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Genspace Notebook

July 5

- Grew out BBa_k325219 and BBa_k325909 in media C.

- Restriction digest of BBa_k231000 and BBa_k231001 with EcoRI and PstI

July 15

- CDS7 isolation and ligation

July 19

- NcoI and BamHI digest of pET Vector

July 20

- Another attempt at NcoI and BamHI digest of pET Vector

July 21

- Gel Purified pET Vector Digest

- Grew out more pET vector

July 24

- Received new NcoI and BamHI enzymes

- pET Vector digest with new NcoI and new BamHI

- pET Vector digest with old NcoI and old BamHI

- pET Vector digest with new NcoI and another with old NcoI

- pET Vector digest with BamHI and anther with old BamHI

July 25

- Ran a gel with all pET Vector digests from July 24th

- Fermentas transformation on pET Vector

July 28

- CDS7 digest with NcoI and BamHI

- Prepared plates of kanamycin and pET Vector

- Oligo annealed with Silver Lab protocol

- Maxiprep using ~50mL CDS7 growth

- pET Vector inoculation

July 29

- pET Vector digest with BamHI and NcoI

- Gel purification of pET Vector digest

- Inoculated more pET Vector

- Inoculated C-media for transformations

- Miniprep of pET Vector

- Ligated 4 oligos

- Prepare transformation ready cells using C-media culture prepared the previous day

July 30

- Miniprep of pET Vector

- Digestion of pET Vector with NcoI and BamHI

- Oligo PCR

-Digest pUC19 with EcoRI and PstI

August 1

- Ran gels to verify pET and PUC double digests

- Restriction digest of annealed oligo sequences with EcoRI and PstI

- Oligo PCR verification

- Digested pSB1C3 with EcoRI and PstI

August 2

- pUC19 and pET Vector were isolated and gel purified

- Miniprep of pUC19, pET Vector, and pSB1C3

August 3

- Ligated pUC19 and all the oligos

August 5

- PCR with new CDS7 was successful. Sung has “never seen a brighter 70 bp lane in [his] life”

- pUC19 – quantum dot synthesis oligos worked

- Miniprep, digest with EcoRI and PstI, and gel run of CDS7

August 6

- Gel extraction of pUC19 and pET Vector compatible CDS7 amplicon

August 7

- Inoculated oligo ligation cells in ampicillin media

- Digested and ran more of oligo ligations

- Inoculated more of the ligations

- Inoculated 6 CDS7 colonies

August 8

- Optimized PCR

August 22

- Oligo annealed

- Ethanol precipitation of DNA

August 23

- Precipitation of CDS7 and restriction digest with EcoRI and PstI

- Oligo annealed

August 24

- Dephosphorylated backbone and created ligation mixtures

- Annealed biobrick part

- Made master mix for biobrick backbone

- Oligo annealed

- Digested pUC19 with EcoR1 and PstI

August 30

- Attempted ligation of biobrick

August 31

- Tested previous day’s ligation in a gel and it failed

September 1

- pUC19 digest with EcoRI and PstI

- Gel purification of pUC19 digest

- Alcohol precipitated CDS7

September 2

- Received new primers

- Ran three PCR reactions, CDS7 forward with RBS, reverse, and control

- Ran 1/100 dilution of gel

September 7

- Another CDS7 PCR

September 8

- Restriction digest of J140 with EcoRI and PstI

September 9

- Ran gel of pET Vector, j140, pSB1C3, CDS7, and CDS7S

September 13

- Restriction digest of master mix with EcoR1 and Pst1

- Made a ligation mix

- Transformed with ligation mix

September 14

- pET Vector digest with Nco1 and BamH1


- CDS7 digest with EcoRI and PstI

- CDS7 digest with XbaI and PstI

- Gel verification and purification of pET Vector and CDS7

- Ligation of CDS7 EcoRI and PstI cut with pSB1C3

September 20

- VF and VR PCR verification

- Restriction digest on pSB1C3 with Pst1 and EcoR1

- Gel verification of digest

September 21

- Verified base pair numbers of primers and verified they work

- PCR of CDS7

- Gel purification of CDS7

- Digest CDS7 with EcoR1 and Pst1

- Phosphotased pSB1C3

- Made ligation mix with all the parts

- Transformed with the ligation mix

September 23

- Ligation mix from previous day failed

- Re-did everything from September 21st except this time made a new master mix with different PCR settings

- Ligated and transformed with the new master mix

September 24

- Grew colonies overnight from previous days ligation and transformation

- Grew out colonies with LB chloramphenicol

- Made minipreps of CDS7

September 26

- PCR a sequencing segment from plasmid with VF2 and VR

- Sent segment off for sequencing

- Reviewed other transformations

September 28

We characterized the ability of the CD7 peptide to nucleate the formation of QDs in E. coli by incubating the bacteria with CdCl and sodium sulfide. Although the E. coli treated in this manner showed visible fluorescence, the intensity did not appear to change in the presence of the inducer IPTG, nor was the brightness greater than that of E. coli carrying the expression vector with no peptide insert. The possible explanations for this are:

  1. The peptide does not nucleate QDs under the conditions described in the literature (the literature is in error)
  2. We deviated in some unknown way from the protocol in the literature.
  3. The QDs might not emit at the wavelengths that the microscope was able to recognize due to its set of filters. We did not have access to a fluorimeter.
    1. But we were amazed that after 3 hours of cadmium treatment the bacteria appeared alive and well!