Difference between revisions of "Team:TUDelft/NotebookTest"

Revision as of 16:26, 19 October 2019

Sci-Phi 29

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Orthogonal Replication

Cloning

Testing of Plasmid

April

May

June

July

August

Goal:	Assembly all the different parts with MoClo
Protocols Used:	Nanodrop DNA quantification, Plasmid Isolation, MoClo, E.coli Heat Shock Transformation, KOD Extreme PCR, Colony Polymerase PCR (GoTaq), E. coli Glycerol Stock, DNA Gel Electrophoresis, Sequencing
Plan:	For Orthogonal Replication, first, all the level 0's were constructed and then assembled in level 1 to make the following constructs: 0.1 T7 p - U RBS - DNAP - WT T7 t 0.5 T7 p - U RBS - DNAP - WT T7 t WT T7 p - U RBS - DNAP - WT T7 t 0.1 T7 p - U RBS - TP - WT T7 t 0.5 T7 p - U RBS - TP - WT T7 t WT T7 p - U RBS - TP - WT T7 t 0.1 T7 p - U RBS - p5 - WT T7 t 0.5 T7 p - U RBS - p5 - WT T7 t WT T7 p - U RBS - p5 - WT T7 t 0.1 T7 p - U RBS - p6 - WT T7 t 0.5 T7 p - U RBS - p6 - WT T7 t WT T7 p - U RBS - p6 - WT T7 t 0.1 T7 p - U RBS - GFP - WT T7 t 0.5 T7 p - U RBS - GFP - WT T7 t WT T7 p - U RBS - GFP - WT T7 t
Results:	All composite parts are confirmed by sequencing.

September

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

Predictable and Transferable Expression

Cloning

Testing of Plasmid

April

May

June

July

August

Goal:	Assembly all the different parts with MoClo
Protocols Used:	Nanodrop DNA quantification, Plasmid Isolation, MoClo, E.coli Heat Shock Transformation, Colony Polymerase PCR (GoTaq), E. coli Glycerol Stock, DNA Gel Electrophoresis, Sequencing, Xgal Stock
Plan:	For Predictable & Transferable Expression, constructs were assembled by golden gate assembly based modular cloning systesm. First, individual parts were cloned in respective level 0s backbones. T7 promoter 0.5 T7 promoter 0.1 T7 promoter P _BHR promoter T7sp1 promoter 0.5 T7sp1 promoter 0.1 T7sp1 promoter P_BHRsp1 promoter SarJ RiboJ Universal RBS TALEsp1 Harmonized GFP T7 terminator variant The level 0 parts were then assembled into transcriptional units level 1 MoClo backbones: L1_A:P_BHRsp1-RiboJ-UniRBS-Juniper GFP-T7term L1_B:T7_sp1 p - RiboJ-UniRBS - Harmonized GFP - T7term L1_C:Pbhr - Sarj-UniRBS - TALEsp1 - T7termvar L1_D:T7 p - SarJ-UniRBS- TALEsp1 - T7termvar /li> L1_E:T7 p - RiboJ-UniRBS - Harmonized GFP -T7term L1_F:0.5 T7 p - SarJ-UniRBS- TALEsp1 - T7termvar L1_G:0.1 T7 p - SarJ-UniRBS - TALEsp1- T7term L1_H:0.5 T7 p - RiboJ-UniRBS- Harmonized GFP - T7t The following multiple transcriptional units, were assembled in MoClo level M backbones: L1_C+L1_A L1_D+L1_B L1_D+L1_E L1_F+L1_H L1_G+L1_B
Results:	To be added

September

October

@@ Line 832: / Line 832: @@
 The level 0 parts were then assembled into transcriptional units level 1 MoClo backbones:
                                                  <ul>
-                                                     <li>P<sub>BHR</sub>sp1-RiboJ-UniRBS-Juniper GFP-T7term</li>
+                                                     <li>L1_A:P<sub>BHR</sub>sp1-RiboJ-UniRBS-Juniper GFP-T7term</li>
-                                                     <li>T7_sp1 p - RiboJ-UniRBS - Harmonized GFP - T7term</li>
+                                                     <li>L1_B:T7_sp1 p - RiboJ-UniRBS - Harmonized GFP - T7term</li>
-                                                     <li>Pbhr - Sarj-UniRBS - TALEsp1 - T7termvar</li>
+                                                     <li>L1_C:Pbhr - Sarj-UniRBS - TALEsp1 - T7termvar</li>
-                                                     <li>T7 p - SarJ-UniRBS- TALEsp1 - T7termvar /li>
+                                                     <li>L1_D:T7 p - SarJ-UniRBS- TALEsp1 - T7termvar /li>
-                                                     <li>T7 p - RiboJ-UniRBS - Harmonized GFP -T7term</li>
+                                                     <li>L1_E:T7 p - RiboJ-UniRBS - Harmonized GFP -T7term</li>
-                                                     <li>0.5 T7 p - SarJ-UniRBS- TALEsp1 - T7termvar</li>
+                                                     <li>L1_F:0.5 T7 p - SarJ-UniRBS- TALEsp1 - T7termvar</li>
-                                                     <li>0.1 T7 p - SarJ-UniRBS - TALEsp1- T7term</li>
+                                                     <li>L1_G:0.1 T7 p - SarJ-UniRBS - TALEsp1- T7term</li>
-                                                     <li>0.5 T7 p - RiboJ-UniRBS- Harmonized GFP - T7t</li>
+                                                     <li>L1_H:0.5 T7 p - RiboJ-UniRBS- Harmonized GFP - T7t</li>
 </ul>
 <br>
                                                  The following multiple transcriptional units, were assembled in MoClo level M backbones:
                                                  <ul>
-                                                     <li>T7p TALEsp1 - Dummy 2 - Dummy 3 - T7p TALEsp1</li>
+                                                     <li> L1_C+L1_A </li>
-                                                     <li>T7p TALEsp1 - Dummy 2 - Dummy 3 - T7p GFP</li>
+                                                     <li> L1_D+L1_B </li>
-                                                     <li>0.5 T7p TALEsp1 - Dummy 2 - Dummy 3 - 0.5 T7_sp1 p GFP</li>
+                                                     <li> L1_D+L1_E </li>
-                                                     <li>0.5 T7p TALEsp1 - Dummy 2 - Dummy 3 - 0.5 T7p GFP</li>
+                                                     <li> L1_F+L1_H </li>
-                                                     <li>0.5 T7p TALEsp1 - Dummy 2 - Dummy 3 - T7_sp1 p GFP</li>
+                                                     <li> L1_G+L1_B </li>
-                                                     <li>T7p TALEsp1 - Dummy 2 - Dummy 3 - 0.5 T7_sp1 p GFP</li>
-                                                    <li>T7p TALEsp1 - T7p TALEsp1</li>
-                                                    <li>T7p TALEsp1 - T7p GFP</li>
-                                                    <li>0.5 T7p TALEsp1 - 0.5 T7_sp1 p GFP</li>
-                                                    <li>T7p Universal RBS TALEsp1 - T7_sp1 p Universal RBS GFP</li>
-                                                    <li>Pbhr Universal RBS TALEsp1 - Pbhr_sp1 Universal RBS GFP</li>
-                                                    <li>Pbhr Universal RBS TALEsp1 - Pbhr_sp1 v2 Universal RBS GFP</li>
-                                                    <li>Pbhr Universal RBS TALEsp1 - Pbhr Universal RBS GFP</li>
-                                                    <li>Pbhr SarJ B00032 TALEsp1 - Pbhr Universal GFP</li>
                                                  </ul>

Difference between revisions of "Team:TUDelft/NotebookTest"

Revision as of 16:26, 19 October 2019

Orthogonal Replication

Gibson Assembly of Gene of Interest Plasmid

Gibson Assembly of Support Plasmid

Brute Force Experiments 1

MoClo Assembly

Toxicity Experiments 1

Toxicity Experiments 2

Characterization of Level 1's

Brute Force Experiments 2

Orthogonal Replication

Predictable and Transferable Expression

Uber Plasmid

MoClo Assembly

Copy Number Independence

Transcriptional Variance Independence

Translation Variance Independence