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src="https://static.igem.wiki/teams/4378/wiki/img/terminator-glasses.png">Cloning</button>
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<p>Three attempts of Gibson assembly, which would produce a functional plasmid containing all the TPADO genes, and one attempt to join the DNA segments through PCR reaction, were performed.
- The protocol used for the Gibson assembly is stated in (link to General Protocols) and was done with equimolar amounts of the three inserts and pET24a vector (when included).</p>
+ The protocol used for the Gibson assembly is stated in <a href="{{ url_for('pages', page='general_protocols') }}">General protocols</a> and was done with equimolar amounts of the three inserts and pET24a vector (when included).</p>
<h3>Attempt 1 - Gibson assembly of inserts and BioBrick ligation</h3>
- <p>The fragments containing tphA1, tphA2 and tphA3 genes came in a concentration of 63.7 ng/µliter, 76.3 ng/µliter and 38 ng/µliter, out of which 1 µliter was taken from each, for Gibson assembly, performed according to protocol.
- This resulted in an estimated concentration of 7.5 ng/µliter, accounting for the overhangs.
+ <p>The fragments containing tphA1, tphA2 and tphA3 genes came in a concentration of 63.7 ng/µL, 76.3 ng/µL and 38 ng/µL, out of which 1 µL was taken from each, for Gibson assembly, performed according to protocol.
+ This resulted in an estimated concentration of 7.5 ng/µL, accounting for the overhangs.
This low amount of overall tphA1A2A3 fragment would cause more issues further down the experiment, as all calculations had to be adjusted to make up for this issue.
<br><br>
To insert these genes into a pET24a vector, the construct resulting from the Gibson assembly and the vector were digested using EcoRI and PstI.
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<p>A third Gibson assembly was performed with the three inserts and pET24a vector, with newer reaction components.
The vector was prepared from a pET24a plasmid which was digested with EcoRI-HF and PstI-HF in rCutSmart buffer (all Thermo Fisher).
Digested DNA was then separated on a 1% agarose gel and extracted with GeneJET Gel Extraction Kit from Thermo Scientific, with the modifications of 11 μl of elution buffer to concentrate the DNA as much as possible, as well as ~5 min incubation at room temperature with elution buffer before the final centrifugation step.
- The concentration of the digested vector (measured with Nanodrop) was very low, which led to a total volume of 30.62 uL Gibson assembly reaction mixture instead of the 20 uL in the protocol.
+ The concentration of the digested vector (measured with Nanodrop) was very low, which led to a total volume of 30.62 µL Gibson assembly reaction mixture instead of the 20 µL in the protocol.
<br><br>
Screening transformation was done with NEB 5-alpha competent <em>E. coli</em> (high efficiency) cells (from New England BioLabs) that were transformed with 3 μl of the two samples, according to the protocol from the manufacturer of the cells.
- Controls for the transformation were 5 uL dH2O (negative) and 8.78 ng pET24a (positive). 100 μL of each incubated mixture were plated on kanamycin plates (except for Gibson positive control, plated on kanamycin + ampicillin), and 50 μL and 20 μL (+30 μL SOC) of the assembly mix were plated on kanamycin plates as well.
+ Controls for the transformation were 5 µL dH2O (negative) and 8.78 ng pET24a (positive). 100 μL of each incubated mixture were plated on kanamycin plates (except for Gibson positive control, plated on kanamycin + ampicillin), and 50 μL and 20 μL (+30 μL SOC) of the assembly mix were plated on kanamycin plates as well.
The result showed functioning controls but only two colonies in total for the plates with the assembly mix (see Figure 2).
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<img style="width: 75%;" src='https://static.igem.wiki/teams/4378/wiki/img/lab-enzymes/tpado/tpado-gibson-assembly-transformation.png' alt="Transformation of Gibson assembly products in NEB 5-aplha cells">
<p><em>Figure 2: Transformation of Gibson Assembly products.
- From left to right; Gibson Assembly PC, transformation positive control, 100 μL Assembly mix and 20 μL Assembly mix.
+ From left to right; Gibson Assembly positive control, transformation positive control, 100 μL Assembly mix and 20 μL Assembly mix.
The colonies on the two rightmost plates are marked with black circles.
The negative control and 50 μL Assembly mix were both without colonies.</em></p>
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<p>The programme used was the colony PCR with 2 min extension time, combined with said annealing temperature gradient.
Reaction mixture was done as for colony PCR but template DNA consisted of 0.7 ng of each of the three fragments.
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- The PCR results were visualised on a 1 % agarose gel, which showed no bands around 3.6kb and therefore that this attempt to connect TphA1A2A3 also failed.
+ The PCR results were visualised on a 1 % agarose gel, which showed no bands around 3.6 kb and therefore that this attempt to connect TphA1A2A3 also failed.
Note that unfortunately neither negative (e.g. dH<sub>2</sub>O) or positive control (e.g. pET24a as DNA template) were used in the experiment so the success of the actual PCR programme could not be determined.
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