Revision as of 01:33, 17 October 2014

Cambridge iGEM 2014

Assembly

Gibson and LCR...

Overview

Ligase cycling reaction

** LCR When dealing with a eukaryotic chassis such as marchantia, it becomes increasingly important to be able to assemble constructs with a range of parts. For us, in particular, we wanted to assemble the following construct: Now, we figured out that for the above construct, we'd need to do a 7 part Gibson assembly for it to work. It was attempted, but no colonies were achieved. We were drawn in to Ligase Cycling Reaction [[...]] by its simplicity and promised efficiency. *** Test construct Deciding that it could be extremely profitable to establish a protocol, we decided to design a simple, two part construct using chromoproteins to assay whether it would be feasible to include it. The construct inserted amilCP into a cassette with a different resistance, containing mRFP1 instead of amilCP. That is, red colonies would be template, blue would be something sensible, and white would be misassembly of some sort. We contacted [[CamBIO]], the UK distributor of the Ampligase thermostable ligase enzyme, to see if we could use it. We received, generously, from them 2500U of the enzyme. For LCR, fragments need to be 5' phosphorylated. This can be done in two ways: either by ordering phosphorylated primers [currently costing 3x the amount of non-phosphorylated primers] OR by ordering normal primers, and then kinasing PCR amplified fragments using T4 Polynucleotide Kinase. The optimized protocol was followed as per the reference above. A Dpn1 digest following PCR was not done, as template would appear red. The results were the following: *** Building the actual construct The construct was split into 7 fragments, and nonphosphorylated primers were used. Initially, reactions were done in a 25ul volume, and a Dpn1 digest was performed. The results yielded no colonies following transformation. We ran out the PCR purified fragments on a gel, and saw that no bands were present. After having discussed with the authors of the paper, we found also that Ampligase performs NAD-dependent ligation, and that the T4 PNK also has NAD dependent kinase activity. In the subsequent attempt, we ran out PCR products on a gel, and then performed an extraction. This way, it was clear from the outset that all of the PCR's had worked. The buffer used in commercial gel extraction kits means that concentrations are very inaccurate on a spectrophotometer. We used the Qubit fluorescent probe based assay to measure the concentration, but unfortunately, it was not high enough to continue with the reaction. Using the gel extracted fragments as a template, we repeated PCRs, and subsequently performed a PCR-purification. This yielded accurate measurements of concentration, and enabled us to continue with the assembly. Following the reactions, colonies were observed on all of the hammerhead ribozyme constructs. A colony PCR was done with 4 primers, and all four fragments were observed by gel electrophoresis, on some of the colonies. It was repeated with 2 primers, and the bands were observed on the same colonies. We miniprepped DNA from the colonies for which the colonyPCR was positive, and performed a restriction digest. Two colonies' DNA appeared to produce the correct fragments upon gel electrophoresis. These were sent for sequencing. Unfortunately, sequencing did not produce reads on any of the samples sent (which included samples from other constructs later verified). We continued with the Marchantia transformation. *** Final protocol + volumes We followed the protocol in the paper above for the PNK treatment. You might find the following useful: - Have a total reaction volume of 15ul. You only transform up to 5ul, and so it saves on valuable enzyme to use a total volume smaller than 25ul. - Recommended concentrations for reagents: + Betaine: 9M, 0.75ul, final concentration: 0.45M. Make a suspension, or warm the tube if you're struggling to dissolve. + Bridging oligos: make a mix of the oligos, and dilute this with ddH2O until the final volume you add is 1.6ul. Final concentration: 30nM. + Ampligase: 5U/ul, 0.45ul + Ampligase buffer: 10x, 0.5ul [note: the kinase is done in the Ampligase buffer, so you need to add accordingly less] + DMSO: 100%, 1.2ul + NAD: 15mM, 0.5ul + PNK mix: 10ul

A Title Title

second part here

Figure 1: Illustration of the idea behind mӧsbi's modularity

A Title Title Title

Third part here

@@ Line 29: / Line 29: @@
 	                    <h2 class="section-heading">Overview</h2>
 	                    <div>
-<h4>A Title</h4>
+<h4>Ligase cycling reaction</h4>
 <p>
-First part here
+** LCR
+When dealing with a eukaryotic chassis such as marchantia, it becomes increasingly important to be able to assemble constructs with a range of parts. For us, in particular, we wanted to assemble the following construct:
+Now, we figured out that for the above construct, we'd need to do a 7 part Gibson assembly for it to work. It was attempted, but no colonies were achieved.
+We were drawn in to Ligase Cycling Reaction [[...]]  by its simplicity and promised efficiency.
+*** Test construct
+Deciding that it could be extremely profitable to establish a protocol, we decided to design a simple, two part construct using chromoproteins to assay whether it would be feasible to include it.
+The construct inserted amilCP into a cassette with a different resistance, containing mRFP1 instead of amilCP. That is, red colonies would be template, blue would be something sensible, and white would be misassembly of some sort.
+We contacted [[CamBIO]], the UK distributor of the Ampligase thermostable ligase enzyme, to see if we could use it.
+We received, generously, from them 2500U of the enzyme.
+For LCR, fragments need to be 5' phosphorylated. This can be done in two ways: either by ordering phosphorylated primers [currently costing 3x the amount of non-phosphorylated primers] OR by ordering normal primers, and then kinasing PCR amplified fragments using T4 Polynucleotide Kinase.
+The optimized protocol was followed as per the reference above. A Dpn1 digest following PCR was not done, as template would appear red.
+The results were the following:
+*** Building the actual construct
+The construct was split into 7 fragments, and nonphosphorylated primers were used.
+Initially, reactions were done in a 25ul volume, and a Dpn1 digest was performed.
+The results yielded no colonies following transformation. We ran out the PCR purified fragments on a gel, and saw that no bands were present.
+After having discussed with the authors of the paper, we found also that Ampligase performs NAD-dependent ligation, and that the T4 PNK also has NAD dependent kinase activity.
+In the subsequent attempt, we ran out PCR products on a gel, and then performed an extraction. This way, it was clear from the outset that all of the PCR's had worked.  The buffer used in commercial gel extraction kits means that concentrations are very inaccurate on a spectrophotometer. We used the Qubit fluorescent probe based assay to measure the concentration, but unfortunately, it was not high enough to continue with the reaction.
+Using the gel extracted fragments as a template, we repeated PCRs, and subsequently performed a PCR-purification. This yielded accurate measurements of concentration, and enabled us to continue with the assembly.
+Following the reactions, colonies were observed on all of the hammerhead ribozyme constructs. A colony PCR was done with 4 primers, and all four fragments were observed by gel electrophoresis, on some of the colonies. It was repeated with 2 primers, and the bands were observed on the same colonies.
+We miniprepped DNA from the colonies for which the colonyPCR was positive, and performed a restriction digest. Two colonies' DNA appeared to produce the correct fragments upon gel electrophoresis.
+These were sent for sequencing. Unfortunately, sequencing did not produce  reads on any of the samples sent (which included samples from other constructs later verified).
+We continued with the Marchantia transformation.
+*** Final protocol + volumes
+We followed the protocol in the paper above for the PNK treatment. You might find the following useful:
+- Have a total reaction volume of 15ul. You only transform up to 5ul, and so it saves on valuable enzyme to use a total volume smaller than 25ul.
+- Recommended concentrations for reagents:
+  + Betaine: 9M, 0.75ul, final concentration: 0.45M. Make a suspension, or warm the tube if you're struggling to dissolve.
+  + Bridging oligos: make a mix of the oligos, and dilute this with ddH2O until the final volume you add is 1.6ul. Final concentration: 30nM.
+  + Ampligase: 5U/ul, 0.45ul
+  + Ampligase buffer: 10x, 0.5ul [note: the kinase is done in the Ampligase buffer, so you need to add accordingly less]
+  + DMSO: 100%, 1.2ul
+  + NAD: 15mM, 0.5ul
+  + PNK mix: 10ul
 </p>
                              </div>

Team:Cambridge-JIC/Marchantia/Assembly

From 2014.igem.org