Difference between revisions of "Part:BBa K4390006"

Revision as of 21:57, 30 September 2022

JUMP N-filler

This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM

Usage and Biology

DNA assembly is the cornerstone of synthetic biology, and fast and reliable assembly is a necessity for this. Modular cloning with Type IIS restriction enzymes allows us to quickly assemble many complex multipart constructs from libraries of basic parts. The JUMP vector platform is compatible with the PhytoBrick standard, and vectors are compatible with BioBrick architecture as well as Standard European Vector Architecture (SEVA), and uses single stranded DNA overhangs (fusion sites) generated by BsmBI and BsaI digestion for ordered assembly. There are six different JUMP part types, corresponding to different elements of a transcriptional unit, the Promoter, Ribosome Binding Site (RBS), N-terminus, Open Reading Frame (ORF), C-terminus, and terminator. Figure 1 shows the fusion sites between these part types, and that basic parts can also take up more than one part by adopting a 5’ fusion site of one part and the 3’ fusion site of another.

Figure 1: JUMP fusion sites. Adapted from Valenzuela-Ortega and French 2020. The second and third lines demonstrate how a basic part can adopt fusion sites for different basic parts and be used in assembly as such.

We have designed a series of parts, so called fillers, which allow for assembly compatibility when one particular part is not desired. The parts are flanked with fusion sites for the part type, and two nucleotides to prevent a frameshift, by having the 3’ fusion site’s 4 nucleotides generate two codons together. The N-filler, for N-type parts, has a structure as seen in figure 2.

Figure 2: The structure of the JUMP N-filler in various stages of assembly. a) JUMP N-filler before domestication (level 0 assembly) represents what the N-filler looks like as a linear DNA part, if it were ordered in synthetically. b) JUMP N-filler after domestication (level 0 assembly) represents the part in a level 0 acceptor vector, for example pJUMP18-Uac. The three dots represent the flanking DNA in plasmid. c) JUMP N-filler after level 1 assembly represents what the N-filler looks like as part of a level 1 (composite) part.

As the first translated part in a transcriptional unit, the ATG in the R-N fusion site functions as the start codon. Then, since fusion sites are always 4 nucleotides long, we inserted two nucleotides to prevent a frameshift mutation for downstream parts. This means there is a single amino-acid scar generated by this part, in addition to the two amino acid scars normally in the flanking JUMP fusion sites. We chose GG as the inserted nucleotides, as this would generate a glycine when combined with the first nucleotide in the downstream fusion site, and glycine does not tend to disrupt protein structure nor function.

One example of how this is useful is with tags. Usually, an untagged control would need to be produced in the experiment as well, so instead of ordering multiple parts with different types, the fillers can be used. We used this to drastically reduce the number of parts we needed to order, and hence improved our modularity by using few parts to make many assemblies. For example one of our experiments involved expressing M. edulis metallothionein, purifying it via His-tagging, and immobilizing on a Cellulose hydrogel via a Cellulose Binding Domain (CBD). We needed entirely untagged controls for the Nickel affinity purification due to the metal chelating nature of metallothioneins, sfGFP controls to show that the His-tag with TEV protease site could bind the Nickel column, and controls with a His-tag without a CBD to show cellulose immobilization was a result of the CBD, resulting in a large combination of assemblies required (see table 1)

Table 1: JUMP assemblies performed in the aforementioned experiments

The N- and O-fillers allowed us to reuse the same parts and still have a large combination of assemblies. These fillers are universal for JUMP assembly, and hence allow for much larger levels of modularity.

Characterization

To confirm the N-filler worked as intended, we did blue-white colony screening, colony PCR of the plasmid insert, and also a simple GFP expression experiment. To further characterize the part, we performed several assemblies with varying N-filler concentration, to see the impact on assembly efficiency.

Blue-white screening

One of the techniques we frequently used to see if an assembly worked was Blue-white screening, as the Level 1 acceptor vector we used was pJUMP29-1A(lacZ), expressing lacZ as a cloning reporter. After assembly and transformation into TOP10 cells, we plated on Kanamycin, IPTG and X-gal plates. The beta-galactosidase encoded by lacZ cleaves X-gal, forming a molecule which dimerizes and turns a bright blue. This means that cells that only took up an acceptor vector without the insert would turn blue. The IPTG is there to prevent lacI from inhibiting lacZ expression on pJUMP29-1A(lacZ). We produced the untagged M. edulis Metallothionein assembly with the N- and O-fillers, which when transformed and plated produced a mixture of white and blue colonies, indicating that some assemblies had been a success (see Figure 3).

Figure 3: Kanamycin plate of TOP10 cells transformed with untagged M. edulis MT

Colony PCR

It is also possible that a white colony in Blue-white screening would have taken up an acceptor vector where the insert had been cut out by BsaI, and T4 ligase would ligate non-complementary sticky ends. To ensure there was an insert in our white colonies, we performed colony PCR, using Q5 polymerase and primers targeting the the T1 and T0 terminator regions (see Table 2). These regions are standard in all JUMP vectors, and flank the insert.

Table 2: Primers used in colony PCR to amplify inserts in a JUMP vector.

Figure 4: Colony PCR of Untagged ME MT, His-tagged ME MT, and His, CBD-tagged ME MT, using PS1 and PS2 as primers, Q5 polymerase, and a normal PCR cycle with an annealing temperature of 72°C. Because of the position of T0 and T1 in JUMP vectors, PCR amplicons will be 308bp longer than the insert.

GFP expression and further characterization

The aim of this experiment was to confirm the N-filler works, and to see if using the N-filler had a negative impact on assembly efficiency. To do this, we performed four assemblies (see Table 3), one without the N-filler and instead using an RBS ending in the N-O fusion site (an RN part), and the rest using the N-filler with varying concentrations. To do the assembly, we followed the protocols outlined in [JUMP paper citation], using pJUMP29-1A(lacZ) as an acceptor plasmid, changing amount of N-filler added as described in Table 3, using the 60-cycle digestion/ligation.

Table 3: Table on the different assemblies done to characterize the N-filler. sfGFP functions as a reporter, as cells transformed with successful assemblies will produce green colonies, and cells transformed with unsuccessful assemblies will produce white colonies. We used a CT part for the terminator to reduce the total number of parts in the assembly.

We kept the identity of the promoter, RBS, reporter and terminator constant. It is also assumed that B0034 as an R part and as an RN part is effectively identical in terms of assembly burden, differing only in the 3’ fusion site. We then transformed a 100 ul aliquot of competent BL21(DE3) cells with the product of this assembly, and plated 100 ul on Kanamycin plates. To measure the assembly efficiency, we simply counted the total number of green colonies on each plate and got results as seen in Figure 6. Figure 5 shows in binary terms that the N-filler works, by successfully producing assemblies that when transformed into cells produce GFP. Variations in the number of cells plated were controlled by keeping the volume of cells plated constant (100 ul), using aliquots of competent cells produced from the same batch, incubating plates in the same incubator for the same period of time (37°C for 24 hours), and plating in triplicate.

Figure 5: Kanamycin plate of a P-R-N(20fmol)-O-CT trial under UV light, showing the presence of GFP expressing colonies.

Figure 6: Results of experiment on N-filler changing assembly uncertainty. Experiments were done in triplicate and error bars were calculated by using standard deviation of trials.

The results of this experiment tell us if using the N-filler changes assembly efficiency. It was expected that the N-filler would reduce assembly efficiency, as compared to the control (P-RN-O-CT), there would be one more part in the level 1 assembly, and more parts tend to decrease assembly efficiency. It was also expected that the assembly efficiency would be low compared to the control, as the N-filler is a very small part, with the insert being only 10 base pairs, 8 of which are single stranded DNA after BsaI digestion. However, we did not observe any burden on assembly efficiency using this part. It is recommended by the JUMP assembly manual to use 20 fmol of each part, which is why we are directly comparing the assembly with 20 fmol of N-filler and assembly without N-filler, where each other part is kept at 20fmol. Increasing the amount of N-filler used also improves assembly efficiency, with a 2.5x molar increase resulting in approximately 2.5x increase in successful assemblies, with diminishing returns going to 5x molar increase.

Overall, we have found that you can use a small “filler” sequence of DNA to perform assemblies with parts with incompatible fusion sites, and the N-filler part has no burden on assembly efficiency.

Sequence and Features