For the assembly of our library we annealed the Fragments v1.1+v1.2 to v1 and v2.1+v2.2 to v2, and then v1+v2 to the variable region for the Monobody construct. The correct size of the assembled construct was estimated by a gelelectrophoresis (Figure 1) and then sequenced. To get further insight into the theoretical assembly take a look here. The size of the annealed v1 fragment is 114 bp, the size of v2 is 132 bp. These are the fragments inserted into our Monobody construct (BBa_K2082000) to build the library. Figure 1: Gelelectrophoresis of assembled construct for Nanobodies. Photography of 1% agarose gel dyed with ethidium bromide. Loading of the samples (from right to left): Monobody construct with variable region (MB), 1kb DNA Ladder (by New England Biolabs).
We further annealed F2.1 and F2.2 to the region containing the variable region called F2 for the Nanobody construct. To get more insight take a look here. The size of the annealed F2 fragment is ought to be 143 bp, this was checked by gelectrophoresis (Figure 2) first and sequencing later. This then can be inserted into our Nanobody construct (BBa_K2082001) to create the Nanobody library.
Figure 2: Gelelectrophoresis of assembled construct for Nanobodies. Photography of 1% agarose gel dyed with ethidium bromide. Loading of the samples (from right to left): 1kb DNA Ladder (by New England Biolabs, NB construct with variable region.
As we were sure our constructs were right, we strieved for having as much clones as possible. To create enough room for the high quantities of colonies we produced, we used big agar plates (picture below). They consisted of 750ml LB-Agar and provided space for the amount of about 15 normal agar plates. We totally recommend larger plates for every team with a library approach!
Comparison of Polymerases
As our variable regions (for each Monobodies and Nanobodies) were ordered through oligonucleotide synthesis, the strands naturally were single stranded. For the correct assembly of the variable regions into the construct, they had to be annealed. For this purpose we tried using the Klenow Fragment (3'→5' exo-) by New England Biolabs at first. However, the cloning of the Klenow annealed fragments was not optimal.
For example, when inserting the variable regions for the Nanobodies, the primers called F2.1 and F2.2 had to be annealed, to then be cloned into the Nanobody construct (missing the variable regions). When separating the samples (annealed oligonucleotides) by gel electrophoresis the reason why was hinted as no fragments of 143 bp were found in the bands on which the annealed sample were loaded (see Figure 1).
Figure 1: Gelelectrophoresis of annealed oligonucleotides. Photography of 1% agarose gel dyed with ethidium bromide. Loading of the samples (from right to left): Primer F2-1, Primer F2-2, 100bp plus ladder (by New England Biolabs), empty space, three times the by the Klenow Fragment annealed parts, empty space, 100bp plus ladder (by New England Biolabs).
We then concluded to use other Polymerases to anneal the oligonucleotides. For that we annealed two oligonucleotides called V1-1 and V1-2 to a fragment called V1 as well as V2-1 and V2-2 to a fragment called V2. These were used to bring in the variable region of the Monobody construct, inhabitating the randomized regions essential for binding proteins in the following.
For this purpose we used the following polymerases: Q5 High-Fidelity DNA Polymerase (by New England Biolabs), KOD DNA Polymerase (by Merck Millipore), Phusion High-Fidelity DNA Polymerase (by New England Biolabs), GoTaq G2 (by Promega).
In Figure 2.1 one can see the result of the annealing of the oligonucleotides V1-1 and V1-2 to the fragment V1 and V2-1 and V2-2 to the fragment V2 by the four polymerases.
This was also used with the same four Polymerases mentioned above for the annealing of the oligonucleotides F2-1 and F2-2 to fragment F2 containing the variable region for the Nanobody. The gel photography can be viewed in figure 2.2.
After choosing the Q5 High-Fidelity DNA Polymerase (by New England Biolabs) for the annealing of the oligonucleotides we once again checked for the right size of the fragments mentioned above (V1, V2 and F2) on an 1% agarose gel (see Figure 3).
Figure 4: Comparison of averaged number of colonies. After the transformation of assembled constructs, grown colonies were counted. The averaged values with standard error are shown in this bar graph.
