Stanford/BIOE44:Module 5:Day1

M5: Day 1

Getting ready for parts to arrive

Parts Request

YOU MUST MAKE YOUR FINAL PARTS REQUESTS ASAP Please fill in this google doc with the information on the parts you need. In case there is overlap on the parts different groups need, there is a column for "other groups that need this part." Please fill this column in so we know how much of each part we need to make. Even if you are using a part that you think we already have, you should make a formal request.

RBS Design

A nice discussion of | RBS design can be found here.

Primer Design Guidelines

• Homology region should be about 20bp.
• Total length should be less than 100bp (it is not possible to order one longer than 100bp). Longer primers take longer to be made, so make your primers as short as possible.
• If you cannot make your promoter+rbs+cds less than or equal to 100bp, then you need to plan on doing a double ligation.
• You should include a spacer between your RBS and the beginning of your coding sequence. It should be 6-12bp (and in frame). The scar sequence is convenient for this purpose.
• It is also probably a good idea to put a spacer sequence between the promoter and RBS.
• You don't need to include the entire prefix or suffix if you are planning on cutting at one of the inner sites (XbaI or SpeI)

Example Primer design

• Blue = Biobrick prefix or suffix
• Pink = Ribosome binding site
• Green = gene sequence (aka homology region)
• Yellow = Promoter

Adding a ribosome binding site

Forward primer: 5'-ATAGAATTCGCGGCCGCTTCTAGAGAAAGAGGAGAAATACTAGATGGACTATATTGTTGGTTTCG-3'

Reverse primer: 5'-ATACTGCAGCGGCCGCTACTAGTATCACCTCACTTTGGGGATAAC-3'

Adding a promoter and a ribosome binding site

Forward primer: 5'-ATAGAATTCGCGGCCGCTTCTAGAGCCAATGCTGGGAACGGCCAGGGCACCTAATACTAGAAAGAGGAGAAATACTAGATGGACTATATTGTTGGTTTCG-3'

Reverse primer: 5'-ATACTGCAGCGGCCGCTACTAGTATCACCTCACTTTGGGGATAAC-3'

Primer Designs

Chris & Karina:

RBS + merR Forward Primer:5'- atagaattcgcggccgcttctag TCTAGAGATTAAAGAGGAGAAATACTAG ATGGTGATGAACTCCTGGGCC -3'

merR Reverse Primer: 5'- ctgcagcggccgctactagtaTTACTAGACCCTGGAGCGTG -3'

Daniel & Aaditya:

1) fnrNatural forward primer:

5'-

GAATTC GCGGCCGC T TCTAGA

GAG CAGACCT

ATGATCCCGGAAAAGCGAATTATACGGC

- 3'

2) fnrSynthetic forward primer

5' -

GAATTC GCGGCCGC T TCTAGA

AAAGAGGAGAAA

TACTAG

ATGATCCCGGAAAAGCGAATTATACGGC

- 3'

3) Generic fnr reverse primer

5' -

CTGCAGCGGCCGCTACTAGTATTATTAG

- 3'

Scott & Travis: Coding region w/ promoter J23106 (medium) and biobrick parts,

With rbs b0030 (stronger) Forward: 5'-TAGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGC ATTAAAGAGGAGAAATACTAGATGAGTAATCAGGAACCGGC-3’

With rbs b0033 (weaker) Forward: 5'-TAGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGC TCACACAGGACTACTAGATGAGTAATCAGGAACCGGC-3’

Reverse: 5'-ATACTGCAGCGGCCGCTACTAGTATTAGAAAATGCGCTCCTGATGC-3’

Frank, Claire, Sanjay (FCS):

(1) arsR forward primer (64 bp):

5' GTTTCTTC GAATTCGCGGCCGCTTCTAGAG TTACGTTGCATTGTGGGTCGTATCAGCGAGCTTG 3'

(2) B0030 - arsR reverse primer (53 bp):

5' GTTTCTTC TACTAGTA attaaagaggagaaa tactag ATGCGGGCACAGCGGC 3'

(3) J23101 - B0030 - arsR reverse primer (96 bp):

5' GTTTCTTC TACTAGTA tttacagctagctcagtcctaggtattatgctagc ctctagta attaaagaggagaaa tactag ATGCGGGCACAGCGGC 3'

(4) J23101 - B0034 - glnK forward primer (99 bp):

5' ATA TTCTAGAG tttacagctagctcagtcctaggtattatgctagc tactaga aaagaggagaaa tactag ATGAAGCTAGTCACAGCCATCATCAAGC 3'

(5) glnK reverse primer (96 bp):

5' GTTTCTTC CTGCAGCGGCCGCTACTAGTA TTAGATCGCGTCGGTATCGGTTTCGCC 3'

Sunny & Alex:

1) Part 5:

Forward Primer (Prefix + RBS + todS)

5'-

ATA

GAATTCGCGGCCGCTTCTAGAG

AAAGAGGAGAAA

TACTAG

ATGAGCTCCTTGGATAGAAAAAAGCCTCAAAATAG

-3' (78bp)

Reverse primer (Suffix + todS):

5'-

ATA

CTGCAGCGGCCGCTACTAGTA

ATACACGGCCTCGGGACAGACCGTCCCTATGAAT

-3' (58bp)

2) Part 6

Forward Primer (Prefix + RBS + todT):

5'-

ATA

GAATTCGCGGCCGCTTCTAGAG

AAAGAGGAGAAA

TACTAG

ATGCCCGCCCGCTGGGGGTGCTTGTTTCCTGGTAA

-3' (78bp)

Reverse primer (Suffix + todT):

5'-

ATA

CTGCAGCGGCCGCTACTAGTA

AATAAGGTCCGATAGGAACTCGGCGAGTCAATGAG

-3' (59bp)

3) Part 3

Forward Primer (Prefix + Promoter + RBS + todS):

5'-

ATA

TCTAGAG

TTTACAGCTAGCTCAGTCCTAGGGACTGTGCTAGC

TACTAG

AAAGAGGAGAAA

TACTAG

ATGAGCTCCTTGGATAGAAAAAAGCCTCAA

-3' (99bp)

Reverse primer (Suffix + todS):

5'-

ATA

CTGCAGCGGCCGCTACTAGTA

ATACACGGCCTCGGGACAGACCGTCCCTATGAAT

-3' (58bp)

4) Part 4

Forward Primer (Prefix + Promoter + RBS + CG (BBa_K274002)):

5'- ATA

TCTAGAG

TTTACAGCTAGCTCAGTCCTAGGGACTGTGCTAGC

TACTAG

TTAAGGAGGTAAAAAAA

ATGAAACATTCTTCCGATATCTGCATTGTTG

-3' (99bp)

Reverse primer (Suffix + CG):

5'-

ATA

CTGCAGCGGCCGCTACTAGTA

AATCGCGAACCGGCGCTTTTGGCGCAGGCCCTAG

-3' (58bp)

I'm Moss-ay

To do list for Tuesday (5/18):

• Finish the Moss Datasheet. Remember we're trying to make a sheet that someone could look at and understand why moss is a useful organism, what you would use it for and how. It needs to include more than what we've done in the class.
• Make BCD liquid media with mannitol.
• Look at our plates.

pSB4A2 troubleshooting