Part:BBa_K4390007
JUMP O part spacer
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.
This part already includes the 5' and 3' JUMP N-part fusion sites, so when making composite parts using this part do not add the 5' and 3' JUMP C-part fusion sites (TTCG and GCTT).
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.
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.
Because of that, this part is part of the parts
1. Part:BBa_K4390055
2. Part:BBa_K4390056
3. Part:BBa_K4390057
4. Part:BBa_K4390058
5. Part:BBa_K4390059
6. Part:BBa_K4390060
7. Part:BBa_K4390061
8. Part:BBa_K4390062
9. Part:BBa_K4390109
10. Part:BBa_K4390110
11. Part:BBa_K4390112
Characterization
To confirm the O-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 O-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 2).
Figure 2: 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 1). These regions are standard in all JUMP vectors, and flank the insert.
| Name | Sequence | Targeted region | Melting Temperature (°C) |
|---|---|---|---|
| PS1 | AGGGCGGCGGATTTGTCC | T1 terminator | 72 |
| PS2 | GCGGCAACCGAGCGTTC | T0 Terminator | 71 |
Table 1: Primers used in colony PCR to amplify inserts in a JUMP vector.
Figure 3: 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
Finally, we confirmed that this part functioned as intended with the untagged sfGFP construct (Part:BBa_K4390056). Only correctly assembled plasmids would produce green fluorescence, and as seen in Figure 4.
Figure 5: Kanamycin plate of untagged sfGFP trial under a blue light box, showing the presence of GFP expressing colonies. This indicates the assembly has worked, and the O-part functions as intended.
Additionally, the Blue-White screening experiment and the sfGFP expression experiment confirmed that assembly can be done with multiple fillers, as using the N- and O-filler produce successful assemblies.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |