloner
  • target melting temperature between 60 and 70°C (if possible - not a critical parameter)
  • if you make mutagenesis the sequence is not complementary anymore!
  • Evaluate PCR chacks for complementary parts and gives product length
• Annotation
  • Features of sequence in serial cloner
  • in gene bank files

Ribosomal binding sites (Konrad)

• Current Plan:
  • add medium RBS for every part
• RBS in Del Cluster
  • RBS from Data base in file (Mail Konrad)
  • We can see the direction of transcription according to the primers

Use of J5 for Gibson assembly (Nikos)

• Parts in device editor all at once
  • Plasmid backbone
  • RBS
  • Several parts for Insert - every mutation is a single bp part
• Define how tot obtain the part
  • most of the PCR amplified
  • other option would be digestion
  • RBS would be embedded in a primer
  • Choose none for very small parts since it will choose lowest cost strategy
• Controls
  • Choose parameters
  • Choose Assembly meths
• Result is Vector Editor
  • Can show restriction sites
  • Primer sequences in csv file
  • Gives you cloning strategy

Agenda for next meeting (13.05.2013 18:00)

• One point per sub group
  • NRPS
  • Software
  • Del
  • Indi
• Define agenda for 15.5.

Group picture