Restriction Ligation cycle 1
Design
Part of our two-plasmid system contains a sevenfold repetitive sequence, referred to below as GFP11ß. This repetitive structure significantly complicates both primer design and PCR implementation. Thanks to Biomatik, the gene has already been delivered in a pET-16b plasmid with BamHI and NdeI restriction sites. The pNit plasmids we selected also have these restriction sites. For this reason, BamHI and NdeI were chosen as restriction enzymes to integrate the GFP11ß gene into the pNit plasmids.
Build
The following figures show the plasmid maps of pET16b-GFP11ß and pNit QT/RT.
An attempt was made to cut out the GFP11ß gene from the plasmid pET16b-GFP11ß using BamHI and NdeI restriction enzymes. The fragment was then to be integrated into our empty pNit vector by restriction and ligation. For digestion, both slow digestion and fast digestion were performed in NEB's CutSmart and rCutSmart buffers, respectively. Initially, 1 µL pET16b-GFP11ß was used as a template.
Test
To test whether the restriction was successful, an agarose gel was run. This was done using a 1% agarose gel and run for 45 minutes at 100V.
Learn
The restriction digestion was successful, as the product of the single digestion runs higher on the gel than that of the double digestion. In addition, the missing DNA region corresponds approximately to the expected size of our target fragment (~700 bp). This suggests that the GFP11ß fragment was successfully excised, but at a low concentration. Since the fragment accounts for about one-tenth of the total size of the plasmid, it can be assumed that it accounts for only about 1/10 of the amount of DNA used, i.e., approximately 100 ng.
Restriction Ligation cycle 2
Design
In the previous experiment, the restriction digestion was successful, but the concentration of the excised fragment was too low. For this reason, higher starting concentrations of the plasmid were used in the further cycles to achieve a sufficient fragment yield.
Build
For the Fast Digest, 1 µg and 5 µg of the plasmid pET16b-GFP11ß were used as templates. BamHI and NdeI were also used as restriction enzymes.
Test
To test whether the restriction was successful, an agarose gel was run. This was done using a 1% agarose gel and run for 45 minutes at 100V.
Learn
Even an increased plasmid concentration did not lead to the desired result. In addition, the intensity of the bands in the gel decreased significantly. Various other restriction enzymes were then tested in order to produce longer fragments and thus facilitate the isolation of the GFP11ß gene. However, these attempts were also unsuccessful.
Gibson assembly 1
Design
After our failed attempts at creating our desired constructs using restriction/ligation, we decided to make another attempt at creating new primers to enable amplification by PCR. The primers were created with overhangs with an approximate length of 15-20 base pairs. These overhangs are homologous and should create sticky ends that can be used for Gibson assembly.
Build
The primers were used to amplify the GFP11ß gene from the pET16b-GFP11ß plasmid. The corresponding primers were also used to amplify pNit_QT/RT plasmids.
Test
The resulting PCR products were subsequently analyzed by running them through a 1% agarose gel for 45 min at 100 V.
Figure Agarosegel of Gibson Overhang PCR-products. From right to left marker (M); GFP11ß (gene fragment of the 11th GFP ß-strand gene fragment); GFP1-10 (gene fragment of the GFP ß-strands 1-10); RT (pNit_RT1); RC (pNit_RC1); RC1 (pNit_RC1 from a plasmid prep as a control).
Learn
The Gel showed bands at the correct size we expected for our product which suggests that our amplification was successful and that no unspecific binding had occurred. We proceeded to utilize these PCR products in subsequent Gibson assembly experiments.
Gibson assembly 2
Design
Gibson assembly allows two fragments to be ligated together using overlapping overhangs. Three enzymes work simultaneously in the reaction. T5 exonuclease digests the 5' end of a DNA double strand, creating sticky ends that are complementary to each other. Hybridization of the complementary double strands occurs. DNA polymerase separates double strands and synthesizes missing pieces, and ligase connects the DNA sequences to each other, resulting in the ligated construct. In our case, the GFP11ß gene would be inserted into the pNit-QT1/RT1 backbone.
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The DNA fragments were mixed and assembled using a Hifi Master Mix that contained the necessary nucleases and ligases (see MaM for more details). The resulting mixture was subsequently incubated at 50 °C for 60 minutes.
Test
The products of the assembly were thereafter introduced into E.coli Dh5α via heat shock, plated on ampicillin infused LB-Agar plates and incubated at 37 °C overnight. No colony growth was observed.
Learn
The lack of colony growth after heatshock and incubation suggested that something went wrong during assembly, as a result of which E. coli was not able to take up an intact plasmid and was subsequently eliminated by the ampicilin.
Gibson assembly 3
Design
Our previous attempt at Gibson assembly implied that something during the assembly process had to have gone wrong to not yield an intact construct. Because of this we reviewed our experiment design and noticed that our overhangs of 15-18 bp were on the short end of what was described in literature. Shorter overhangs are weaker than longer overhangs due to the fact that they are not able to produce as many hydrogen bonds holding them together. We hypothesized that maybe the assembly temperature could be too high resulting in our overhangs perhaps overlapping but instantly separating from each other again as a result of which no proper ligation could be performed. As a result of this we decided to perform the assembly at lower temperatures and over a longer period of time. We chose room temperature, 30 °C and 37 °C because these temperatures were easiest to accommodate overnight with the facilities we had at our disposal.
Build
The DNA fragments were mixed and assembled by mixing them together with a Hifi Master Mix that contained the necessary nucleases and ligases (see MaM for more details). The resulting mixtures were subsequently incubated at 30 °C, 37 °C and room temperature overnight.
Test
The products of the assembly were thereafter introduced into E.coli Dh5α via heat shock, plated on ampicillin infused LB-Agar plates and incubated at 37 °C overnight. We observed colony growth. Colonies were picked and used to perform a colony PCR that confirmed insert integration into the MCS for 1 colony from the 30 °C assembly and 1 colonies from the 37 °C assembly.
Figure Agarosegel of colony PCR products of Gibson assembly heatshock colonies. Assemblies were carried out over night at 30 °C, 37 °C and room temperature (RT). All available colonies were tested. Marker (M); Colony (C).
Learn
The results of the colony PCR suggested that assembly at lower temperatures was successful in inserting the target gene into the multiple cloning site of the pNit_QC Vector. Integration was subsequently also confirmed via sequencing by MicroSynth and confirmed our previous results.
Figure Sequencing results alignment of pNit_QC_GFP1-10 from MicroSynth with the designed sequence. The alignment shows that the insert is correctly integrated into the MCS.
DFO production & quantification
Design
To verify potential DFOB production, a reliable method for its extraction and quantification was required. The design phase began with a literature review to identify a suitable approach. This led us to the Chrome Azurol S (CAS) assay, which enables the detection of siderophores like desferrioxamine B (DFOB) in the absence of unbound iron ions. Additionally, the CAS assay was selected to evaluate different methods for removing iron ions bound to DFOB.
Build
DFOB was produced using Streptomyces coelicolor and purified with a combination of XAD-4 and XAD-16 resins. Following elution with methanol, the DFOB was dried and resuspended in ddH₂O. A CAS assay was then performed on the DFOB samples, alongside an EDTA standard curve, to confirm the presence of chelators and to quantify the concentration produced.
Test
Upon extraction, the DFOB solution exhibited a brownish coloration, indicating the presence of bound iron ions. To address this, CAS assays were conducted using DFOB samples treated with ascorbic acid, sodium carbonate, and sodium hydroxide in an effort to remove the bound iron. The results suggested ascorbic acid, sodium carbonate and sodium hydroxide were effective in releasing iron ions. However, similar color changes were measured in control samples containing only the additives without DFOB
Learn
The successful purification of DFOB demonstrated that the extraction method could be applied in future experiments if DFOB production in Rhodococcus opacus 1CP is achieved. The CAS assay proved useful for determining DFOB concentrations. However, its sensitivity limits became apparent when evaluating iron ion removal methods. For such applications, a more specific or less interference-prone assay should be researched. Furthermore, removing the reducing agent or neutralizing the pH before performing the CAS assay could be beneficial.