On this page we have documented our weekly wet lab progress during our project. The purpose of this Notebook is to outline which experiments were done when, and also to present some of the preliminary results. To see the final results in detail, please visit the Results Biology page.

To learn more about our engineering approach, go to the Engineering page. To view our full timeline please refer to the following PDF.

August 7 - 13

• Made sure everything we need is in the lab
• Made media, autoclaved tips, and other materials to prepare for experiments

August 14 - 20

• We completed the interlabs calibration protocol and prepared experiment 2 of interlabs
• Obtained all designed and ordered plasmids/parts

August 21 - 27

• Finished experiment 2 of interlabs and sent the results
• Finished cloning strategy for fluorescent biosensor plasmid
• Made & tested chemically competent E. coli DH5-alpha cells
• Mini-prepped the luciferase plasmid + promoter
• PCR of YhjH front and back, AGE backbones, CUG_Fleq, CUG_promoter, Luciferase cassette

August 28 - September 3

• PCR & Cloning luciferase plasmid (2x) + promoter
• Analyzed results of last week’s PCR’s on agarose gel
• First attempt of Gibson assembly of the CUG promoter in an empty plasmid
• Second attempt at previously failed Luciferase cassette PCR
• Band purification using the Thermo Scientific GeneJET PCR purification kit
• Made more competent cells
• Colony PCR CUG promoter
• Gibson assembly of the c-di-GMP hydrolase
• Received a dilution streak of a TG1 glycerol stock
• PCR_phusion of the backbones
• Tested competency of both DH5-alpha and TG1 cells
• Digestion of backbones
• PCR cleanups
• Colony PCR

September 4 - 10

• PCR + Cloning (Gibson Assembly) of hydrolase plasmid + promoter
• Sent promoter for sequencing
• Hydrolase plasmid: Transform into end-point cells (TG1)
• Colony PCR of promoter and hydrolase
• Send hydrolase and CUG promoters for sequencing
• Design new primers for golden gate for CUG promoter that didn’t work with Gibson Assembly
• Perform standard assembly to bind the promoter (constitutive) to the GFP and RFP Redigested GFP E+S and ligation
• Biobrick inoculation

September 11 - 17

• Transformation of the GFP and RFP construct + colony PCR
• PCR the luciferase cassette
• Sent the RFP and GFP constructs for sequencing
• Transformed AGE bricks and UspB in E. coli DH5-alpha
• Analyze sequencing results
• Perform Golden Gate for CUG promoter
• Electroporation for dispersin B
• Mini-prepped AGE biobricks
• Restriction digest GFP

September 18 - 24

• Mini-prepped Dispersin B transformation and sent for sequencing
• Golden gate assemblies of CUG promotor (pSB1A3_CUG) didn’t work
• Sent results for sequencing and analyzed them
• Made new overnights and mini-prepped them
• Transformation and colony PCR CG_CUG 1:5 and 1:10 ratios, were unsuccessful

September 25 - October 1

• Restriction digests and ligation, GFP and RFP+C7 were successful
• Ligation of psBIC3_GFP_Pce+RFP transformation in E. coli DH5-a
• Discussed with supervisors why assembly of CUG promoter didn’t work. Need to redesign plasmid maps. Will try to build a construct with a different inducible promoter due to time constraints.
• Made electrocompetent E. coli DH5-a cells
• Isolated GFP to measure with the electronic device, the device was able to detect the isolated GFP!
• Set overnight cultures of more biobricks and mini-prepped them
• PCR of new biobricks
• Digests, ligation and transformation to obtain new constructs

October 2 - 8

• Sent GFP+C7 samples for sequencing
• Analyzed sequencing results of GPR
• Q5 PCR BBa_K180000+DprI and PCR clean-up
• Digested BBa_B1006 with E+X, Digested BBa_E0040 with E+S and ligation (pSBIC3_GFP+Term)
• Colony PCR of ligation 02-10 (pSBIC3_GFP+Term)
• Mini-prepped YhjH overnight cultures, performed restriction digest and analyzed results on agarose gel, obtained correct construct
• Sent GFP + Term DNA samples for sequencing
• Digestion of several constructs, ligation of correct construct fragments and transformation in E. coli DH5-a
• Fragments used for ligations:
  • 1. Ptac + BBa_B0034 > K1800000
  • 2. Linear Ptac + pSBIC3_GFP_Pte+RFP > pSBIC3_Ptac+GFP_Pce+RFP
  • 3. CUG-FleQ + pSBIC3_GFP+Term > pSBIC3_CUG-FleQ_GFP+Term
  • 4. pSBIC3_GFP+Term + pSBIA3_RFPdevice > pSBIA3_GFP+Term_RFPdevice
  • 5. FleQ + CUG + pSBIK3 > pSBIK3_CUG
• Colony PCR for colonies of transformation with possible constructs 1, 2, 3, 4 & 5.
• Colony PCR results were ambiguous due to malfunctioning of agarose gel electrophoresis
• Made overnights of colony material or new colonies from all material containing possible constructs
• Mini-prepped overnights and performed digestion to analyze identity of constructs. Sent promising looking results for sequencing.
• Performed ligations to obtain possible new constructs and transformed them in E. coli DH5-alpha
• Fragments used for ligations:
  • A: pSBIK3_Ptac+RBS + pSBIC3_GFP+Term* > pSBIC3_Ptac+RBS+GFP+Term
  • B: Ptac (K180000) + pSB3T5 (E8) > pSB3T5_Ptac
  • C: pSBIK3_CUG + pSBIC3_GFP+Term* > pSBIC3_CUG_GFP+Term
• Ligations were done with multiple samples of a fragment to have a higher chance of getting the desired results.
• The c-di-GMP sensor was not obtained. Ptac+GFP+Pcl+RFP was not obtained (RFP was likely removed by recombination, because of the repeating sequence of terminators). Pcl+Ppel+GFP was obtained, but not pure (dilution streak with colony screening required).