Team:Guelph/Improve
Our major improvement to the amilCP biobrick involved the addition of the tetracycline-responsive promotor. This allowed the amilCP to be directly controlled by the addition of tetracycline.
To create a biosensor we first wanted to create a basic sense and respond gene circuit that would produce a visible colour change in response to tetracycline. We needed a basic reporter gene and decided to use the chromoprotein AmilCP. We initially wanted to do work with BBa_K592009, the BioBrick created from the iGEM Uppsala 2011 team that has been well characterized. However this part in our distribution kit came without a RBS that we needed for our biosensor to transcribe amilCP. So instead we PCR amplified the AmilCP from the Biobrick BBa_K1343022 from Distribution Kit Plate 5 Well 22I.
We realise that this means that the functional unit we are improving is the AmilCP BBa_K592009 in BBa_K1343022, but to not deviate from the iGEM rules, we have listed our improvement under the Biobrick BBa_K1343022. We placed, the Amil chromoprotein gene (amilCP) from BBa_K1343022, downstream of the BBa_K3189001, our tetracycline-responsive promoter. Initially, we attempted to transform the part in pSB1C3 as found in the distribution kit into E. coli DH5α, but no colonies grew on the LB+chloramphenicol plates (not shown). The amilCP of the BBa_K1343022 composite part, including the ribosome binding site immediately upstream (position 84-820), was then amplified by PCR, to put compatible restriction enzyme ends on it. The amilCP was then digested, along with pTet using EcoRI and PacI restriction enzymes according to our protocol listed to remove the GFP and give both DNA pieces sticky ends.The AmilCP amplicon was then ligated into pTet in the place of GFP.
The identity of the newly created BioBrick, BBa_K3189015, was confirmed by transforming it into E. coli DH5α, selecting colonies that grew on LB+Ampicillin. A colony PCR that amplified our BBa_K3189001 and AmilCP from BBa_K1343022 to give an amplicon of 759bp was done using Frogga Taq. We then ran the samples in a 1% agarose gel electrophoresis. This confirmed that the AmilCP was present in the right orientation, downstream of BBa_K3189001, our pLTetO sequence, creating the new plasmid pTA or our composite part BBa_K3189015.
To see whether BBa_K3189015 was functional, we grew up the cells in 50 mL LB flasks with 50 ng/mL tetracycline (as described above with GFP and in Experiments). After 6h of growth, the cells were washed with PBS and the characteristic brightblue colour was not observed.
We tried multiple tests with many PCR confirmed colonies however, we never saw the blue. To see why our part was not working, we sequenced an archive we made the day after we PCR confirmed the mutants from Figure 5 of strains with BBa_K3189015, using Sanger sequencing. What we found was a premature stop codon introduced at Gly 129, that mutated the first G in Glycine’s GGA to a TGA, resulting in a premature stop codon. This stop codon would result in protein structure of the AmilCP chromoprotein to be truncated, potentially explaining our results from Figure 6.
These findings lend support to what has already characterized about BBa_K592009 by iGEM Uppsala in 2018. They have observed that the amilCP protein is mildly toxic to bacteria, and thus the functional gene imposes a selective pressure that encourages the survival of nonfunctional mutants. However, this theory can be challenged by the fact that this mutation was observed when the gene was not suppose to be induced and expressing the toxic amilCP. The sequencing was done on strains that were archived after confirming their initial transformation (Figure 5). So another reasoning may be that the amilCP that was amplified out of BBa_K1343022 innately possesses a stop codon.
A more sensitive expression assay was then conducted on the functioning BBa_K3189015 by, growing E. coli BBa_K3189015 transformants in a 96-well plate in varying concentrations of tetracycline labeled in Figure 9. The plate was left at 16 °C for one day, and then moved to 4 °C. After two days, pigments were visible, but they intensified after one week at 4°C. A dark blue pigment was seen at 100 ng/mL tetracycline, with less colour visible at 50 ng/mL for some of the samples, and no colour visible in the absence of tetracycline.
Improve
Improvement of BBa_K1343022: Addition of BBa_K3189001 to make BBa_K3189015
The forward and reverse sequences can be respectively found here and here
After several failed expression tests with our first version of BBa_K3189015 (based on BBa_K1343022) from iGEM plate we tried to circumvent the problem by synthesizing the sequence found in the registry from IDT. We repeated the same work in order to recreate a new BBa_K3189015, and this was sent for Sanger sequencing which returned an error-free sequence.
After PCR confirming and sequencing our BBa_K3189015, used a positive E. coli strain and commenced our initial expression testing.
We took an overnight of BBa_K3189015 and added it to LB-Amp with 100 ng/mL of Tetracycline and used no tetracycline in our negative control. They were grown at 37°C, shaking at 250 rpm.
After 24 hours, a dark blue pigment was observed that increased in intensity after longer incubation. Figures 8a show the tubes that were incubated and Figure 8b shows a dark blue pellet that is observed when 2mL of each culture is spun down in the presence of tetracycline.
