Week Nine - 12 August to 18 August


Tae and Sarah transformed the ligated RBS and constitutive promoter.


Mike and Grant PCR’d out the Pgad promoter from the CUHK team’s plasmid they sent us
Yuan digested following the usual protcol
citrobacter phytase (red and white) X/P
citrobacter phytase (red and white) controls

Lajja and Sarah ran a gel on the digested products Yuan ran this morning along with the PCR Pgad products

Lajja and Sarah made more kanamycin and chloramphenicol plates

Tae and Grant performed a digestion following the usual protocol


Yuan digested EC phy, EC clc, and the lysis part (miniprepped from the transformation) to prep for an analytical gel.

EC phy

  • digested with X/P
  • 500 ng needed / concentration of 102.8 ng/uL = 4.86 uL miniprep used
  • 37.6 uL H2O used

EC clc

  • digested with X/P
  • 500 ng needed / concentration of 76.8 ng/uL = 6.51 uL miniprep used
  • 36.0 uL H2O used


  • digested with X/P
  • 500 ng needed / concentration of 83.5 ng/uL = 5.98 uL miniprep used
  • 36.5 uL H2O used

Yuan also digested a kanamyacin backbone in preparation for ligation of the digested citrobacter phytase into a backbone (again).

kan bb

  • digested with E/P
  • 500 ng needed / concentration of 24.3 ng/uL = 20.58 uL pcr purified used
  • 21.9 uL H2O used

Ligation with kan bb and citrobacter phy (both digested). This ligation is the first time we used the Open Wetware ligation protocol.

  • 3.87 uL H2O used
  • Insert (citrobacter phy) mass: 37.74 ng = 3.63 uL of citrobacter DNA
  • Vector (kanamyacin) mass: 10 ng = 1.0 uL of kanamyacin backbone
  • 1.0 uL T4 ligase buffer used
  • 0.5 uL T4 ligase used

Yuan ran the gel backwards.

Yuan started another colony PCR for the citrobacter phytase considering we’re almost out of digest.

Sarah and Tae digested the constitutive promoters for assembly with the RBS (cut with E and X)
Sarah and Tae digested the Pgad promoter (both the lower ligation and higher ligation temperature PCR products) for assembly with the RBS
stronger c.p. E/S
strong c.p. E/S
Pgad(55 degrees) E/S
Pgad(60 degrees) E/S

Grant made a gel and the digested products (Pgad and c.p.) were run to make sure the digest worked

The Pbad and constitutive promoters were ligated with the strong and stronger RBS’s
Brian transformed the ligated Pbad and Constitutive promoter constructs


Tae and Lajja digested the newly PCR’d citrobacter phytase with DpnI by using 1uL of DpnI and 50uL of sample. Later, they PCR purified the sample of citrobacter phytase using PCR protocol online. Yuan is planning to run both the old (digested) citrobacter phytase, the new PCR’d citrobacter phytase, and the T4 lysis part on an analytical gel.

Brian transformed promoter-less GFP and the citrobacter part ligated into a backbone yesterday.
GFP: (Plate 1 Well 14K)

We will use the promoter-less GFP and assemble it with pGAD as a reporter protein. This will allow us to tell if the clc-pGAD part works or not.

Yuan ligated the “Blue” pGAD to a kan backbone. And citro phy to a kan backbone.

pGAD digest

  • cut with E/P
  • 6.6 uL DNA used
  • 35.9 uL water used

kan bb digest

  • cut with E/P
  • 6.5 uL DNA used
  • 36.0 uL water used

citro phy digest

  • cut with E/P
  • 10.86 uL DNA used
  • 31.6 uL water used

pGAD + kan ligation

  • kan used: 1 uL
  • pGAD used: 3.41 uL
  • water used: 4.09 uL

citro + kan ligation

  • kan used: 1 uL
  • citro used: 3.54 uL
  • water used: 3.96 uL

After ligation, Yuan transformed the pGAD+kan and citro+kan ligations.

Tae and Grant ran a digest of the strong constitutive promoter, EC Phy, and Pbad parts for assembly
The strong CP and EC Phy parts will be changed from the ampicillin backbone to a kanamycin backbone
Strong constitutive promoter 256.8 (E/P) and control (E)
1.95 uL DNA
40.55 uL water

ECPhy 102.8 (E/P) and control (E)
4.86 uL DNA
37.64 uL water

Pbad 92.6 (E/S) and control (E) (not enough entirely for control)
5.40 uL DNA
37.10 uL water

Grant and Tae made and ran the gel of the digested C.P, EC Phy, and Pbad parts

Grant and Tae ligated the C.P and EC Phy into kanamycin backbones. The Pbad part was ligated to a strong RBS


The Pbad, strong and strongest constitutive promoters and RBS’s were miniprepped from last night’s overnights

The strong and strongest constitutive promoters in ampicillin backbones were digested (E/P) and religated into a kanamycin backbone

The PCR purified Pgad part(cut with E and S) was assembled with the lysis part (amp backbone cut with X and P) into a kanamycin backbone

The Pbad part was digested (E/P) and religated into a kanamycin backbone

The kanamycin backbone stock used was 8/15 cut with E and P

The ligated constitutive promoters and Pbad parts were transformed into e. coli by Tae following the usual protocol; however, the medium constitutive promoter only had 15 uL of cells placed in it due to an aliquoting error.


Grant updated the notebook and tried to fill in missing recent days=

Lajja and Yuan made more 5x TBE buffer for gels

Tae and Mike miniprepped the overnighted Pgad, citrophy in kana backbone (august 14h/15th), gfp no promoter

Grant digested the miniprepped Pgad, citrophy, and gfp without a promoter

Yuan transformed more constitutive promoters and RBS’s
weak constitutive promoter: kit plate 1 well 20I

NEWstrong constitutive promoter(digest of previous one did not work): kit plate 2 well 2I

Medium RBS: kit plate 1 well 2I

Double terminator:

Grant and Mike had a modeling meeting with Leonard

Grant and tae transformed the GFP with Pgad


Tae and Lajja miniprepped