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.
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.
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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.
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.
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
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MoClo Assembly
Goal:
Assembling the parts of the replication machinery separately with MoClo
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:
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
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Toxicity Experiments 2
Goal:
Determinig whether the orthogonal replication constructs could be toxic to the cell
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
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Characterization of Level 1
Goal:
Characterize the different promoters strengths in the assembled constructs
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.
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:
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.
Level M constructs (L1D + L1E, L1G + L1B) were both cloned in low and medium copy backbones. The constructs were then co-transformed with MK1283 which contains a plasmid harbouring a T7 RNAP mixed forward loop. Fluorescence was detected by FACS.
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:
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.
