Cloning
Testing of Plasmid
April
Gibson Assembly of Gene of Interest Plasmid
Goal:
Construction of the Gene of Interest Plasmid
Protocols Used:
Nanodrop DNA quantification , Plasmid Isolation , Phusion PCR , Restriction Digestion ,
E.coli Heat Shock TransformationKOD Extreme PCR , Colony Polymerase PCR (GoTaq) , E. coli Glycerol StockGibson Assembly ,
DNA Gel Purification ,
DNA Gel Electrophoresis
Plan:
The plasmid we had contained OriL and OriR flanking GFP. We wanted to clone a kanamycin gene inside this to use as a selection marker.
Results:
There were no plates with colonies, therefore Gibson Assembly has not work.
Gibson Assembly of Support Plasmid
Goal:
Construction of the Support Plasmid
Protocols Used:
Nanodrop DNA quantification , Plasmid Isolation , Phusion PCR , Restriction Digestion ,
E.coli Heat Shock TransformationKOD Extreme PCR , Colony Polymerase PCR (GoTaq) , E. coli Glycerol StockGibson Assembly ,
DNA Gel Purification ,
DNA Gel Electrophoresis
Plan:
We had two different plasmids. One plasmid with p5 and p6 on it and one plasmid with phi 29 DNAP and phi 29 TP. We wanted to put this on one plasmid through Gibson Assembly
Results:
There were no plates with colonies, therefore Gibson Assembly has not work.
May
June
July
Brute Force Experiments 1
Goal:
Putting all separate plasmids in and see what survives
Protocols Used:
Transformation ,
IPTG stock ,
Plan:
As construction of the plasmids did not work, we just wanted to put all the separate constructs in, to see whether something would grow or not. We induced it with no IPTG, 10 µM IPTG and 50 µM IPTG. We put in the following constructs:
p02+p04+oriL-GFP-oriR
p02+oriL-GFP-oriR
oriL-GFP-oriR
p01+p02+p04
Results:
Some plates grew, some not. No conclusive result.
August
MoClo Assembly
Toxicity Experiments 1
September
Toxicity Experiments 2
Goal:
Determinig what parts are toxic to the cell
Protocols Used:
E.coli Heat Shock TransformationOD600 Measurement ,
IPTG stock ,
Plan:
To determine which plasmids were toxic, we put transformed the plasmids separately in BL21 pLysS and set an overnight measurement to measure OD600 to see if the cells were alive.
p01 (control)
p02
p03
p04
Results:
Add link
Characterization of Level 1's
Goal:
Add characterization for the parts page and see if the different promoters strength are visible
Protocols Used:
PURE System ,
Protein Gel ,
Mass Spectrometry ,
Plan:
The following constructs were added to the PURE system and then run on a protein gel:
0.1 T7 p - U RBS - DNAP - WT
0.5 T7 p - U RBS - DNAP - WT
WT T7 p - U RBS - DNAP - WT
0.1 T7 p - U RBS - TP - WT
0.5 T7 p - U RBS - TP - WT
WT T7 p - U RBS - TP - WT
0.1 T7 p - U RBS - p5 - WT
0.5 T7 p - U RBS - p5 - WT
WT T7 p - U RBS - p5 - WT
0.1 T7 p - U RBS - p6 - WT
0.5 T7 p - U RBS - p6 - WT
WT T7 p - U RBS - p6 - WT
After this, a remainder of the sample was analyzed with Mass Spectrometry.
Results:
Add link
October
Brute Force Experiments 2
Goal:
Putting all the needed components for orthogonal replication and see which cells replicate
Protocols Used:
Transformation ,
IPTG stock ,
Plan:
To test whether orthogonal replication works in vivo, we transformed all the plasmid with the components needed in pLysS together with a final construct, which is either OriL-Kan-OriR or OriL-GFP-Kan-OriR. This was done with different IPTG concentrations.
oriL-Kan-OriR with
WT T7p TP, DNAP, p5, p6
0.5 T7p TP, DNAP, p5, p6
0.1 T7p TP, DNAP, p5, p6
oriL-GFP-Kan-OriR
WT T7p TP, DNAP, p5, p6
0.5 T7p TP, DNAP, p5, p6
0.1 T7p TP, DNAP, p5, p6
Results:
To be done
Orthogonal Replication
Some text in the Modal Body
Some other text...
Cloning
Testing of Plasmid
April
May
June
Uber Plasmid
July
August
MoClo Assembly
Goal:
Assembly all the different parts with MoClo
Protocols Used:
Nanodrop DNA quantification , Plasmid Isolation , MoClo ,
E.coli Heat Shock TransformationColony Polymerase PCR (GoTaq) , E. coli Glycerol StockDNA Gel Electrophoresis
Sequencing
Xgal Stock
Plan:
For Predictable & Transferable Expression, first, all the level 0's were constructed and then assembled in level 1 to make the following constructs:
0.5 T7 p - SarJ + B0032 - TALEsp1 - WT T7 t
T7 p - SarJ + B0032 - TALEsp1 - WT T7 t
0.5 T7 p - RiboJ + B0032 - Juniper GFP - WT T7 t
T7_sp1 p - RiboJ + B0032 - Juniper GFP - WT T7 t
T7 p - RiboJ + B0032 - Juniper GFP - WT T7 t
0.5 T7_sp1 p - RiboJ + B0032 - Juniper GFP - WT T7 t
Pbhr - SarJ + B0032 - TALEsp1 - WT T7 t
Pbhr - RiboJ + B0032 - Juniper GFP - WT T7 t
0.5 T7 p - SarJ + B0032 - TALEsp1 - WT T7 t
T7 p - SarJ + B0032 - TALEsp1 - WT T7 t
Pbhr - Sarj + B0032 - TALEsp1 - WT T7 t
0.5 T7 p - RiboJ + B0032 - GFP - WT T7 t
0.1 T7 p - RiboJ + B0032 - GFP - WT T7 t
19bp T7 p - RiboJ + B0032 - GFP - WT T7 t
19bp 0.5 T7 p - RiboJ + B0032 - GFP - WT T7 t
19bp 0.1 T7 p - RiboJ + B0032 - GFP - WT T7 t
T7sp_sp1 p - RiboJ + B0032 - GFP - WT T7 t
Pbhr_sp1 - RiboJ + Universal RBS - GFP - WT T7 t
T7 p - SarJ + Universal RBS - GFP - WT T7 t
After assembling the level 1's, the following level M's were assembled:
T7p TALEsp1 - Dummy 2 - Dummy 3 - T7p TALEsp1
T7p TALEsp1 - Dummy 2 - Dummy 3 - T7p GFP
0.5 T7p TALEsp1 - Dummy 2 - Dummy 3 - 0.5 T7_sp1 p GFP
0.5 T7p TALEsp1 - Dummy 2 - Dummy 3 - 0.5 T7p GFP
0.5 T7p TALEsp1 - Dummy 2 - Dummy 3 - T7_sp1 p GFP
T7p TALEsp1 - Dummy 2 - Dummy 3 - 0.5 T7_sp1 p GFP
T7p TALEsp1 - T7p TALEsp1
T7p TALEsp1 - T7p GFP
0.5 T7p TALEsp1 - 0.5 T7_sp1 p GFP
T7p Universal RBS TALEsp1 - T7_sp1 p Universal RBS GFP
Pbhr Universal RBS TALEsp1 - Pbhr_sp1 Universal RBS GFP
Pbhr Universal RBS TALEsp1 - Pbhr_sp1 v2 Universal RBS GFP
Pbhr Universal RBS TALEsp1 - Pbhr Universal RBS GFP
Pbhr SarJ B00032 TALEsp1 - Pbhr Universal GFP
Results:
To be added
September
Copy Number Independence
Some text in the Modal Body
Some other text...
Transcriptional Variance Independence
Some text in the Modal Body
Some other text...
Translation Variance Independence
Some text in the Modal Body
Some other text...
October
