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The focused contributions of this year's Alma iGEM team were two-fold. Firstly, we looked to provide additional data for the registry regarding the BioBricks I13521, K123002, and K123003. Secondly, we looked to improve upon the golden gate assembly methodology by creating universal golden gate overhangs that could be added to any BioBricks in the iGEM registry.

In addition, we also made an improvement to the pSB1C3 backbone, to allow it to replicate in Vibrio natriegens.

Figure 1. Schematic showing the theorized binding and use of our universal Golden Gate primers.

Universal Primers for Golden Gate Cloning

The use of universal golden gate primers solve several issues seen with standard 3-A assembly, as well as traditional golden gate cloning. The use of these primers virtually erases the time wasted on designing individual primers for 3-A assembly as well as the time spent trying to match traditional golden gate primers to each individual BioBrick. Therefore, the construction of a small set of primers to be used for golden gate assembly of any BioBrick will greatly reduce cost and time as well as increase assembly flexibility by seamlessly swapping the order of assembled BioBricks with ease.

The benefit of this is twofold. By allowing a small set of primers to be used for several BioBricks the process becomes time efficient. Moreover, it will provide a basis for many high school teams and low budget biology departments to enhance their biochemistry and biology departments by teaching them Golden Gate Assembly, a highly effective method, in a cost efficient manner.

The precedent of universality in terms of a primer came from "One Primer To Rule Them All : Universal Primer that Adds BBa_B0034 Ribosomal Binding Site to any Coding Standard 10 BioBrick." by Bryksin et al. The primers they designed allowed the addition of a ribosome binding site using universal primers. This was done by including the new sequence in between two regions of primer annealing. Instead of a ribosome binding sequence, we created similar primers that include a BsaI site and a few different overhangs.

We were able to display that Golden Gate overhangs could be added to BioBricks via PCR with universal primers – primers that will anneal to the Prefix and Suffix that is present on every BioBrick.

Figure 2. The intended way in which our primers are intended to overhang and bind the pSB1C3 backbone.

The 2019 Alma iGEM team pioneered this method of Golden Gate cloning. This approach worked, but was inconsistent. This year, we sought to test a variation on this approach which features more homology between the primer and the template. Although this introduces a larger scar sequence between parts, it appears to be a much more robust version of this method.

Measuring Expression of I13521

Transformation of I13521 was successfully conducted in two separate strains of E. coli: DH5ɑ and HL2483. The HL2483 E. coli strain is engineered to constitutively produce TetR; therefore, this E. coli strain was used in comparison to the DH5ɑ strain as a way to show regulation of I13521 through its TeT repressible promoter. Following successful transformation, each strain was streaked onto a LB (Miller) agar plate with chloramphenicol and incubated at 37°C overnight.

Figure 3. Streaked plate showing I13521 and J04450 transformed into both DH5ɑ and HL2483 E. coli.

Following incubation, the plates were pulled out and as can be seen in the image below, the HL2483 strain was able to successful inhibit the production of RFP, while normal RFP production was seen in the DH5ɑ strain. Additionally, constitutive RFP production from the J04450 BioBrick was used as a positive control in both DH5ɑ as well as HL2483, as can be seen below.

Figure 4. Bar graph showing the fluorescence of RFP production by I13521 in both Hl2483 and DH5ɑ when in LB media, when exposed to IPTG, and when exposed to aTc.

This bar graph shows the fluorescent data for the same strains of E. coli discussed above. Additional information was garnered regarding the function of the I13521 by adding both IPTG as well as aTc to the plates and measuring the fluorescence compared to cells in LB only. IPTG acts to induce the transcription of genes under control of the Lac promoter. As J04450 is under the control of the LacI promoter, it would be expected to increase its expression in the presence of IPTG in comparison to cells containing only LB, which is what is seen above. Additionally, aTc acts as an inducer to TetR; therefore, in the presence of aTc we would expect an increase in the fluorescence of HL2483 strains with I13521 compared to the LB culture. This result is also seen in the Fig. 4 above.

These results were validated by a colony PCR that tested for the presence of the TetR promoter (R0040) and other components of the BioBrick, indicating that our strains did indeed harbor the plasmid we suspected. The annotated gel from colony PCR gel electrophoresis of these clones can be seen in Fig. 5.

Figure 5. Colony PCR gel electrophoresis of the clones measured for RFP fluorescence. The arrow indicates a length of approximately 923 base pairs: the length of I13521.

Measuring Expression of K123002

Assembly of K123002 was conducted through the use of gibson assembly, and transformation of the assembled plasmid was done in NEB stable strain E. coli. Following transformation, the cells were isolated and suspended in fresh LB + Chloramphenicol. These cultures were incubated for 6 hours at 37°C in a shaking incubator. Cells were pelleted by centrifugation. The pellet was then resuspended in SDS sample loading buffer and loaded onto a SDS PAGE gel. The gel was run and stained with coomassie stain, followed by a destaining period of several hours. No TetR expression was observed by this BioBrick, as is seen in the SDS PAGE below. Therefore, further experimentation will be taken in the future to evaluate this problem, as mentioned in our Engineering page.

SDS PAGE gel analysis of K123002. The arrow indicates 25 kDa, the approximate size of the TetR protein.

Toxicity of K123003

While efforts were made to characterize K123003, issues were encountered during the transformation stage of the process. Upon attempted transformation of the assembled plasmid into NEB stable strain cells, no cell growth was seen in our experimental samples; however, as is displayed in the image below, colonies were noted on the positive control plates for all other Gibson assembly reactions. Therefore, this part appears to inhibit proper transformation into E. coli. Additionally, the possibility of this part being cytotoxic to E. coli cannot be excluded. In order to improve upon this observed dysfunction, codon harmonized sequences for multiple estrogen receptor sequences in E. coli have been generated with the intention of continuing to attempt transformations of an estrogen receptor sequence into K123003. These generated sequences can be seen here.

Figure 7.
Left: Plate showing no colony growth following an attempted Gibson assembly and transformation of K123003 into NEB stable strain E. coli.
Right: Positive control of a Gibson assembly followed by transformation into NEB stable strain E. coli.

Backbone Improvement

In an effort to speed up our cloning, early on we attempted to use Vibrio natriegens to assembly our BioBricks. This organism grows faster than E. coli, and was pioneered for use in iGEM projects by Marburg 2018. We had difficulty transforming this plasmid into Vibrio natrigens (ATCC strain 14048) and the commercially available Vmax. However, our control transformations worked fine. Further investigation revealed that a single point mutation (T280C) in the normal pUC19 origin exists in the pSB1C3 backbone. We hypothesized that this could be the reason why we were not getting any colonies, since it is in a critical region for the origin sequence, and sought to revert this mutation.

Figure 8. A view of the ColE1 origin of replication, with the revelant point mutation highlighted.

By introducing the reversion, C280T, into the origin of part BBa_J04450, we improved this part by making it suitable for growth in Vibrio. We never obtained colonies with the original part, but on several occasions were successful in transforming Vibrio with our improved version (BBa_K3445002). The cells even display the red color of RFP! We hope this improvement opens the doors for more teams to utilize Vibrio as their chassis or assembly host in the future.