<p>This feedback loop is essential for improving future designs and assemblies.</p>
<p>This feedback loop is essential for improving future designs and assemblies.</p>
<p>This feedback loop is essential for improving future designs and assemblies.</p>
<p>This feedback loop is essential for improving future designs and assemblies.</p>
<p><b> GA 1 & 2 </b></p>
<p>As it was the teams first time building a construct we missed out a few steps, such as the DpnI cleanup of the plasmid. We implemented this step into all future Gibson assembly attempts. </p>
<p><b> GA 3 & 4 </b></p>
<p>We learnt while testing these assemblies that our primers had multiple binding sites, leading to different possible bands. Testing with primers that had no binding sites or few sites was started. After this attempt, we had a discussion with our PI and an expert who had worked with Gibson before hand. They recommended shortening the PCR amplification cycles from 30 to 10 for the fragments, as our fragments were large, this would lead to less possible mutation, and have an insignificant impact on the concentration. They also recommended changing the ratio of vector to fragment from 1:1 to 1:2, as having an excess of fragments could lead to a higher chance of overhangs to bind. It was also recommended switching from milliQ water to nuclease free water. This made our electrophoresis gels much clearer and partially no contamination. We decided to increase the amount of assembly added into the competent cells, as our assembly possibly had a low concentration.</p>
<p><b> GA 5 & 6 </b></p>
<p>We learnt, from this and the previous attempt, that changing the ratio of the vector to the fragment was not necessary. After debriefing with our PI, we doubted whether the PCR was having an effect on the binding sites of the fragments, not allowing them to form the construct. We decided that it might not be effective to amplify the fragments and use the original g-blocks, even if they were at a much lower concentration.</p>
<p><b> GA 7 & 8 </b></p>
<p>As no growth was observed from these plates, it was clear that the concentration of plasmid and g-blocks were too low, or that too small of a concentration of the assembly was added to the competent cells. We decided to go back to amplifying the fragments. We chose to follow the protocol and guidance from our PI, and past attempts to ensure the best possible outcome. This meant doing 10 cycles, and taking the best fragments and pSEVA based on the gel electrophoresis, however this time applying it with the high fidelity Gibson Assembly master mix.</p>
<p>As no growth was observed from these plates, it was clear that the concentration of plasmid and g-blocks were too low, or that too small of a concentration of the assembly was added to the competent cells. We decided to go back to amplifying the fragments. We chose to follow the protocol and guidance from our PI, and past attempts to ensure the best possible outcome. This meant doing 10 cycles, and taking the best fragments and pSEVA based on the gel electrophoresis, however this time applying it with the high fidelity Gibson Assembly master mix.</p>
<p><b> GA 9 & 10 </b></p>
<p>After it was concluded that the assembly had failed, we reasoned that our inserts might be too large, making it hard for the construct to form. As well as the possibility that the fragments' overhangs were being altered by the PCR.</p>
<p><b> GA 1 & 2 </b></p>
<p>After performing 10 Gibson Assembly with no success, we considered that the full construct was too large to form. We decided to pivot and create a smaller construct using the first two fragments. Additionally, we were advised to pick more single colonies from the transformed plates, as there was a possibility that one might have a successful assembly.</p>
