Cloning strategy

July

06.07.09

Four forward primers were designed to amplify:
1.Promoter T7, RBS, CBP and LOVTAP:

gtttcttcgaattcgcggccgcttctagagtaatacgactcactataggggaattgtg

2.RBS, CBP and LOVTAP:

gtttcttcgaattcgcggccgcttctagagtgtttaactttaagaaggag

3.CBP and LOVTAP:

gtttcttcgaattcgcggccgcttctagatgaagcgacgatggaaaaagaatttcatag

4.LOVTAP:

gtttcttcgaattcgcggccgcttctagatgctactacacttgaacgtattgagaagaac

One reverse primer were designed:

gtttcttcctgcagcggccgctactagtatcaatcgcttttcagcaacacctcttc

The recipient IGEM part have been chosen: [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_B0010 BBa_B0010], well 13D in the received kit plate 1

07.07.09

To design plasmids : software Vector NTI

08.07.09

Inducible LOVTAP biobrick strategy

09.07.09

Partial digestion strategy.

10.07.09

13.07.09

Restriction enzymes on [http://www.neb.com/nebecomm/products/category1.asp?#2 Biolabs website] and [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp clevage oligonucleotides]

TRP promoter biobrick strategy

14.07.09

Primers designed for LOVTAP read-out and RBphP project:

1.Forward primer Trp promoter:

gtttcttc gaattcgcggccgcttctagagtggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaactagtacgc

2.Reverse primer Trp promoter:

ctagctagctaggtcgataccctttttacgtgaacttgcgtactagttaactagttcgatgattaattgtca

3.1st Forward primer Inverter TetR:

aatcatcgaactagttaactagtacgcaagttcacgtaaaaagggtatcgacaaagaggagaaatactagatgtcc

4.2nd Forward primer Inverter TetR:

gtttcttcgaattcgcggccgcttctagagtggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaactagta

5.Reverse Primer Inverter TetR:

ctagctagctag tttctcctctttctctagtagtgc

6.Forward primer ppsR1 R.Palustris CGA009:

gtttcttc*gaattcgcggccgcttctag*atgctggaggatatttgccctggtg

7.Reverse primer ppsR1 R.Palustris CGA009:

gtttcttc*ctgcagcggccgctactagta*ttactcatcggctccgtctccttc

8.Forward primer ppsR2 R.Palustris CGA009:

gtttcttc*gaattcgcggccgcttctag*atggcgtcaaagtccgttcatgcc

9.Reverse primer ppsR2 R.Palustris CGA009:

gtttcttc*ctgcagcggccgctactagta*tcaatcctctgcgtcgtctgagg

10.Forward primer BrBphP Bradyrhizobium ORS278:

gtttcttc*gaattcgcggccgcttctag*atgcccgttccgctgacgac

11.Reverse primer BrBphP Bradyrhizobium ORS278:

gtttcttc*ctgcagcggccgctactagta*tcactcctcgctctgcgagc

12.Forward primer ppsR1 Bradyrhizobium ORS278:

gtttcttc*gaattcgcggccgcttctag*atgagggcgttcagagctcc

13.Reverse primer ppsR1 Bradyrhizobium ORS278:

gtttcttc*ctgcagcggccgctactagta*ctattccaactgactgtcttcttcgc

14.Forward primer ppsR2 Bradyrhizobium ORS278:

gtttcttc*gaattcgcggccgcttctag*atggccgagtttcacggtccac

15.Reverse primer ppsR2 Bradyrhizobium ORS278:

gtttcttc*ctgcagcggccgctactagta*ctagctccccttttcggtttcctc

15.07.09

16.07.09

17.07.09

August