iGEM 2019 — Team TUDelft

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# Team:TUDelft/Notebook

Sci-Phi 29

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[**Home**](https://2019.igem.org/Team%3ATUDelft)

**Project**

[Description](https://2019.igem.org/Team%3ATUDelft/Description)
[Design](https://2019.igem.org/Team%3ATUDelft/Design)
[Results](https://2019.igem.org/Team%3ATUDelft/Results)
[Demonstrate](https://2019.igem.org/Team%3ATUDelft/Demonstrate)

**Wetlab**

[Experiment Protocols](https://2019.igem.org/Team%3ATUDelft/Experiments)
[Safety](https://2019.igem.org/Team%3ATUDelft/Safety)
[Notebook](https://2019.igem.org/Team%3ATUDelft/Notebook)

**Modeling**

[Model](https://2019.igem.org/Team%3ATUDelft/Model)

[Reaction Kinetics](https://2019.igem.org/Team%3ATUDelft/Model#Kinetics)

[Copy Number independence](https://2019.igem.org/Team%3ATUDelft/Model#Kinetics)
[Transcription Variation](https://2019.igem.org/Team%3ATUDelft/Model#TranscriptionalVariations)
[Translation Variation](https://2019.igem.org/Team%3ATUDelft/Model#TranslationalVariations)

[Codon Harmonization](https://2019.igem.org/Team%3ATUDelft/Model#CodonUsage)

**Parts**

[Parts Overview](https://2019.igem.org/Team%3ATUDelft/Parts)

**Human Practices**

[Human Practices](https://2019.igem.org/Team%3ATUDelft/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ATUDelft/Public_Engagement)

**Team**

[Team Members](https://2019.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2019.igem.org/Team%3ATUDelft/Collaborations)

**Acknowledgements**

[Attributions](https://2019.igem.org/Team%3ATUDelft/Attributions)
[Sponsors](https://2019.igem.org/Team%3ATUDelft/Sponsors)

[Judging Form](https://igem.org/2019_Judging_Form?id=2918)
[Medal Requirements](https://2019.igem.org/Team%3ATUDelft/Medals)

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![Design](https://static.igem.org/mediawiki/2019/e/e4/T--TUDelft--Notebook_logo.png)

## Orthogonal Replication

|  | Cloning | | Testing of Plasmid | |
| --- | --- | --- | --- | --- |
| April | Gibson Assembly of Gene of Interest Plasmid  ×Gibson Assembly of Gene of Interest Plasmid  |  |  | | --- | --- | | Goal: | We wanted to construct a Gene of Interest Plasmid. The Gene of Interest of Plasmid should have OriL, gene of interest, selection marker and OriR. The gene of interest was GFP in our case. We had a plasmid with OriL-GFP-OriR and therefore we designed primers to clone Kanamycin in. The Gene of Interest Plasmid is the plasmid that will be replicated with the phi29 machinery. | | Protocols Used: | [Nanodrop DNA quantification](https://2019.igem.org/Team%3ATUDelft/Experiments#Nanodrop), [Plasmid Isolation](https://2019.igem.org/Team%3ATUDelft/Experiments#PlasmidIsolation), [Phusion PCR](https://2019.igem.org/Team%3ATUDelft/Experiments#PCR), [Restriction Digestion](https://2019.igem.org/Team%3ATUDelft/Experiments#Restriction), [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments#Ecoli), [KOD Extreme PCR](https://2019.igem.org/Team%3ATUDelft/Experiments#PCR), [Colony Polymerase PCR (GoTaq)](https://2019.igem.org/Team%3ATUDelft/Experiments#PCR), [*E. coli* Glycerol Stock](https://2019.igem.org/Team%3ATUDelft/Experiments#Ecoli), [Gibson Assembly](https://2019.igem.org/Team%3ATUDelft/Experiments#GA), [DNA Gel Purification](https://2019.igem.org/Team%3ATUDelft/Experiments#CleanUp), [DNA Gel Electrophoresis](https://2019.igem.org/Team%3ATUDelft/Experiments#Electrophoresis) | | Plan: | Our plasmid p01 contained GFP flanked by OriL and OriR. We wanted to clone a kanamycin gene from plasmid p03 inside this to use as a selection marker. After Gibson Assembly, the plasmid was transformed into *E. coli* top 10 cells and plated on LB + Kan. | | Results: | There were no plates with colonies, therefore Gibson Assembly has not worked as no cells expressed the Kanamycin gene. | | Gibson Assembly of Support Plasmid  ×Gibson Assembly of Support Plasmid  |  |  | | --- | --- | | Goal: | We wanted to construct the Support Plasmid. We have two plasmids each containing two of the four replication machinery. We wanted to put them on one plasmid to minimize the amount of plasmids to be transformed into our construct. | | Protocols Used: | [Nanodrop DNA quantification](https://2019.igem.org/Team%3ATUDelft/Experiments), [Plasmid Isolation](https://2019.igem.org/Team%3ATUDelft/Experiments), [Phusion PCR](https://2019.igem.org/Team%3ATUDelft/Experiments), [Restriction Digestion](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments), [KOD Extreme PCR](https://2019.igem.org/Team%3ATUDelft/Experiments), [Colony Polymerase PCR (GoTaq)](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Glycerol Stock](https://2019.igem.org/Team%3ATUDelft/Experiments), [Gibson Assembly](https://2019.igem.org/Team%3ATUDelft/Experiments), [DNA Gel Purification](https://2019.igem.org/Team%3ATUDelft/Experiments), [DNA Gel Electrophoresis](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | We wanted to assembly the replication machinery on one plasmid through Gibson Assembly. We possess one plasmid (p04) with p5 and p6 and one plasmid with DNAP and TP (p02) and therefore they were cloned together. After Gibson Assembly, the plasmid was transformed into *E. coli* top 10 cells and plated on LB + Amp. | | Results: | There were no plates with colonies, therefore Gibson Assembly has not worked as no cells expressed the Ampicilin gene. | |  | |
| May |  | |
| June |  | |
| July | Brute Force Experiments  ×Brute Force Experiments  |  |  | | --- | --- | | Goal: | Testing orthogonal replication in vivo | | Protocols Used: | [Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments), [IPTG stock](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | Although the construction of the plasmids using Gibson Assembly did not succeed, we transformed a combination of constructs to determine whether in vivo replication would occur. We induced it with no IPTG, 10 µM IPTG and 50 µM IPTG to enhance protein expression. We transformed in the following constructs into BL21(DE3) Rosetta 2 cells:  * p02 (DNAP + TP) + p04 (p5 +p6) + oriL-GFP-oriR * p02 (DNAP - TP) + oriL-GFP-oriR * oriL-GFP-oriR * p01 (GFP) + p02 (DNAP + TP) + p04 (p5 + p6) | | Results: | There were no colonies on most of the plates. Inconclusive result. | | |
| August | MoClo Assembly  ×MoClo Assembly  |  |  | | --- | --- | | Goal: | Assembling the parts of the replication machinery separately with MoClo | | Protocols Used: | [Nanodrop DNA quantification](https://2019.igem.org/Team%3ATUDelft/Experiments), [Plasmid Isolation](https://2019.igem.org/Team%3ATUDelft/Experiments), [MoClo](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments), [KOD Extreme PCR](https://2019.igem.org/Team%3ATUDelft/Experiments), [Colony Polymerase PCR (GoTaq)](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Glycerol Stock](https://2019.igem.org/Team%3ATUDelft/Experiments), [DNA Gel Electrophoresis](https://2019.igem.org/Team%3ATUDelft/Experiments), [Sequencing](https://2019.igem.org/Team%3ATUDelft/Experiments), [IPTG stock](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | First all the level 0's were constructed for the orthogonal replication module. Subsequently, the level 0's were used to assembly the following level 1 constructs:  * [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [DNAP](http://parts.igem.org/Part%3ABBa_K2918034) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [DNAP](http://parts.igem.org/Part%3ABBa_K2918034) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [DNAP](http://parts.igem.org/Part%3ABBa_K2918034) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [TP](http://parts.igem.org/Part%3ABBa_K2918001) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [TP](http://parts.igem.org/Part%3ABBa_K2918001) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [TP](http://parts.igem.org/Part%3ABBa_K2918001) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p5](http://parts.igem.org/Part%3ABBa_K2918002) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p5](http://parts.igem.org/Part%3ABBa_K2918002) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p5](http://parts.igem.org/Part%3ABBa_K2918002) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p6](http://parts.igem.org/Part%3ABBa_K2918003) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p6](http://parts.igem.org/Part%3ABBa_K2918003) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p6](http://parts.igem.org/Part%3ABBa_K2918003) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - GFP - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - GFP - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - GFP - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [OriL-GFP-Kan-OriR](http://parts.igem.org/Part%3ABBa_K2918020) | | Results: | All composite parts are confirmed by sequencing | | | Toxicity Experiments 1  ×Toxicity Experiments 1  |  |  | | --- | --- | | Goal: | Determinig whether the orthogonal replication constructs could be toxic to the cell | | Protocols Used: | [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments), [OD600 Measurement](https://2019.igem.org/Team%3ATUDelft/Experiments), [IPTG stock](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | The following plasmids were transformed into BL21(DE3) Rosetta 2 separately:  * p01 (GFP as control) * p02 (DNAP + TP) * p03 (Kan) * p04 (p5 + p6)    Transformation was carried out to determine whether the failure of the Gibson Assembly constructions were caused by toxicity of the orthogonal replication proteins. | | Results: | The OD measurements of p02 and p04 dropped very quickly after 3 hours. However, the results are inconclusive due to lack of selection | | |
| September | Toxicity Experiments 2  ×Toxicity Experiments 2  |  |  | | --- | --- | | Goal: | Determinig whether the orthogonal replication constructs could be toxic to the cell | | Protocols Used: | [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments), [OD600 Measurement](https://2019.igem.org/Team%3ATUDelft/Experiments), [IPTG stock](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | A different method and type of cell were used to determine how this would influence the suspected toxicity. The cells used in the experiment were BL21 (DE3) pLysS which have less leaky expression of T7 RNAP than BL21 (DE3) and therefore are suspected to survive longer. To determine whether this type of cell works better, we transformed the plasmids separately in BL21 (DE3) pLysS and measured the OD600 overnight to determine the viability of the cells containing the following plasmid:  * p01 (GFP as control) * p02 (DNAP + TP) * p03 (Kan) * p04 (p5 + p6) | | Results: | The cells containing the plasmids did not grow overnight | | Characterization of Level 1  ×Characterization of Level 1  |  |  | | --- | --- | | Goal: | Characterize the different promoters strengths in the assembled constructs | | Protocols Used: | [Expressing phi29 proteins in PUREfrex system](https://2019.igem.org/Team%3ATUDelft/Experiments), [Purifying phi29 proteins for SDS PAGE](https://2019.igem.org/Team%3ATUDelft/Experiments), [Mass Spectrometry Preparation](https://2019.igem.org/Team%3ATUDelft/Experiments), [SDS Page](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | The following constructs were expressed in vitro and run on a protein gel to determine whether there was amplification of the gene:  * [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [DNAP](http://parts.igem.org/Part%3ABBa_K2918034) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [DNAP](http://parts.igem.org/Part%3ABBa_K2918034) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [DNAP](http://parts.igem.org/Part%3ABBa_K2918034) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [TP](http://parts.igem.org/Part%3ABBa_K2918001) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [TP](http://parts.igem.org/Part%3ABBa_K2918001) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [TP](http://parts.igem.org/Part%3ABBa_K2918001) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p5](http://parts.igem.org/Part%3ABBa_K2918002) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p5](http://parts.igem.org/Part%3ABBa_K2918002) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p5](http://parts.igem.org/Part%3ABBa_K2918002) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p6](http://parts.igem.org/Part%3ABBa_K2918003) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p6](http://parts.igem.org/Part%3ABBa_K2918003) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [p6](http://parts.igem.org/Part%3ABBa_K2918003) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015)   Subsequently, a fraction of the sample was analyzed with Mass Spectrometry. | | Results: | The strength of the promoter correlates with the amount of proteins transcribed. Read more [here](https://2019.igem.org/Team%3ATUDelft/Results). | |
|  | |
| October |  |  | Orthogonal Replication  ×Orthogonal Replication  |  |  | | --- | --- | | Goal: | To replicate our constructs (OriL-GFP-Kan-OriR or OriL-GFP-OriR) in vitro | | Protocols Used: | [DNA gel electrophoresis](https://2019.igem.org/Team%3ATUDelft/Experiments), [Coupled IVTT and DNA replication](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | Our construct (OriL-GFP-Kan-OriR) combined our MoClo DNAP and TP plasmid were added with purifed p5 and p6 to determine whether replication would occur. In parallel, we added a plasmid containing DNAP and TP, in a mix containing a shorter construct: OriL-GFP-OriR together with pruified p5 and p6. | | Results: | Replication in vitro occurs! Read more [here](https://2019.igem.org/Team%3ATUDelft/Results). | |  |

## Controllable Expression

|  | Cloning | Testing of Plasmid | | |
| --- | --- | --- | --- | --- |
| April |  |  | | |
| May |  |  | | |
| June | UBER Plasmid  ×UBER Plasmid  |  |  | | --- | --- | | Goal: | Construction of the UBER Plasmid | | Protocols Used: | [Nanodrop DNA quantification](https://2019.igem.org/Team%3ATUDelft/Experiments), [Plasmid Isolation](https://2019.igem.org/Team%3ATUDelft/Experiments), [Phusion PCR](https://2019.igem.org/Team%3ATUDelft/Experiments), [Restriction Digestion](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments), [Ligation](https://2019.igem.org/Team%3ATUDelft/Experiments), [Colony Polymerase PCR (GoTaq)](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Glycerol Stock](https://2019.igem.org/Team%3ATUDelft/Experiments), [DNA Gel Purification](https://2019.igem.org/Team%3ATUDelft/Experiments), [DNA Gel Electrophoresis](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | We wanted to insert GFP in the UBER plasmid by restriction digestion and ligation. | | Results: | Check PCR did not result in the expected size on the gel | |  | | |
| July |  |  | | |
| August | MoClo Assembly  ×MoClo Assembly  |  |  | | --- | --- | | Goal: | Assembly all the different parts with MoClo | | Protocols Used: | [Nanodrop DNA quantification](https://2019.igem.org/Team%3ATUDelft/Experiments), [Plasmid Isolation](https://2019.igem.org/Team%3ATUDelft/Experiments), [MoClo](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments), [Colony Polymerase PCR (GoTaq)](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Glycerol Stock](https://2019.igem.org/Team%3ATUDelft/Experiments), [DNA Gel Electrophoresis](https://2019.igem.org/Team%3ATUDelft/Experiments), [Sequencing](https://2019.igem.org/Team%3ATUDelft/Experiments), [Xgal Stock](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | For Predictable & Transferable Expression, constructs were assembled by golden gate assembly based modular cloning systesm. First, individual parts were cloned in respective level 0 backbones.  * T7 promoter * 0.5 T7 promoter * 0.1 T7 promoter * P BHR promoter * T7sp1 promoter * 0.5 T7sp1 promoter * 0.1 T7sp1 promoter * PBHRsp1 promoter * SarJ * RiboJ * Universal RBS * TALEsp1 * Harmonized GFP * T7 terminator variant    The level 0 parts were assembled into transcriptional units level 1 MoClo backbones:    * [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [Universal RBS](http://parts.igem.org/Part%3ABBa_K2918014) - [Harmonized GFP](http://parts.igem.org/Part%3ABBa_K2918037) -[WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_A: [PromoterBHRsp1](http://parts.igem.org/Part%3ABBa_K2918011) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Juniper GFP](http://parts.igem.org/Part%3ABBa_J97005) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_B: [T7sp1 promoter](http://parts.igem.org/Part%3ABBa_K2918010) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Harmonized GFP](http://parts.igem.org/Part%3ABBa_K2918037) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_C: [Pbhr](http://parts.igem.org/Part%3ABBa_K2918000) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008) -[WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016) * L1\_D: [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008) - [WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016) * L1\_E: [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Harmonized GFP](http://parts.igem.org/Part%3ABBa_K2918037) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_F: [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008) - [WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016) * L1\_G: [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008)- [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_H: [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Harmonized GFP](http://parts.igem.org/Part%3ABBa_K2918037) - [WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016)    The following multiple transcriptional units were assembled in MoClo level M backbones:   * [L1\_C+L1\_A](http://parts.igem.org/Part%3ABBa_K2918050) * [L1\_D+L1\_B](http://parts.igem.org/Part%3ABBa_K2918046) * L1\_D+L1\_E * [L1\_F+L1\_H](http://parts.igem.org/Part%3ABBa_K2918048) * [L1\_G+L1\_B](http://parts.igem.org/Part%3ABBa_K2918040) | | Results: | Read more [here](https://2019.igem.org/Team%3ATUDelft/Results). |  Codon Harmonization  ×Codon Harmonization  |  |  | | --- | --- | | Goal: | Demonstration that the harmonized coding sequence generated from our modelling still | | Protocols Used: | [Nanodrop DNA quantification](https://2019.igem.org/Team%3ATUDelft/Experiments) [Plasmid Isolation](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | The harmonized GFP construct (WT T7 promoter - Uni RBS - Harmonized GFP - WT T7 terminator) was transformed into BL21 pLysS cells together with a plasmid with regular GFP (positive control). As a negative control a cell without plasmid was used. | | Results: | The harmonized GFP works. Read more [here](https://2019.igem.org/Team%3ATUDelft/Results). | |  | | |
| September | Copy Number Independence  ×Copy Number Independence  |  |  | | --- | --- | | Goal: | To demonstrate stable GFP expression despite change in copy number | | Protocols Used: | [Nanodrop DNA quantification](https://2019.igem.org/Team%3ATUDelft/Experiments), [Plasmid Isolation](https://2019.igem.org/Team%3ATUDelft/Experiments), [MoClo](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments), [Colony Polymerase PCR (GoTaq)](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Glycerol Stock](https://2019.igem.org/Team%3ATUDelft/Experiments), [DNA Gel Electrophoresis](https://2019.igem.org/Team%3ATUDelft/Experiments), [Sequencing](https://2019.igem.org/Team%3ATUDelft/Experiments), [Xgal Stock](https://2019.igem.org/Team%3ATUDelft/Experiments), [OD600 Measurement](https://2019.igem.org/Team%3ATUDelft/Experiments), [FACS](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | Level M constructs (L1D + L1E, L1G + L1B) were both cloned in low and medium copy backbones. The constructs were then co-transformed with [MK1283](https://www.addgene.org/71428/) which contains a plasmid harbouring a T7 RNAP mixed forward loop. Fluorescence was detected by FACS.  * L1\_A: [PromoterBHRsp1](http://parts.igem.org/Part%3ABBa_K2918011) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Juniper GFP](http://parts.igem.org/Part%3ABBa_J97005) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_B: [T7sp1 promoter](http://parts.igem.org/Part%3ABBa_K2918010) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Harmonized GFP](http://parts.igem.org/Part%3ABBa_K2918037) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_C: [Pbhr](http://parts.igem.org/Part%3ABBa_K2918000) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008) -[WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016) * L1\_D: [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008) - [WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016) * L1\_E: [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Harmonized GFP](http://parts.igem.org/Part%3ABBa_K2918037) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_F: [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008) - [WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016) * L1\_G: [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008)- [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_H: [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Harmonized GFP](http://parts.igem.org/Part%3ABBa_K2918037) - [WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016) | | Results: | Read more [here](https://2019.igem.org/Team%3ATUDelft/Results). | | Transcriptional Variantions  ×Transcriptional Variantions  |  |  | | --- | --- | | Goal: | To demonstrate stable GFP expression despite change in transcriptional rates | | Protocols Used: | [Nanodrop DNA quantification](https://2019.igem.org/Team%3ATUDelft/Experiments), [Plasmid Isolation](https://2019.igem.org/Team%3ATUDelft/Experiments), [MoClo](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments), [Colony Polymerase PCR (GoTaq)](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Glycerol Stock](https://2019.igem.org/Team%3ATUDelft/Experiments), [DNA Gel Electrophoresis](https://2019.igem.org/Team%3ATUDelft/Experiments), [Sequencing](https://2019.igem.org/Team%3ATUDelft/Experiments), [Xgal Stock](https://2019.igem.org/Team%3ATUDelft/Experiments), [OD600 Measurement](https://2019.igem.org/Team%3ATUDelft/Experiments), [FACS](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | Level M constructs (L1\_C+L1\_A, L1\_D+L1\_B, L1\_D+L1\_E, L1\_F+L1\_H, L1\_G+L1\_B) were characterized using FACS to demonstrate stable GFP expression when the ratio of transcriptional rates between GFP and TALE remained the same. Here a quick reminder of what the constructs were:  * L1\_A: [PromoterBHRsp1](http://parts.igem.org/Part%3ABBa_K2918011) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Juniper GFP](http://parts.igem.org/Part%3ABBa_J97005) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_B: [T7sp1 promoter](http://parts.igem.org/Part%3ABBa_K2918010) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Harmonized GFP](http://parts.igem.org/Part%3ABBa_K2918037) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_C: [Pbhr](http://parts.igem.org/Part%3ABBa_K2918000) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008) -[WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016) * L1\_D: [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008) - [WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016) * L1\_E: [WT T7 promoter](http://parts.igem.org/Part%3ABBa_K2918007) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Harmonized GFP](http://parts.igem.org/Part%3ABBa_K2918037) - [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_F: [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008) - [WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016) * L1\_G: [Weak T7 promoter](http://parts.igem.org/Part%3ABBa_K2918005) - [SarJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918036) - [TALEsp1](http://parts.igem.org/Part%3ABBa_K2918008)- [WT T7 terminator](http://parts.igem.org/Part%3ABBa_K2918015) * L1\_H: [Medium T7 promoter](http://parts.igem.org/Part%3ABBa_K2918006) - [RiboJ-Universal RBS](http://parts.igem.org/Part%3ABBa_K2918038) - [Harmonized GFP](http://parts.igem.org/Part%3ABBa_K2918037) - [WT T7 terminator variant](http://parts.igem.org/Part%3ABBa_K2918016)    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: | Read more [here](https://2019.igem.org/Team%3ATUDelft/Results). | | Translational Variantions  ×Translational Variantions  |  |  | | --- | --- | | Goal: | To demonstrate stable GFP expression despite change in translational rates | | Protocols Used: | [Nanodrop DNA quantification](https://2019.igem.org/Team%3ATUDelft/Experiments), [Plasmid Isolation](https://2019.igem.org/Team%3ATUDelft/Experiments), [MoClo](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Heat Shock Transformation](https://2019.igem.org/Team%3ATUDelft/Experiments), [Colony Polymerase PCR (GoTaq)](https://2019.igem.org/Team%3ATUDelft/Experiments), [*E. coli* Glycerol Stock](https://2019.igem.org/Team%3ATUDelft/Experiments), [DNA Gel Electrophoresis](https://2019.igem.org/Team%3ATUDelft/Experiments), [Sequencing](https://2019.igem.org/Team%3ATUDelft/Experiments), [Xgal Stock](https://2019.igem.org/Team%3ATUDelft/Experiments), [OD600 Measurement](https://2019.igem.org/Team%3ATUDelft/Experiments), [FACS](https://2019.igem.org/Team%3ATUDelft/Experiments) | | Plan: | Gene expression independent of translational rates was demonstrated by cloning constructs in which the RBSs for TALE and GFP were switched out RBSs (BBa\_B0032 and Universal RBS) of different strengths. The GFP expression was measured by FACS. To overcome translational variations associated with codon usage, eGFP sequence was predicted using our codon harmonizer tool. The function of harmonized eGFP was confirmed by detecting output fluorescence. | | Results: | Read more [here](https://2019.igem.org/Team%3ATUDelft/Results). | |
| October |


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:TUDelft/Experiments

Sci-Phi 29

![Top](https://static.igem.org/mediawiki/2019/6/6d/T--TUDelft--Top.png)

[![](https://static.igem.org/mediawiki/2019/e/e7/T--TUDELFT--logonavbar-white.png)](https://2019.igem.org/Team%3ATUDelft)
[**Home**](https://2019.igem.org/Team%3ATUDelft)

**Project**

[Description](https://2019.igem.org/Team%3ATUDelft/Description)
[Design](https://2019.igem.org/Team%3ATUDelft/Design)
[Results](https://2019.igem.org/Team%3ATUDelft/Results)
[Demonstrate](https://2019.igem.org/Team%3ATUDelft/Demonstrate)

**Wetlab**

[Experiment Protocols](https://2019.igem.org/Team%3ATUDelft/Experiments)
[Safety](https://2019.igem.org/Team%3ATUDelft/Safety)
[Notebook](https://2019.igem.org/Team%3ATUDelft/Notebook)

**Modeling**

[Model](https://2019.igem.org/Team%3ATUDelft/Model)

[Reaction Kinetics](https://2019.igem.org/Team%3ATUDelft/Model#Kinetics)

[Copy Number independence](https://2019.igem.org/Team%3ATUDelft/Model#Kinetics)
[Transcription Variation](https://2019.igem.org/Team%3ATUDelft/Model#TranscriptionalVariations)
[Translation Variation](https://2019.igem.org/Team%3ATUDelft/Model#TranslationalVariations)

[Codon Harmonization](https://2019.igem.org/Team%3ATUDelft/Model#CodonUsage)

**Parts**

[Parts Overview](https://2019.igem.org/Team%3ATUDelft/Parts)

**Human Practices**

[Human Practices](https://2019.igem.org/Team%3ATUDelft/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ATUDelft/Public_Engagement)

**Team**

[Team Members](https://2019.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2019.igem.org/Team%3ATUDelft/Collaborations)

**Acknowledgements**

[Attributions](https://2019.igem.org/Team%3ATUDelft/Attributions)
[Sponsors](https://2019.igem.org/Team%3ATUDelft/Sponsors)

[Judging Form](https://igem.org/2019_Judging_Form?id=2918)
[Medal Requirements](https://2019.igem.org/Team%3ATUDelft/Medals)

![Email](https://static.igem.org/mediawiki/2019/b/b4/T--TUDELFT--mailicon.png)
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![Protocol](https://static.igem.org/mediawiki/2019/0/06/T--TUDelft--Protocol_logo.png)

* [General](#General)
* [Strain Specific](#Specific)

## General Protocols

* Annealing primers for G-block **﹀**

  The primers to create G-blocks are provided with a concentration of 100 µM. The working stock for MoClo is 100 fmol/µL. Carry out a dilution to have a concentration of 0.1 µM. In order to anneal the primer for the G-block, follow the instructions below:

  1. Prepare the reaction mix:

     | Component | Volume (µL) |
     | --- | --- |
     | Primer 1 | 9 |
     | Primer 2 | 9 |
     | Buffer\* 10X | 2 |
     | Total | 20 |
  2. Heat the reaction to 94 °C for 5 minutes.
  3. Let the reaction cool down to room temperature.

  The buffer 10X used for the annealing consists of:

  | Component | Final concentration (mmol/L) |
  | --- | --- |
  | Tris | 100 mmol/L |
  | NaCl | 500 mmol/L |
  | EDTA | 10 mmol/L |

  The pH of the buffer should be between 7.5 - 8 and filter sterilize the buffer.
* Antibiotic Stock Solution **﹀**

  1. Weigh X gram of the antibiotic of interest.
  2. Dissolve the antibiotic of interest in Milli-Q water. The volume of Milli-Q water needed is determined by the desired antibiotic concentration.Note: Dissolve in 95% ethanol in case of chloramphenicol. We used the following antibiotic stock concentrations:

  | Antibiotic | Stock Solution (mg/mL) |
  | --- | --- |
  | Ampicillin | 50 |
  | Chloramphenicol | 34 |
  | Kanamycin | 50 |
  | Spectinomycin | 50 |
* Coupled IVTT and DNA replication **﹀**

  1. For coupled *In Vitro* Transcription and Translation (IVTT), pipette the following volumes in a single PCR tube:

  | Component | Volume (µL) |
  | --- | --- |
  | Solution I (Buffer) | 7.5 |
  | Solution II (Enzymes) | 0.75 |
  | Solution III (Ribosomes) | 1.5 |
  | Ammonium Sulfate (200mM) | 1.5 |
  | Purified p5 (10mg/mL) | 1.125 |
  | Purified p6 (7mg/mL) | 0.45 |
  | DNAP (2nM) | X |
  | TP (2nM) | Y |
  | Linear DNA (1nM) | Z |
  | dNTPs | 0.45 |
  | RNAse inhibitor | 0.375 |
  | RNAse free Milli-Q | 15 - X - Y - Z |
  | Total | 15 |

  2. Incubate the nuclease-free PCR tubes at 30 °C for 8 hours

  To visualize the amplification of the linear DNA construct, you have to get rid of the RNA and DNA. This can be done by following the next steps:

  3. Treat with 0.5 µL RNAase A
  4. Treat with 1 µL RNAase I
  5. Incubate at 30 °C for 30 minutes
  6. Add 3 µL of EDTA stop solution
  7. Add 0.5 µL of Proteinase K
  8. Add 0.5 µL of Proteinase K
  9. Incubate at 30 °C for 30 minutes
  10. Incubate at 30 °C for 30 minutes
  11. Column purification
  12. Dry for 5 minutes with open lid
  13. Elute with 14 µL
  14. Load half (7 µL) with 3 µL loading dye on a 1% agarose gel
* DNA Gel Electrophoresis **﹀**

  The protocol for DNA gel electrophoresis includes mutagenic chemicals such as ethidium bromide (EtBR), SYBR Safe or another DNA staining. Carry out the protocol in an assigned area and wear gloves.

  Gel preparation:
  1. Prepare 0.8% (weight/volume) agarose in Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE) buffer.
  2. Heat the gel solution in the microwave to dissolve the agarose. Swirl the solution a few times while heating to ensure that the agarose powder is dissolved. The agarose is dissolved when the solution has bubbles and is clear.
  Casting of gel:
  1. Pick an appropriate gel stand and gel comb and place in the gel casting stand. Make sure to fasten the stand in place. Perform the next few steps quickly to prevent the gel from solidifying.
  2. Spread 15 µL (for a large stand) or 4-5 µl (for a small stand) of SYBR Safe or EtBr on the stand.
  3. Carefully pour the gel into the stand. Ensure that 3/4 of the teeth of the comb are submerged in the solution.
  4. Allow the gel to solidify. This will take approximately 30 minutes.
  Sample loading:
  1. After the gel has solidified, carefully remove the comb and transfer the gel to a gel box filled with TAE or TBE buffer.
  2. Cut a strip of parafilm. Pipette a drop of loading dye onto the parafilm, then pipette the sample on top of the loading dye. The ratio should be 1:5:dye:sample (e.g. 1 µL of dye and 5 µL of sample). Mix the solutions by pipetting up and down a few times. Repeat this step for each sample.
  3. Load the samples carefully in the slots. Make sure not to poke the gel.
  4. Load 3 µL of DNA ladder in a separate slot.
  5. Set the voltage to 100 volt.
  6. Run the gel until the DNA has moved across 2/3 of the gel. This will take approximately 45 minutes.
  7. Visualize the gel using a gel documentation system (gel doc).
* DNA Gel Purification **﹀**

  Protocol for DNA gel purification using Promega Wizard® SV Gel and PCR Clean-Up System:

  Dissolving the gel slice:

  1. Following electrophoresis, excise a DNA band from the gel and place this gel slice in a 1.5 mL microcentrifuge tube.
  2. Add 10 µL of Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50–65 °C until the gel slice is completely dissolved.

  Binding of DNA:

  1. Insert a Wizard® SV Minicolumn into a Collection Tube.
  2. Transfer the dissolved gel mixture to the Minicolumn
     assembly. Incubate at room temperature for 1 minute.
  3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert the Minicolumn into the Collection Tube.

  Washing:

  1. Add 700 µL of Membrane Wash Solution. Membrane Wash Solution should be prepared beforehand with 15 mL of 95% ethanol added per 3 mL of concentrated Membrane Wash Solution. Centrifuge at 16,000 × g
     for 1 minute. Discard flowthrough and reinsert the Minicolumn into the Collection
     Tube.
  2. Repeat step 1 with 500 µL of Membrane Wash Solution. Centrifuge at 16,000 × g
     for 5 minutes.
  3. Empty the Collection Tube and centrifuge the column assembly again for 1 minute at 16,000 x g with the microcentrifuge lid open or off to allow evaporation of any residual
     ethanol.

  Elution:

  1. Carefully transfer the Minicolumn to a clean 1.5 mL microcentrifuge tube.
  2. Add 50 µL of nuclease-free water to the Minicolumn. Incubate at room temperature for 1 minute. Then centrifuge at 16,000 × g for 1 minute.
  3. Discard the Minicolumn and store the microcentrifuge tube holding the DNA at 4 °C or –20 °C.
* [Expressing phi29 proteins in PURE*frex* system **﹀**](#PURE)

  1. Thaw DNA and Pure*frex* 2.0 on ice
  2. Pipette the following volumes in a single PCR tube

     | Component | Volume (µL) |
     | --- | --- |
     | Solution I (Buffer) | 5 |
     | Solution II (Enzymes) | 0.5 |
     | Solution III (Ribosomes) | 1 |
     | DNA (5nM) | X |
     | Green Lys | 0.5 |
     | RNAse free Milli-Q | 3 - X |
     | Total | 10 |
  3. Incubate the nuclease-free PCR tubes at 37 °C for 3 hours
  4. Treat the expressing sample with RNAase (RNaseA Solution, Promega), place the sample at 37 °C for 30 min
  5. Denaturate the protein in the sample for 10 minutes at 90 °C in 2x SDS loading buffer with 10 mM dithiotreitol (DTT)
  6. Load 8 µL on a [SDS PAGE](#sdspage)
  7. **SDS PAGE visualization:** To visualize the fluorescently labeled phi29 proteins a fluorescence gel imager (Typhoon, Amersham Biosciences) was used with a a 488-nm laser and a band pass emission filter of 520 nm.
* [FACS **﹀**](#FACS)

  1. Pick a single colony of a clone and inoculate in 5 mL LB with respective antibiotic in a 50 mL falcon tube.
  2. Incubate the culture overnight (for *E. coli* at 37 ° celsius overnight and 30 ° celsius for *P. putida*) at 250 rpm.
  3. Dilute the overnight culture 1:100 in 2 mL eppendorf tubes.
  4. Incubate the culture at 37 or 30 ° celsius and 250 rpm for 2 hours.
  5. After 2 hours, dilute the culture 1:100 in 5 mL medium in 50 mL falcon tubes.
  6. Incubate the culture at 37 ° celsius and 250 rpm for 4 hours.
  7. After 4 hours of incubation, keep the cells in the fridge for an hour.
  8. Dilute the sample 1:10 in PBS. The total sample volume can be 20 µl.
  9. Analyze your sample on FCSalyzer v.0.9.18-alpha with settings appropriate for bacterial cultures.
* Gibson Assembly **﹀**

  The Gibson Assembly is based on the protocol provided by New England Biolabs.

  1. Thaw 10 µL of 2X Gibson Assembly Master Mix on ice.
  2. Add DNA vector and insert. The recommended ratio of mol vector to mol insert is 1:3. The volume of vector and insert together should not exceed 10 µL.

     | Component | Volume (µL) |
     | --- | --- |
     | Gibson Assembly Master Mix 2X (NEB) | 10 |
     | Vector | X |
     | Insert | Y |
     | Milli-Q | 10 - X - Y |
     | Total | 20 |
  3. Incubate the assembly reaction at 50 °C for 1 hour.
* GFP Measurement in Gel Doc **﹀**

  1. Measure the OD of the cell culture and dilute the cell cultures to an OD of 1.
  2. Centrifuge the cell for 1 minute at max rpm.
  3. Discard the supernatant.
  4. Measure the fluorescence with the GFP fluorescence measurement setting installed in the gel doc.
* IPTG Induction **﹀**

  Day 1:
  1. Inoculate 10 mL of liquid LB medium containing the right selection marker with the cells of interest from the glycerol stock.
  2. Incubate the liquid media at 37 ° C and 250 rpm overnightDay 2:
  1. Inoculate 1 mL of fresh LB media into an Eppendorf tube containing the right selection marker with 1 µL of the overnight grown culture.
  2. Incubate the cells at 37 ° C and 250 rpm till the OD of the cell culture reaches 0.4
  3. Add 2 µL of 1 mM IPTG to the cell culture and incubate the cells overnight at 20 ° C and 250 rpm.
* IPTG Stock **﹀**

  1. Mix 238 mg of IPTG with 900 µl of ddH2O (we used Milli-Q).
  2. Add ddH2O until final volume is 1 mL.
  3. Filter sterilize with a 0,22 micro syringe filter (the ones that we have are 25 and that is fine)
     Store at -20 °C.
* Ligation **﹀**

  1. Thaw the ligase buffer on ice.
  2. Add the following components together:

     | Component | Volume (µL) |
     | --- | --- |
     | 10X ligase buffer | 2 |
     | T4 DNA ligase | 1 |
     | DNA vector | X (~100 ng) |
     | DNA fragment | Y\* |
     | Milli-Q | 17 - X - Y |
     | Total | 20 |

  \*The recommended vector to insert ratio is 1:3.

  3. Incubate at 4 °C for at least 1 hour. Optimally, incubate overnight at 4 °C.
* Mass Spectrometry Preparation **﹀**

  To prepare PURE*frex* reaction for Mass Spectrometry follow these steps:

  1. Add the following to a tube

  | Component | Volume (µL) |
  | --- | --- |
  | Cell free expression sample | 5 |
  | 100 mM Tris pH 8 | 6.25 |
  | 250 mM DTT | 0.4 |
  | 20 mM CaCl2 | 1.25 |
  | Milli-Q | 9.775 |
  | PURE buffer | 0 |
  | Sample after using following  [Expressing phi29 proteins inPUREfrex system](#PURE) | 2 |

  2. Incubate for 10 minutes on 90 °C
  3. Add 1.5 µl 250 mM IAM to the tube
  4. Incubate for 30 minutes in a dark space
  5. Add 0.4 µl 250 mM DTT and 0.625 Trypsin (1 mg/ml) to the tube
  6. Incubate overnight at 37 ° C
  7. Add 2.52 10% TFA (or formic acid) to the tube
  8. Centrigue for 10 minutes at max rpm
  9. Transfer to glass vial with insert
  10. Sample is ready to be put in Mass Spectrometer
* [Modular Cloning (MoClo) **﹀**](#MoClo)

  Modular cloning (MoClo) based on the Golden Gate cloning protocol.

  Pipette the following volumes in a single PCR tube for MoClo level 0:

  | Component | Volume (µL) |
  | --- | --- |
  | DNA insert (40 fmol) | 1 |
  | Vector (40 fmol) | 1 |
  | T4 DNA ligase buffer (NEB) | 1 |
  | T4 DNA ligase (NEB) | 0.5 |
  | BpiI | 0.5 |
  | Nuclease-free water | 6 |
  | Total | 10 |

  Pipette the following volumes in a single PCR tube for MoClo level 1 and 2:

  | Component | Volume (µL) |
  | --- | --- |
  | Each DNA insert (40 fmol) | 1 |
  | Vector (40 fmol) | 1 |
  | T4 DNA ligase buffer (NEB) | 1 |
  | T4 DNA ligase (NEB) | 0.5 |
  | Level 1: BsaI V2 (NEB)   Level 2: BpiI | 0.5 |
  | Nuclease-free water | Amount needed for a total volume of 10 µL |
  | Total | 10 |

  Place the PCR tube in a thermocycler with the following protocol for level 0 assembly:

  | Step | Temperature ( °C) | Time |
  | --- | --- | --- |
  | 1 | 37 | 2 minutes |
  | 2 | 16 | 5 minutes |
  | Repeat step 1 and 2 50 times. |  |  |
  | 3 | 50 | 10 minutes |
  | 4 | 80 | 10 minutes |

  Place the PCR tube in a thermocycler with the following protocol for level 1 assembly:

  | Step | Temperature ( °C) | Time |
  | --- | --- | --- |
  | 1 | 37 | 20 minutes |
  | 2 | 16 | 3 minutes |
  | 3 | 37 | 1.5 minutes |
  | Repeat step 1 and 2 15 times. |  |  |
  | 4 | 37 | 20 minutes |
  | 5 | 50 | 5 minutes |
  | 5 | 50 | 5 minutes |

  Place the PCR tube in a thermocycler with the following protocol for level 2 assembly:

  | Step | Temperature ( °C) | Time |
  | --- | --- | --- |
  | 1 | 37 | 5 minutes |
  | 2 | 16 | 10 minutes |
  | Repeat step 1 and 2 30 times. |  |  |
  | 4 | 37 | 1 hour |
  | 5 | 60 | 5 minutes |
  | 5 | 8 | ∞ |
* Nanodrop DNA Quantification **﹀**

  1. Use a Nanodrop spectrometer to quantify the DNA amount and purity in a DNA purification or extraction sample.
  2. Adjust the settings to measure double-stranded DNA (dsDNA) at 260 nm and impurities at 230 nm and 280 nm.

  Note: DNA should have a 260/280 between 1.8 and 2.0. If the value is below 1.8 it means the sample is contaminated with extraction agents, such as phenol or ethanol.
* * Polymerase Chain Reaction (PCR) **﹀**
    + Colony PCR (GoTaq) **﹀**

      1. Add the following components for a colony PCR in a PCR tube:

      | Component | Volume (µL) | Final concentration |
      | --- | --- | --- |
      | GoTaq 5X buffer | 10 | 1x |
      | 10 mM dNTPs | 1 | 200 µM |
      | Forward primer | 1 | 200 nM |
      | Reverse primer | 1 | 200 nM |
      | Colony\* | NA | NA |
      | GoTaq Polymerase (5 U/µL) | 0.2 | 20 U/mL |
      | Nuclease-free water | 36.8 | NA |
      | Total | 50 |  |

      \*Pick a colony under aseptic conditions and resuspend it in Milli-Q water.

      2. Place the PCR tube in a thermocycler with the following protocol:

      | Step | Temperature ( °C) | Time |
      | --- | --- | --- |
      | Initial denaturation | 98 | 150 sec |
      | Denaturation | 94 | 60 sec |
      | Annealing | Tannealing\* | 60 sec |
      | Extension | 72 | 1 min/kb DNA |
      | Repeat the steps from 'Denaturation' to 'Extension' 29 times. | | |
      | Final extension | 72 | 5 minutes |
      | Hold | 4 | ∞ |

      \*The annealing temperature is dependent on the melting temperature (Tm) of the primers. Tannealing = Tm - 5 °C.

      3. Check the PCR product on an agarose gel **[(see DNA electrophoresis protocol)](#Electrophoresis)**.
    + Phusion PCR **﹀**

      1. Add the following components for a Phusion PCR in a PCR tube:

      | Component | Volume (µL) | Final concentration |
      | --- | --- | --- |
      | 5X Phusion HF buffer | 10 | 1x |
      | 10 mM dNTPs | 1 | 200 µM |
      | Forward primer | 1 | 200 nM |
      | Reverse primer | 1 | 200 nM |
      | Template DNA (10 ng) | X | Varies |
      | Phusion polymerase | 0.5 | 20 U/mL |
      | Nuclease-free water | 36.5 - X | NA |
      | Total | 50 |  |

      2. Place the PCR tube in a thermocycler with the following protocol:

      | Step | Temperature ( °C) | Time |
      | --- | --- | --- |
      | Initial denaturation | 98 | 30 sec |
      | Denaturation | 98 | 10 sec |
      | Annealing | Tannealing\* | 15 sec |
      | Extension | 72 | 30 sec/kb DNA |
      | Repeat the steps from 'Denaturation' to 'Extension' 29 times. | | |
      | Final extension | 72 | 5 minutes |
      | Hold | 20 | ∞ |

      \*The annealing temperature is dependent on the melting temperature (Tm) of the primers. Tannealing = Tm - 5 °C.

      3. Check the PCR product on an agarose gel **[(see DNA electrophoresis protocol)](#Electrophoresis)**.
    + KOD Xtreme PCR **﹀**

      1. Add the following components for a KOD Xtreme PCR in a PCR tube:

      | Component | Volume (µL) | Final concentration |
      | --- | --- | --- |
      | 2x Novagen KOD Xtreme buffer | 2 | 1x |
      | 10 mM dNTPs | 2 | 400 µM |
      | Forward primer | 1.5 | 300 nM |
      | Reverse primer | 1.5 | 300 nM |
      | Template DNA (100 ng) | X | Varies |
      | Novagen KOD polymerase | 1 |  |
      | Nucleas-free water | 36.5 - X | NA |
      | Total | 50 |  |

      2. Place the PCR tube in a thermocycler with the following protocol:

      | Step | Temperature ( °C) | Time |
      | --- | --- | --- |
      | Initial denaturation | 94 | 2 minutes |
      | Denaturation | 98 | 10 sec |
      | Annealing | 68 | 30 sec |
      | Extension | 68 | 4 minutes |
      | Repeat the steps from 'Denaturation' to 'Extension' 29 times. | | |
      | Final extension | 68 | 7 minutes |
      | Hold | 12 | ∞ |

      3. Check the PCR product on an agarose gel **[(see DNA electrophoresis protocol)](#Electrophoresis)**.
  * PCR Clean-up **﹀**

    Protocol for PCR clean-up using Promega Wizard® SV Gel and PCR Clean-Up System:

    Processing PCR amplifications:

    1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

    Binding of DNA:

    1. Insert an SV Minicolumn into a Collection Tube.
    2. Transfer the prepared PCR product to the Minicolumn
       assembly. Incubate at room temperature for 1 minute.
    3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert the
       Minicolumn into the Collection Tube.

    Washing:

    1. Add 700 µL of Membrane Wash Solution. Membrane Wash Solution should be prepared beforehand with 15 mL of 95% ethanol added per 3 mL of concentrated Membrane Wash Solution. Centrifuge at 16,000 × g
       for 1 minute. Discard flowthrough and reinsert the Minicolumn into the Collection
       Tube.
    2. Repeat step 1 with 500 µL of Membrane Wash Solution. Centrifuge at 16,000 × g
       for 5 minutes.
    3. Empty the Collection Tube and centrifuge the column assembly again for 1 minute at 16,000 x g with the microcentrifuge lid open or off to allow evaporation of any residual
       ethanol.

    Elution:

    1. Carefully transfer the Minicolumn to a clean 1.5 mL microcentrifuge tube.
    2. Add 50 µL of nuclease-free water to the Minicolumn. Incubate at room temperature for 1 minute. Then centrifuge at 16,000 × g for 1 minute.
    3. Discard the Minicolumn and store the microcentrifuge tube holding the DNA at 4 °C or –20 °C.
  * Plasmid Isolation **﹀**

    Protocol for plasmid isolation using Promega PureYield™ Plasmid MiniPrep System:

    Prepare lysate:

    1. Pipette 1.5 mL of the bacterial culture in an Eppendorf tube and centrifuge for 30 seconds at maximum speed in a microcentrifuge.
    2. Discard the supernatant.
    3. Repeat steps 1 and 2 until the entire inoculum volume has been spun down.
    4. Add 600 µL of Milli-Q water to the cell pellet and resuspend completely.
    5. Add 100 µL of Cell Lysis Buffer and mix by inverting the tube 6 times. The solution should change from opaque to clear blue, indicating complete lysis.
    **Notes:**
    1. If you have more sample volume, split it. This will help complete this step quickly.
    2. Proceed to step 6 within 2 minutes. Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, process samples in groups of ten or less. Continue with the next set of ten samples after the first set has been neutralized and mixed thoroughly.6. Add 350 µL of cold (4–8 °C) Neutralization Solution and mix thoroughly by inverting the tube. The sample will turn yellow when neutralization is complete, and a yellow precipitate will form. Invert the sample an additional 3 times to ensure complete neutralization.
    7. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
    8. Transfer the supernatant (~900 µL) to a PureYield™ Minicolumn. Do not disturb the cell debris pellet. For maximum yield, transfer the supernatant with a pipette.
    9. Place the minicolumn into a PureYield™ Collection Tube and centrifuge at maximum speed in a microcentrifuge for 15 or 30 seconds.
    10. Discard the flowthrough and place the minicolumn into the same PureYield™ Collection Tube.

    Wash:

    1. Add 200 µL of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds. It is not necessary to empty the PureYield™ Collection Tube.
    2. Add 400 µL of Column Wash Solution to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.

    Elute:

    1. Transfer the minicolumn to a clean 1.5 mL microcentrifuge tube, then add 30 µL of nuclease-free water at neutral pH directly to the minicolumn assembly. Let stand for 1 minute at room temperature.
    **Notes:**
    1. Elution buffer can also be used, but it can interfere with future cloning steps.
    2. For large plasmids (>10 kb), warm the Elution Buffer to 50ºC prior to elution, and increase elution volume to 50 µL. Also incubate the column at room temperature (22–25 °C) for 5–10 minutes before proceeding to the next step.2. Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube and store eluted plasmid DNA at –20 °C.
    3. Follow the Nanodrop DNA quantification protocol to determine the purity and yield of your plasmid isolation.
  * Restriction Digestion **﹀**

    1. Choose the desired enzyme and its required buffer. The CutSmart buffer 10X is used for most NEB restriction enzymes.
    2. Add the following components together:

       | Component | Volume (µL) |
       | --- | --- |
       | 10X CutSmart buffer (NEB) | 2 |
       | Fragment (~1-2 μg) | X |
       | Restriction enzyme(s) | 1 of each enzyme |
       | Milli-Q | 18 - X - added restriction enzyme |
    3. Incubate for 4 hours at 37 °C.
    4. Inactivate the restriction enzyme by heating to 65 °C for 10 minutes.
  * SDS PAGE **﹀**

    **SDS PAGE preparation:** An SDS PAGE gel consist of a stacking gel solution and a resolving gel solution. In this protocol we use a 4% Stack gel and a 18% resolving gel (this is for protein samples between 10 kDa and 100 kDa).

    1. Prepare the following gels:

       | Component | 3x stacking gel solution 4% | 3x resolving gel solution 18% |
       | --- | --- | --- |
       | 1.5M Tris-HCl (pH 8.8) | - | 3.75 mL |
       | 0.5M Tris-HCl (pH 8.8) | 1.25 mL | - |
       | 20% SDS w/v | 0.075 mL | 0.025 mL |
       | AA/BAA 37.5%/0.8% | 7.2 mL | 0.53 mL |
       | 10% APS | 0.075 mL | 0.025 mL |
       | TEMED | 0.01 mL | 0.005 mL |
       | Milli-Q | 3.9 mL | 3.2 mL |

       This is enough to make 3 gels
    2. Assemble the moulds and pour the resolving gel in the mould
    3. When the gel is solidified, the stacking gel can be poured on top and the gel comb can be placed
    4. Let it solidify, then remove the comb. The gel is now ready to use, but can also be stored at 4 °C in hoisted paper towels.

    **Running SDS PAGE gel:** Run the gel at 100 Volt for 15 minutes and after that run it at 180 Volt for 1 hour

## Strain Specific Protocols

* *Bacillus subtilis* **﹀**
  + Electrocompetent Cells Preparation **﹀**

    1. Dilute an overnight culture of *Bacillus subtilis* 16-fold in growth medium and grow at 37 °C to an OD 600 of 0.85-0.95.
    2. Cool the cells on ice water for 10 minutes and harvest by centrifugation at 4 °C and 5000 x g for 5 minutes.
    3. Wash cells four times in an ice-cold electroporation medium.
    4. Suspend the cells in 1/40 of the culture volume of the electroporation solution with a cell concentration of 1.0-1.3 x 1010 cfu/mL.
    5. The competent cells can be stored at –80 °C until use with some decrease in transformation efficiency.
  + Transformation by Electroporation **﹀**

    1. Add 1 µL (50 ng/µL) of plasmid DNA to 60 µL of electrocompetent cells. Homogenize by gently pipetting up and down several times.
    2. Transfer mixture into a pre-chilled cuvette. Incubate for 1.0 - 1.5 minutes.
    3. Wipe moisture from the cuvette and insert the cuvette into an electroporation device.
    4. Electroporation settings:

    |  |  |
    | --- | --- |
    | **Mode** | Prokaryotes "O" |
    | **Voltage** | 2,100 V |
    | **Time constant (τ)** | 5 ms |
    | **No. of pulses (n)** | 1 |

    5. Immediately add 1 mL outgrowth medium and incubate for 3 hours at 37 °C.
    6. Plate onto selective LB agar plates and incubate overnight at 37 °C.
* *Escherichia coli* **﹀**
  + Chemically Competent Cells Preparation **﹀**

    The protocol for chemically competent cells has a duration of three days, starting from the preparation of the reagents. Throughout the protocol, it is mandatory to work under aseptic conditions to prevent contamination risks as much as possible.

    Day 1 (preparation):
    1. Prepare the necessary reagents and sterilize the reagents and centrifuge bottles.
       - 1 L Luria-Bertani (LB) medium
       - 10 mL Luria-Bertani (LB) medium
       - 400 mL 100mM CaCl2
       - 20 mL 100 mM CaCl2 + 15% glycerol
    2. Store the CaCl2 and the CaCl2 + 15% glycerol stock solution at 4°C for at least 24 hours.
    3. Take the strain of interest from the -80°C freezer and store it on ice.
    4. Streak out the desired  *E. coli* strain on an LB plate with antibiotic selection marker and incubate at 37°C overnight.

    Day 2 (preparation):
    5. Inoculate one single colony from the overnight grown plate in 10 mL LB medium with antibiotic selection marker overnight at 37°C while shaking at 200 rpm.

    Day 3:
    6. Inoculate 10 mL from the overnight grown liquid cell culture in 1 L of LB medium with antibiotic selection marker. Grow the inoculated medium at 37°C while shaking at 200 rpm until the OD 600 is between 0.4 and 0.6. The OD is measured for the first time after 1.5 hours after inoculation. (Important: the OD should not rise above 0.6 in order to obtain good competent cells)
    **Notes:**
    1. From this point on be very careful with your cells. The cells are very fragile.
    2. Everything that touches the cells should not be warmer than 4°C.
    3. Always keep the cells on ice. Resuspension steps also need to be performed on ice.
    4. Work clean and fast, the cells heat up very quickly which influences the quality of the competent cells a lot.
    7. Divide the 1 L of LB medium with cells into centrifuge cylinders. Weigh them to ensure equal weight distribution during centrifugation. Prepare a counterweight if necessary.
    8. Harvest the cells by centrifugation for 5 minutes at 4000 rpm at 4°C. Carefully resuspend the pellets in 200 mL ice-cold CaCl2 using an electronic pipet.
    9. Incubate the resuspended cells on ice for 20 minutes.
    10. Harvest the resuspended cells by centrifugation for 5 minutes at 3000 rpm at 4°C. Gently resuspend the new pellet in 100 mL ice-cold CaCl2 using an electronic pipet.
    11. Incubate the resuspended cells on ice for 1 hour. In the meantime, mark approximately 75 1.5 mL Eppendorf tubes and store them on ice.
    12. Harvest the resuspended cells by centrifugation for 5 minutes at 3000 rpm at 4°C. Gently resuspend the new pellet in 10 mL ice-cold CaCl2 + 15% glycerol using an electronic pipet.
    13. Aliquot 50 μL into sterile 1.5 mL microfuge tubes that you marked previously and snap freeze with liquid nitrogen. Since the cells are very fragile, cut the pipet tips in order to reduce stress on the cells.
    14. Store the frozen cells in the -80°C freezer.
    15. Immediately perform a heat shock transformation to determine the transformation efficiency.
  + Glycerol Stock Preparation**﹀**

    1. Incubate ~50 µL of cells in 5 mL LB medium with selection marker overnight.
    2. Pipette 1.5 mL in an Eppendorf tube and spin down the bacterial culture.
    3. Discard the supernatant.
    4. Repeat step 2 and 3.
    5. Resuspend the pellet in 0.9 mL liquid bacterial culture.
    6. Add the liquid bacterial culture from the Eppendorf tube to a cryotube.
    7. Add 0.9 mL 50% glycerol to the cryotube and mix well.
    8. Store at -80°C.
  + Luria-Bertani (LB) Agar Preparation **﹀**

    1. Dissolve 14.24 g of LB agar in 400 mL of Milli-Q water.
    2. Autoclave the LB agar solution.
  + Luria-Bertani (LB) Medium Preparation **﹀**

    1. Dissolve 20 g/L Luria-Bertani (LB) powder in water as described in the instructions provided by the manufacturer.
    2. Autoclave the medium at 121 °C to heat sterilize.
    3. Store the sterilised LB medium at room temperature.
  + Heat Shock Transformation **﹀**

    1. Thaw 50 µL of chemically competent cells on ice for 10 minutes.
    2. Add 0.1-10 ng of plasmid DNA or 20-50 ng of ligate to the competent cells. Incubate on ice for 30 minutes.
    3. Heat shock for 45 seconds at 42 °C.
    4. Incubate on ice for 2 minutes.
    5. Add 1 mL of LB medium and mix gently.
    6. Incubate for 1 hour at 37 °C.
    7. Prepare agar plate with the right corresponding antibiotic marker. Use 25 mL agar with 200 µL of antibiotic stock solution.
    8. Plate 100 µL of cell suspension on the agar plate and grow it overnight at 37 °C.
* *Pseudomonas putida* **﹀**
  + Electrocompetent Cells Preparation **﹀**

    1. Inoculate 50 mL LB medium with 7 mL of a fresh overnight culture of *Pseudomonas putida*. Grow cells at 30 °C
       to an OD 600 of 0.8.
    2. Harvest by centrifugation at 4000 rpm for 5 minutes at 4 °C.
    3. Wash twice with 50 mL of ice-cold glycerol. Centrifuge at 3000 rpm for 5 minutes at 4 °C.
    4. Resuspend cells in 0.8 mL of ice-cold glycerol. Keep on ice.
  + Transformation by Electroporation **﹀**

    1. Add 4 µL of plasmid DNA (1 µg) to 40 µL of electrocompetent cells. Homogenize by gently pipetting up and down several
       times. Transfer mixture into a prechilled cuvette.
    2. Wipe moisture from the cuvette and insert the cuvette into an electroporation device.
    3. Electroporation settings:

    |  |  |
    | --- | --- |
    | **Mode** | Prokaryotes "O" |
    | **Voltage** | 2,400 V |
    | **Time constant (τ)** | 5 ms |

    4. Immediately add 1 mL of LB medium. Incubate for 2 hours at 30 °C.
    5. Plate cells on selective plates.
* *Vibrio natriegens* **﹀**
  + Electrocompetent Cells Preparation **﹀**

    1. Grow a 10 mL culture of *V. natriegens* cells in BHIv2 (BHI with v2 salt: 204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2) overnight.
    2. Inoculate 100 mL of BHIv2 with 1 mL of overnight culture.
    3. Place in an orbital shaker at 37 °C at 200 rpm until OD 600 reaches 0.5.
    4. Separate culture into two 50 mL falcon tubes and place on ice for 15 minutes.
    5. Pellet cells at 6500 rpm for 20 minutes at 4 °C.
    6. Resuspend the cells in each tube in 5 mL (10 mL total) of electroporation buffer (680 mM sucrose, 7 mM K2HPO4, pH 7).
    7. Fill each tube to ~35mL of electroporation buffer and invert gently to mix.
    8. Centrifuge cells at 6500 rpm for 15 minutes at 4 °C.
    9. Decant supernatant with a pipette.
    10. Wash the cells with electroporation buffer as described above a total of three times.
    11. After the final wash, resuspend cells in electroporation buffer as to have an OD 600 of 16.
    12. Make aliquots of 100 µL of the cells and store at -80 °C.
  + Transformation by Electroporation **﹀**

    Transformation in *Vibrio natriegens* is based on the following protocol [Vmax™ Express Electrocompetent Cells User Guide, 7 Synthetic Genomics Inc.](https://www.biocat.com/bc/pdf/Vmax_Express_40026_100517-Rev1.6.pdf)

    1. For each transformation reaction,
       • Thaw one vial of Electrocompetent cells on ice for 5 minutes.
       • Warm one vial of BHIv2 (BHI with v2 salt: 204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2) to room temperature (20 °C to 24 °C).
       • Pre-chill a 0.1 cm electroporation cuvette on ice.
    2. As soon as the electrocompetent cells are thawed, add 10 ng – 100 ng of plasmid DNA (in TE buffer or water only) to the competent cells on ice. Briefly mix by flicking tubes.
       Note: Add no more than 2 μL of plasmid DNA to the competent cells. Concentrate the plasmid DNA if necessary.
    3. Incubate the DNA and cell mixture on ice for 3–5 minutes.
    4. During incubation, set the following electroporation parameters: 900 V, 25 uF, 200 Ohms, 0.1 cm cuvette. If further optimization is desired, voltage changes in the range of +/- 200V in 50 V increments can be used. Note: These are the optimized electroporation settings for the Gene Pulser® XcellTM Microbial System using a cuvette with a 0.1 cm gap. If other instruments and/or cuvettes are used, you may need to optimize the conditions for best results.
    5. Transfer cells to a chilled cuvette using a pipette. Verify that the cells completely occupy the bottom of the cuvette and that no air remains between walls of the cuvette and the cells.
    6. Transfer the cuvette to the shock pod and perform electroporation.
       • Note: Time constants typically range from 3.3–3.7 ms.
    7. Add 500 uL of BHIv2 recovery media to the cuvette immediately after pulse.
    8. Transfor the BHIv2 media and cell mixture to an Eppendorf tube and place in an orbital shaker (250 rpm) at 30 °C for 2 hours at to allow recovery of cells and expression of antibiotic resistance marker.
       Note: Recovery can be performed at 37 °C unless kanamycin is used for selection.
    9. During cell recovery, pre-warm LB-Miller agar plates prepared with antibiotics at the appropriate incubation temperature.

    **Plating notes**
    • We recommend incubating plates at 25 °C to 30 °C overnight.
    • For plates containing kanamycin, plates must be incubated at 25 °C to 30 °C.
    • Colonies typically appear after 12–16 hours at 25 °C to 30 °C and after ~8 hours at 37 °C.
    • Store agar plates with established VmaxTM Express at room temperature for up to 72 hours. Due to contact inhibition single colonies will remain well-isolated.
    • Do not store VmaxTM agar plates at 2–8 °C as this can adversely impact propagation and growth.
    • For long-term storage, we recommend preparing glycerol stocks with 25% glycerol and storing at −80 °C.

    | Antibiotic | Solid media | Liquid culture |
    | --- | --- | --- |
    | Ampicillin | 2-50 µg/mL | 2-25 µg/mL |
    | Kanamycin | 100 µg/mL | 200 µg/mL |
    | Tetracycline | 2.5 µg/mL |  |
    | Chloramphenicol | 5–12.5 μg/mL | 12.5–25 μg/mL |

## **Special thanks to our sponsors**

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# Team:TUDelft/Results

Sci-Phi 29

![Top](https://static.igem.org/mediawiki/2019/6/6d/T--TUDelft--Top.png)

[![](https://static.igem.org/mediawiki/2019/e/e7/T--TUDELFT--logonavbar-white.png)](https://2019.igem.org/Team%3ATUDelft)
[**Home**](https://2019.igem.org/Team%3ATUDelft)

**Project**

[Description](https://2019.igem.org/Team%3ATUDelft/Description)
[Design](https://2019.igem.org/Team%3ATUDelft/Design)
[Results](https://2019.igem.org/Team%3ATUDelft/Results)
[Demonstrate](https://2019.igem.org/Team%3ATUDelft/Demonstrate)

**Wetlab**

[Experiment Protocols](https://2019.igem.org/Team%3ATUDelft/Experiments)
[Safety](https://2019.igem.org/Team%3ATUDelft/Safety)
[Notebook](https://2019.igem.org/Team%3ATUDelft/Notebook)

**Modeling**

[Model](https://2019.igem.org/Team%3ATUDelft/Model)

[Reaction Kinetics](https://2019.igem.org/Team%3ATUDelft/Model#Kinetics)

[Copy Number independence](https://2019.igem.org/Team%3ATUDelft/Model#Kinetics)
[Transcription Variation](https://2019.igem.org/Team%3ATUDelft/Model#TranscriptionalVariations)
[Translation Variation](https://2019.igem.org/Team%3ATUDelft/Model#TranslationalVariations)

[Codon Harmonization](https://2019.igem.org/Team%3ATUDelft/Model#CodonUsage)

**Parts**

[Parts Overview](https://2019.igem.org/Team%3ATUDelft/Parts)

**Human Practices**

[Human Practices](https://2019.igem.org/Team%3ATUDelft/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ATUDelft/Public_Engagement)

**Team**

[Team Members](https://2019.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2019.igem.org/Team%3ATUDelft/Collaborations)

**Acknowledgements**

[Attributions](https://2019.igem.org/Team%3ATUDelft/Attributions)
[Sponsors](https://2019.igem.org/Team%3ATUDelft/Sponsors)

[Judging Form](https://igem.org/2019_Judging_Form?id=2918)
[Medal Requirements](https://2019.igem.org/Team%3ATUDelft/Medals)

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![Results](https://static.igem.org/mediawiki/2019/7/74/T--TUDelft--Results_logo.png)

* [**Overview**](#Overview)
* [**Parts Construction**](#PartConstruction)
* [**Orthogonality**](#Orthogonality)
* [Parts Characterization](#PartCharacterization)
* [Replication](#Replication)
* [Toxicity Assay](#ToxicityAssay)
* [**Controllability**](#Controllability)
* [Transcriptional Variations](#Transcription)
* [Portable T7 Expression System](#Portability)
* [Translational Variations](#Translation)
* [Stable Expression Across Species](#stabilization)

![](https://static.igem.org/mediawiki/2019/5/5b/T--TUDelft--Abstract_Results.png)

# Overview

The aim of this project is to develop Sci-Phi 29: a versatile platform to expand the repertoire of bacterial species currently used in synthetic biology.
Sci-Phi 29 enables host independent replication of genetic vectors using the concept of orthogonality and ensures controllable expression of a gene of interest across different bacterial species with the use of an incoherent feed-foward loop.
To achieve this goal, we investigated the bacteriophage [**Φ29 replication system**](https://2019.igem.org/Team%3ATUDelft/Design) (orthogonality module) and engineered an [**incoherent feed-forward loop**](https://2019.igem.org/Team%3ATUDelft/Design) (controllability module).

# Parts Construction

To circumvent the need to discover bacterial host specific parts and re-characterize parts for every host, we created a [collection of parts](https://2019.igem.org/Team%3ATUDelft/Parts). The collection provides essential tools for controllable expression of genes across different bacterial hosts. All parts in our collection have been assembled by golden gate assembly based on a hierarchical cloning system called [Modular Cloning (MoClo)](https://www.addgene.org/kits/marillonnet-moclo/). We extended the traditional golden gate assembly method for [modular cloning](https://2019.igem.org/Team%3ATUDelft/Design) of circuits in host specific vectors. The success of our design has been demonstrated by transfer of a genetic circuit (expressing GFP) between *E. coli* and *P. putida* backbones in a one-pot reaction.

Furthermore, we have successfully cloned ribozyme insulators through modular cloning (Figure 1) as described on our [design page](https://2019.igem.org/Team%3ATUDelft/Design#moclo).

![Snow](https://static.igem.org/mediawiki/2019/6/66/T--TUDelft--ribojunirbs.png)

**Figure 1:** Sequencing results of golden gate assembly of RiboJ and Universal RBS parts.

In addition, to establish optimal conditions for *in vivo* orthogonal replication using phi29 components, a series of 16 composite parts were constructed. Each of these parts were used to determine optimal concentrations of the phi29 components necessary to potentially demonstrate orthogonal replication.

# Orthogonality

## Part Characterization

### *In vitro* characterization of the orthogonal replication parts

To verify that our constructs for orthogonal replication were functionally expressed, we characterized the four proteins needed for orthogonal replication, DNAP, TP, SSB (p5) and DSB (p6) *in vitro*. For expressing these constructs, we used PURE*frex*® (GeneFrontier Corporation, Japan). The PURE (Protein Synthesis Using Recombinant Elements) system contains all the purified components found to be sufficient to sustain the reactions involved in protein synthesis. Specifically, we employed PURE*frex*®2.0 for its high protein yield and its prolonged-expression lifetime in bulk. After cell-free gene expression of each construct (3 hours, 37°C) we verified for the presence of our protein of interest by SDS-PAGE and/or mass spectrometry. For fluorescent labeling, BODIPY-Lys-tRNALys (FluoroTectTM GreenLys, Promega) was added, this binds to the translation products at the lysine residues sites. Since there are a multitude of proteins present in the PURE*frex*® system, for some of our constructs, especially DNAP and TP, it was difficult to properly identify the presence of our expressed protein via SDS-PAGE. Luckily, using mass spectrometry, we were able to determine whether these proteins were present.
To perform mass spectrometry analysis, PURE system samples were first trypsin-digested and then analyzed by liquid chromatography-coupled mass spectrometry (LC-MS). All mass spectrometry data were normalized to the presence of EF-TU, an elongation factor that can be found in the same concentration in all PURE system reactions. The mass spectrometer looks for the mass of unique peptide sequences, and their elution time. The raw data and optimized parameters for the mass spectrometry method can be found [here](https://static.igem.org/mediawiki/parts/0/07/T--TUDelft--RAWDATA.xlsx.zip) and [here](https://static.igem.org/mediawiki/parts/4/4f/T--TUDelft--transitionlist.xls.zip), respectively.

* DNA Polymerase **﹀**

  The expression of DNAP from our construct was confirmed by mass spectrometry, as reported by Figure 2. For DNAP, the mass spectrometer screened for the following peptide sequences: ENGALGFR and LVEGSPDDYTDIK, both were detected by mass spectrometry, so DNAP expression from our construct was successful.

  ![Snow](https://static.igem.org/mediawiki/2019/6/61/T--TUDelft--DNAP1.png)

  ![Forest](https://static.igem.org/mediawiki/2019/9/9e/T--TUDelft--DNAP2.png)

  **Figure 2:** Identification by mass spectrometry of ENGALGFR peptide (left) and of LVEGSPDDYTDIK peptide (right) in the PUREfrex sample expressing DNAP.

* Terminal Protein **﹀**

  The expression of TP from our construct was confirmed by mass spectrometry, as reported by Figure 3. For TP, the mass spectrometer screened for the following peptide sequences: IAEIER, LVDEK, ILSYLEQYR. All three peptides were detected by mass spectrometry, so TP expression from our construct was also successful.

  ![Snow](https://static.igem.org/mediawiki/2019/b/b5/T--TUDelft--TP1.png)

  ![Forest](https://static.igem.org/mediawiki/2019/f/f5/T--TUDelft--TP2.png)

  ![Mountains](https://static.igem.org/mediawiki/2019/9/97/T--TUDelft--TP3.png)

  **Figure 3:** Identification by mass spectrometry of IAEIER peptide (left), of LVDEK peptide (middle) and of ILSYLEQYR peptide (right) in the PUREfrex sample expressing TP.

* Single Stranded Binding Protein (p5) **﹀**

  The p5 expression starting from our three DNA constructs was confirmed by mass spectrometry, as reported by Figure 4. For p5, the mass spectrometer screened for the following peptide sequences: IFNAQTGGGQSFK and TVAEAASDLIDLVTR. The difference in height can be attributed to the strength of the promoters, fewer peptides were measured with decreasing strength. From this data, we can conclude that p5 expression was successful.

  Moreover, the expression of p5 (SSB) was identified by an 18% SDS-PAGE gel. From the 10-μL of expression reaction, 8 μL was loaded on the SDS-PAGE, while the other 2 μL was analyzed by the mass spectrometer for further confirmation. Click [here](https://2019.igem.org/Team%3ATUDelft/Experiments) for the protocols.
  An SDS-PAGE was carried out for the p5 protein with three different promoter strengths (Wild-Type, medium, and weak). As a negative control, one expression reaction was run in the absence of DNA. In Figure 5, the three samples with different promoter strengths and the control can be seen on the protein gel. The presence of a band (depicted with a red star) at the expected molecular weight (13,3 kDa) confirmed the presence of the expressed p5. In the control line with no gene added, a smear background of GreenLys is visible that is distinct from gene-specific bands. The other upper band could be due to contamination in the expression reaction as it is also present in the negative control.

  ![Snow](https://static.igem.org/mediawiki/parts/a/a5/T--TUDelft--p5masspec1.png)

  ![Forest](https://static.igem.org/mediawiki/parts/1/1b/T--TUDelft--p5masspec2.png)

  **Figure 4:** Identification by mass spectrometry of FNAQTGGGQSFK peptide (left) and of TVAEAASDLIDLVTR peptide (middle) in the p5 expressed sample.

  ![Mountains](https://static.igem.org/mediawiki/parts/2/2c/T--TUDelft--p6666.jpg)

  **Figure 5:** SDS-PAGE gels of SSB (p5) after cell-free expression. Translation prodfucts were analyzed by fluorescence imaging at 488 nm of an 18% gel. The bands depicted with an upper red asterisk correspond to the protein of interest with expected molecular weight. In the negative control lane where no DNA was added (most right) no specific band can be observed.

* Double Stranded Binding Protein (p6) **﹀**

  As for p5, the expression of p6 (DSB) was confirmed by mass spectrometry, as reported by Figure 6. For p6, the mass spectrometer screened for the following peptide sequences: GEPVQVVSVEPNTEVYELPVEK and FLEVATVR. The difference in height can be attributed to the strength of the promoters, fewer peptides were measured with decreasing promoter strength.

  Furthermore, the expression was confirmed by an 18% SDS-PAGE gel and mass spectrometry. Click [here](https://2019.igem.org/Team%3ATUDelft/Experiments) for the protocols.
  An SDS-PAGE was carried out for the three p6 encoding constructs with the three different promoter strengths (Wild-Type, medium, and weak). As a negative control, one expression reaction was run in the absence of DNA. In Figure 7, the three samples and the control can be seen on the protein gel. The presence of a band (depicted with a red star) at the expected molecular weight (12 kDa) confirmed the presence of the expressed p6. In the control line with no gene added, a smear background of GreenLys is visible that is distinct from gene-specific bands. The other upper band could be due to contamination in the expression reaction as it is also present in the negative control. This data confirms that we were able to express p6 successfully.

  ![](https://static.igem.org/mediawiki/parts/a/a3/T--TUDelft--p6masspec1.png)

  ![](https://static.igem.org/mediawiki/parts/9/96/T--TUDelft--p6masspec2.png)

  **Figure 6:** Identification by mass spectrometry of FLEVATVR peptide (left) and of GEPVQVVSVEPNTEVYELPVEK peptide (right) in the p6 expressed sample.

  ![Mountains](https://static.igem.org/mediawiki/parts/3/31/T--TUDelft--p55555.jpg)

  **Figure 7:**  SDS-PAGE gels of p6 (DSB) after cell-free expression. Translation products were analyzed by fluorescence imaging at 488 nm of an 18% agarose gel. The bands depicted with an upper red asterisk correspond to the protein of interest with the expected molecular weight. In the control lane where no DNA was added (most right) no specific band was observed.

### Conclusion

We were able to successfully identify DNAP, TP, p5 and p6 after *in vitro* expression of our constructs. The results indicate that our constructs are correct and can lead to the expression of the protein of interest. Furthermore, we were able to observe a difference in expression level when using different promoter strengths, for example, a lower amount of protein was produced when using the weak promoter.

## Orthogonal Replication

For this module we also wanted to demonstrate *in vitro* replication of our linear construct (OriL-GFP-Kan-OriR), to show that our construct can be propagated by the phi29 replication machinery.

* Results -- *In Vitro* Replication **﹀**
  + For this experiment, we used an *in vitro* transcription-translation system (PURE*frex*®2.0) supplemented with dNTPs and purified auxiliary proteins: single-stranded binding protein (SSB) and double-stranded binding protein (DSB), needed for an efficient DNA replication. We used 1 nM as the starting DNA concentration of our OriL-GFP-Kan-OriR with a twofold excess of plasmid DNA encoding for both DNAP and TP. After a 4 hour incubation at 30°C, we purified the DNA from the reaction as described in the [complete protocol](https://2019.igem.org/Team%3ATUDelft/Experiments). The size of our construct was determined by DNA gel electrophoresis. As a control, we used samples with and without dNTPs. On our gel, we observed that our linear construct (~2,3 kb) was successfully amplified, as shown in Figure 8. These results show that the phi29 replication machinery can be used for orthogonal DNA replication *in vitro*. We demonstrated that our origin-flanked linear construct (OriL-GFP-Kan-OriR) could be replicated *in vitro* by the phi29 minimal replication machinery.

    ![](https://static.igem.org/mediawiki/2019/f/fb/T--TUDelft--ivtt.png)

    **Figure 8:** Purified OriL-GFP-Kan-OriR from a coupled DNA transcription-translation and replication reaction with (+) and without (-) dNTPs, amplified *in vitro* using the PUREfrex 2.0 system An intense band in the expected size (~2.3 kb) is observed from the +dNTPs reaction, demonstrating the OriL-GFP-Kan-OriR *in vitro* replication.

### Conclusion

Successful replication of our linear construct has been shown on the agarose gel (Figure 8). A very intense band is visible at a size of 2.3 kb, corresponding to the size of the OriL-GFP-Kan-OriR construct. This indicates that there has been successful *in vitro* amplification of our construct.

## Toxicity Assay

When trying to express the phi29 replication machinery in *E. coli* we came into contact with, [Dr. Chang Liu and Dr. Julian Willis](https://2019.igem.org/Team%3ATUDelft/Human_Practices), who informed us that the expression of these four proteins needs to be tightly controlled to avoid toxic events.
To find conditions where DNAP, TP, p5, and p6 are expressed in cells that can show exponential growth, we conducted a toxicity assay. We individually expressed in *E. coli* BL21 (DE3) pLysS cells all the four components of the phi29 machinery under three different T7 promoter variants with varying strengths. The medium T7 promoter has a relative strength of 0.64 compared to the Wild-Type T7 promoter. The weak T7 promoter has a relative expression of around 0.1. The used pLysS strain used exhibits some leaky expression, allowing limited gene expression without IPTG induction [(Rosano et al., 2014)](#Rosano2014). Furthermore, to better find a window of non-toxicity, we induced these constructs with different IPTG concentrations. *E. coli* BL21(DE3) pLysS cells have their T7 RNA polymerase (RNAP) under the control of a lac promoter. IPTG binds to the lac repressor allowing the expression of the genes which are under the control of the lac operator. Therefore, the higher the IPTG concentrations, the higher the expression of T7 RNAP and the genes under the T7 promoter.

* Results -- Toxicity Assay**﹀**
  + We transformed all 15 constructs from the orthogonal module separately in *E. coli* BL21 (DE3) pLysS cells. All cells transformed with the constructs were plated on LB agar supplemented with the corresponding antibiotics; chloramphenicol and ampicillin. Single colonies for all constructs were grown overnight in LB with the corresponding antibiotics, and diluted the next day to have the same Optical Density (OD) at 600nm. The diluted cells were directly pipetted into a 96 well plate with different IPTG concentrations.
    The OD of the cells was measured over 20 hours in a plate reader. From the [raw data](https://static.igem.org/mediawiki/2019/4/44/T--TUDelft--rawdatatoxicity.zip) the OD over time was plotted. The maximum slope of the exponential phase was calculated from the collected data. This maximum was normalized against the maximum slope of a strain expressing GFP with the corresponding T7 promoter. These results are plotted in bar graphs, as reported in Figure 9.

    ![](https://static.igem.org/mediawiki/2019/4/4c/T--TUDelft--toxicitynoiptg.png)

    ![](https://static.igem.org/mediawiki/2019/d/de/T--TUDelft--toxicity1iptg.png)

    ![Mountains](https://static.igem.org/mediawiki/2019/5/5a/T--TUDelft--toxicity10iptg.png)

    **Figure 9:** The graphs reported in this figure show the maximum growth rate of cells containing the 5 different constructs (DNAP, TP, p5, p6 and GFP as a control). Each construct has been studied in the presence of three different strength promoters, here indicated as weak, medium, and Wild-Type promoter. For all these conditions three different IPTG inductions were applied: Top left) no IPTG induction and Top right) 1 mM IPTG induction and Bottom left) 10 mM IPTG induction

    The graphs reported in Figure 9 show that the maximum growth rate of the cells containing the constructs for the four replication proteins decreases whenever the promoter strength increases. The growth rates of cells expressing GFP were normalized so the relative difference could be compared to each other. When comparing the construct containing cells and the GFP one, we can conclude that expressing our constructs of interest using a higher promoter strength results in a higher display of toxicity by the cells.
    In some conditions, such as cells containing constructs for DNAP and p5, the cells show higher maximum growth rates than the cells containing the GFP construct. This is especially observed when strains are grown in the presence of high IPTG, which increases the concentration of expressed proteins. This can be due to the fact that high levels of GFP expression can lead to some toxicity due to H2O2 formation [(Ganini et al., 2017)](#Ganini2017). Cells containing TP and p6 show a significantly lower slope than cells containing GFP expressed under a Wild-Type and medium strength promoter, indicating that these proteins are more toxic to cells than GFP.
    When higher IPTG concentrations are supplemented to the sample, there is a higher expression of genes under the T7 promoter. This explains why higher IPTG concentrations seem to show higher toxicity for cells containing TP and p6 when normalized to cells containing GFP. In contrast, cells containing DNAP and p5 show lower toxicity at higher IPTG contractions compared to cells containing GFP. As mentioned above, this is probably due to the fact that GFP produces H2O2, and this production is also induced with IPTG.

### Conclusion

Cells containing DNAP, TP, p5, and p6 under the expression of a strong promoter show higher toxicity compared to cells where these proteins are less strongly expressed, when normalized to GFP.
Cells containing DNAP and p5 do not show extra toxicity compared to cells containing GFP, while cells containing TP and p6 do show toxicity.
Overall, our data (Figure 9) suggests that the ideal condition to express this protein *in vivo* is by using medium or low strength promoter in low IPTG induction, especially for TP and p5. Until now, we have studied some conditions for toxicity for every single construct. However, if we want to express all the components together, more fine tuning will be required.

# Control of gene expression levels with iFFL

## Overview

The behavior of genetic parts and circuits in different bacterial species is unpredictable as it is influenced by host-dependent variations [(Yang et al., 2018)](#yang). We engineered a unique implementation of [the iFFL incoherent feed-forward loop motif](https://2019.igem.org/Team%3ATUDelft/Design#control) to achieve gene expression independent of these variables.

We have incorporated T7 promoter variants into our iFFL systems, which have been used to show independence of transcriptional rates as well as being compatible with [portable T7 expression systems](https://2019.igem.org/Team%3ATUDelft/Design#control). Furthermore, a broad host range version of the system was developed and demonstrated in *E. coli* and *P. putida*.

## Transcriptional Variation

Transcriptional variations may be caused by 3 host-dependent variables:

1. Copy number
2. Promoter strength
3. T7 RNAP concentration

* **Results -- Copy Number Independence****﹀**

  The expression levels in a genetic circuit are strongly correlated to the plasmid copy number of the DNA template [(Segall-Shapiro et al., 2018)](#segall). The amount of plasmid copy number can change when the plasmid is transferred between organisms. Therefore, there is a need for expression levels independent of plasmid copy number if the same genetic circuit is used in different organisms. The steady-state solution of our model tells us that when our repressor binding is fully non-cooperative, we have complete independence of plasmid copy number.

  ![TALE system](https://static.igem.org/mediawiki/2019/e/e4/T--TUDelft--copynumber.svg)

  **Figure 11**: Steady-state GOI production for gene plasmid copy number 1 to 600 (genome integration to high plasmid copy number plasmid).

  Figure 11 shows the steady-state level of the gene of interest as predicted by the model.

  ### Conclusion

  The model without assumptions has the same expression level independent of plasmid copy number (Figure 11). We therefore can transfer our circuit between organisms and expect the expression of the gene of interest to be independent of the changes in plasmid copy number of our orthogonal plasmid.

* **Results -- Independence to Promoter Strengths****﹀**

  To validate our model prediction, we designed T7 promoters based iFFL systems with varying promoter strengths. We compared our [wild-type T7 promoter based iFFL system](http://parts.igem.org/Part%3ABBa_K2918040) to a iFFL system based on a T7 promoter variant with 50% strength compared to the wild-type ([medium T7 based iFFL system](http://parts.igem.org/Part%3ABBa_K2918048)) [(Ryo Komura et al., 2018)](#ryo).
  As a control, we express GFP without TALE regulation. We expect our iFFL systems to give the same expression level independent of the promoter strength.

  The output GFP fluorescence in *E. coli* was measured using flow cytometry during logarithmic growth phase after induction with 1mM IPTG. As a reference for background fluorescence, we used *E. coli* BL21(DE3) cells without any plasmid. The most dense region (determined by eye) in the scatter plot (forward and side-scatter) was selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram was gated to discern between fluorescent and non-fluorescent cells, by gating the *E. coli* BL21(DE3) cells without any plasmid. The median of the background was subtracted from the median of the samples, and the resulting values were plotted (Figure 12).

  ### Results

  ![TALE system](https://static.igem.org/mediawiki/2019/b/b3/T--TUDelft--promotervariation.png)

  **Figure 12**: Steady-state GFP fluorescence measurement of promoter variation using flow cytometry. The graph depicts T7 and medium T7 iFFL systems, expected to give the same fluorescence according to the model. As a control, GFP under control of an unrepressed T7 promoter was used.

  In Figure 12, similar GFP fluorescence can be observed for the T7 and medium T7 iFFL systems while the unrepressed control system shows higher fluorescence. This suggests successful insulation of gene expression from change in promoter strengths. This validates our model, which predicts that as long as the ratio of promoter strengths of both genes are kept constant we have stabilized expression.

  Raw data from flow cytometry experiments can be found [here](http://parts.igem.org/Part%3ABBa_K2918048).

* **Results -- Tunability****﹀**

  The predictions made by modeling not only tell us that we can maintain the same level of gene expression but also that we can easily tune the expression levels by changing one of the promoters. By changing one of the promoters to another variant of T7, we can expect a different level of expression, while at the same time expect it to behave similarly when transferred between organisms. Through the use of T7 variants, we could achieve wide ranges of expression levels, which can be used to establish complex genetic circuits, while also expecting it to work similarly in different biological contexts.

  We tested the prediction by changing the promoter controlling the TALE protein to a weak variant of T7, which has been shown to be about 10% in strength compared to the wild-type [(Ryo Komura et al., 2018)](#ryo). According to the model, the expression should increase.

  We measure GFP fluorescence using flow cytometry. As a reference for background fluorescence, we use *E. coli* BL21(DE3) cells without any plasmid. The most dense region (determined by eye) in the scatter plot (forward and side-scatter) is selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram is gated to discern between cells that are "off" or "on" (expressing GFP or not), by gating the *E. coli* BL21D(E3) cells without any plasmid.

  The median of the background is subtracted from the samples and are compared.

  ### Results

  Figure 13 indicates that there is higher fluorescence when a weaker T7 promoter variant is used to express the TALE protein in comparison to the original system.

  ![TALE system](https://static.igem.org/mediawiki/2019/a/a8/T--TUDelft--Tunability.png)

  **Figure 13**: Steady-state GFP fluorescence measurement of *E. coli* BL21(DE3) cells expressing our iFFL systems. The graph depicts a different T7 iFFL systems, one with both promoters T7, one with both medium strength and one where the promoter controlling of the TALE is weak. As a control GFP under control of an unrepressed T7 promoter was used.

  ### Conclusion

  The data suggests that we can increase expression of the gene of interest by decreasing the promoter strength of the TALE gene, through the use of a weaker T7 promoter variant. As orthogonal promoter varients will keep their ratio in strengths independent of biological context this would allow for both tunability and predictability. Therefore, we expect the use of T7 promoter variants to allow for high-precision tuning of expression levels in genetic circuits in a host-independent manner.

* **Results -- Independence to T7RNAP Concentration****﹀**

  Transcriptional variations may also be caused due to changes in RNAP concentrations in expression strains of different species. This is also the case for portable T7 RNAP expression systems where variations are caused by translational differences between organisms. The iFFL loop was tested for different concentrations of IPTG, and thus on the effect of changes in *E. coli* BL21(DE3) T7 RNAP concentrations. In unrepressed systems, the expression of the GOI is a function of IPTG concentrations. However, in our iFFL system, since the transcriptional rates of TALE and GFP are under the control of T7 promoters, both genes have a constant ratio of transcription rates, thus similar GOI expression is expected. As a control, we expressed GFP under the control of the T7sp1 promoter.

  The output GFP fluorescence in *E. coli* was measured using flow cytometry during logarithmic growth phase after induction with different concentrations of IPTG. As a reference for background fluorescence, we used *E. coli* BL21(DE3) cells without any plasmid. The densest region (determined by eye) in the scatter plot (forward and side-scatter) was selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram was gated to discern between cells that are "off" or "on" (expressing GFP or not), by gating wild-type *E. coli* BL21(DE3). The median of the background was subtracted and the samples were compared to each other.

  ### Results

  In Figure 14, the GFP fluorescence of the unrepressed control changes with changing concentrations of IPTG while the iFFL system shows the same GFP expression across different IPTG concentrations. Thus, the iFFL system has been shown to insulate gene expression against changes in transcription rates (achieved by varying IPTG concentrations). There is a high difference in levels of expression between the controlled and uncontrolled samples. The large difference could be due to overexpression of TALE protein which significantly repressed the expression of GFP.

  ![TALE system](https://static.igem.org/mediawiki/2019/a/a9/T--TUDelft--IPTGtitration.svg)

  **Figure 14**: Steady-state GFP fluorescence measurement of IPTG titration using flow cytometry. The graph depicts a T7 iFFL system, induced using different levels of IPTG, which, according to the model, should give the same result. As a control, GFP under control of an unrepressed T7 promoter was used.

  ### Conclusion

  We can infer from the data that with our orthogonal iFFL, we can maintain constant levels of expression in varying T7 RNAP concentrations, which further corroborates with the potential of developing a portable system integrated to our loop. Unfortunately, a great decrease in fluorescence was observed. This is likely due to still fairly high rates of expression of TALEsp1, as its expressed by the same RBS. Therefore, a next step would be the design of RBS sequences that show lower translation rates of TALE or the use of lower strength T7 promoter variants.

### Conclusion

Results above indicate successful implementation of the iFFL system to insulate from transcriptional variations. Varying promoter strengths and different IPTG concentrations do not affect expression levels as long as a constant ratio of transcription rate of TALE and GFP is maintained.
Furthermore, we demonstrated the tunability of the iFFL system to achieve different levels of gene expression. As predicted by our model, we achieved gene expression independent of changes in transcriptional rates by maintaining constant ratios of transcriptional rates of TALE and GFP genes.

## Portable T7 Expression System

Combining our iFFL circuit with [portable T7 expression systems](https://2019.igem.org/Team%3ATUDelft/Design#control) we see the potential of establishing universal systems for controllable expression.

* **Results -- Cotransformation with Portable T7 Expression System** **﹀**

  To test the compatibility of our system with the portable T7 RNAP expression system, we first cloned our T7 promoter based optimized iFFL into low and medium copy number backbones (pICH82113, and pICH82094 respectively) from the MoClo toolkit. These plasmid backbones contain origins of replication compatible with ColE1.
  The clones were then co-transformed with the [plasmid](https://www.addgene.org/71428/) containing the portable T7 expression system into *E. coli* TOP10. Wild-type *E. coli* TOP10 does not endogenously express T7 RNAP.

  ### Results

  ![TALE system](https://static.igem.org/mediawiki/2019/3/34/T--TUDelft--cotransformations.png)

  **Figure 15**: Fluorescence histogram of cotransformations obtained by flow cytometry. Black is regular *E. coli* TOP10 cells, green is cotransformation of our  [**T7 promoter based optimized iFFL**](http://parts.igem.org/Part%3ABBa_K2918010), pink is cotransformation of an iFFL without repression.

  Figure 15 shows the fluorescence histogram obtained by flow cytometry. A higher level of fluorescence is observed when our plasmid is cotransformed with the UBER plasmid.

  ### Conclusion

  We demonstrated the compatibility of our iFFL circuit with orthogonal T7 RNAP expression circuits. Setting the basis for universal systems that establish stable levels of expression across bacteria.

## Translational Variation

Strengths of ribosome binding sites (RBSs) across organisms change due to differing anti-Shine Dalgarno sequences [(Salis, Mirsky, & Voigt, 2009)](#salis) therefore, circuits need to be retuned. The iFFL system can be used to achieve controllable gene expression despite differences in RBS strengths. In order to see the effect of translational variation on the expression levels of the gene of interest (GOI) we modeled our system for a range of translational rates for both genes (TALE and GOI).

* **Results****﹀**

  ![TALE system](https://static.igem.org/mediawiki/2019/4/4c/T--TUDelft--translationvariation.svg)

  **Figure 16**: Steady-state GOI production while translation rates of both TALE and GOI are changed. The lines indicate the constant ratio of the translation rates.

  As can be seen in Figure 16, the kinetic model maintains the same level of GFP expression when the translation rates for both genes remain in a constant ratio. To keep the same ratio in translation rates across organisms we used the same ribosome binding site (RBS) for both genes. Using the same RBS ensures that translation initiation for both genes change in a similar manner [(Salis et al., 2009)](#salis), more on the design choices can be found [here](https://2019.igem.org/Team%3ATUDelft/Design).

  ### Conclusion

  According to our model solution we can maintain the same level of GOI expression when both translation rates (for TALE and for the GOI) remain constant. We therefore designed our system to contain the same RBS in front of both TALE and the GOI.

## Expression across Different Organisms

In order to test our system in different organisms we made a broad host range version of our iFFL. This allows us to test the functioning of our iFFL circuits in  *E. coli* and  *P. putida*. Thereby demonstrating gene expression insulated from variations associated with microbial hosts.

* **Results -- Expression Across Organisms****﹀**

  Broad host range promoter (PBHR) was designed by combining the conserved -10 and -35 regions from  *E.coli* and  *B.subtilis*   [(Yang S et al., 2018)](#yang)  and the promoter was engineered to contain a binding site for TALE repressor (PBHRsp1). Using the PBHR and PBHRsp1, we constructed an iFFL genetic circuit driving GFP expression.
  The circuit was transformed in *E. coli* and  *P. putida* and, output fluorescence was measured by flow cytometry during logarithmic growth phase. To correct for background fluorescence, *E. coli* and  *P. putida* without plasmids were used as blanks. GFP under the control of PBHRsp1 was used as a positive (unrepressed) control. As the cell morphologies of *E. coli* and *P. putida* are different they cannot be compared directly, gating was based on the most dense regions in the scatter plot for each organism. In order to compare the GFP expression levels between each organism, the background fluorescence for each organism was subtracted by its respective blank.

  ### Results

  In Figure 17, the median fluorescence of the gated populations is plotted. A large difference in expression levels is observed between the unrepressed controls and the broad host range promoter based iFFL systems in *E. coli* and *P. putida*. However, similar levels of expression were observed from iFFL systems in *E. coli* and *P. putida*. The difference in expression levels between the unrepressed circuit is significantly higher than the difference in expression levels between the iFFL system (578530 and 2351.2, respectively).

  ![TALE system](https://static.igem.org/mediawiki/2019/5/5d/T--TUDelft--man.png)

  **Figure 17:**  Comparison of expression across *E. coli* and *P. putida*. Blue shows constitutive expression and red shows the expression when our iFFL system is implemented.

  ### Conclusion

  The data suggests that our system succesfully decreased the differences in expression levels between *E. coli* and *P. putida*. More tests would need to be done to truly demonstrate that our system can achieve similar expression across bacterial species. However, we strongly believe that with our wet lab results in combination with modeling predictions we have laid the foundation stabilization of genetic circuits across species barriers.

### References

* [Ganini, D., Leinisch, F., Kumar, A., Jiang, J., Tokar, E. J., Malone, C. Mason, R. P. (2017). Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells. Redox biology, 12, 462–468.](http://www.ncbi.nlm.nih.gov/pubmed/28334681)
* [Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in microbiology, 5, 172.](https://www.frontiersin.org/articles/10.3389/fmicb.2014.00172/full)
* [Ryo Komura, W. A., Keisuke Motone, Atsushi Satomura, Mitsuyoshi Ueda (2018). High-throughput evaluation of T7 promoter variants using biased randomization and DNA barcoding." PLOS ONE.](http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196905)
* [Salis, H. M., et al. (2009). Automated design of synthetic ribosome binding sites to control protein expression. Nature Biotechnology 27(10): 946-950.](http://www.nature.com/articles/nbt.1568)
* [Segall-Shapiro, T. H., et al. (2018). Engineered promoters enable constant gene expression at any copy number in bacteria. Nature Biotechnology 36: 352.](http://www.nature.com/articles/nbt.4111)
* [Yang, S., Liu, Q., Zhang, Y., Du, G., Chen, J., & Kang, Z. (2018). Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology.](http://www.ncbi.nlm.nih.gov/pubmed/29061047)

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