Mechanism

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Split-Resistance Genes

There are many important features, that must be taken into consideration while planning to construct a two-plasmid system with split antibiotics. Otherwise the probability of success decreases. While trying to find the right position to split the resistance gene it must be searched for the right N- and C-terminal extein sequences. In our case we talked to Mr. Albert Cheng, PhD who suggested us to orientate ourselves on Table 1 (Cheriyan, 2013). On this table the relative favourability of each amino acid at each relevant position neighbouring the inteins is documented. Additionally, it is important to make sure that the split point is not interrupting an important secondary structure of the protein and that the exteins are not hydrophobic. Thanks to Dr Francine Perler reminding us about the hydrophobicity of the exteins, we did not forget to take it into consideration. Instead of analysing ourselves the DNA sequence of the resistance genes we decided to do a model, that will be able to output the right split points.
As a positive reference to our selected split points we contacted Prof. Dr. Barbara Di Ventura. She provided us with the plasmids that contain the protein fusions, so that we can use them to clone our new backbones. Two plasmids encoding an active TEM-1 lactamase for ampicillin resistance and other two plasmids encoding the aph(3’)-Ia for kanamycin resistance (not published jet, Navaneethan Palanisam, Freiburg University).

An important aspect, when constructing and working with a split-resistance gene system is to make sure, that one plasmid does not provide to the cells the ability to grow on medium with the corresponding antibiotic. Therefore, from every cell collection containing one of the plasmid pairs, we plated different on plates with low antibiotic concentration.
Normal working concentrations for chloramphenicol are between 25 and 170 µg/mL (ATCC_recommendations_antibiotic_concentrations, 2003). As Figure 11 shows the chloramphenicol plate was divided into four segments. Cells were plated on the upper left site containing pSB1Ca3, upper right site containing pSB1Cc3, lower left site containing pSB3Cb5 and on the lower right site containing pSB3Cd5. On none of the segments growing colonies can be seen and that proves, that only one plasmid does not provide antibiotic resistance. Similar is the case with hygromycin.
Normal working concentrations for hygromycin B is between 200 and 500 µg/mL (Hygromycin B, Thermofisher). As Figure 12 shows the hygromycin plate was divided into two segments. Cells were plated on the upper site containing pSB1Ha3 and on the lower site containing pSB3Hb5. Like before, none of the plated colonies grew.

After proving that one plasmid does not provide an antibiotic resistance, the next experiment was to analyse the growth efficiency of the double-plasmid containing cells. Therefore, we poured plates with diverse concentrations of chloramphenicol and hygromycin B (Figure 13, 14).
Regarding the chloramphenicol resistance the concentrations that were prepared are from the upper left to the lower right site 0.1, 1, 7, 10, 20, 40, 60, 80, 100 μg/mL. Each plate was divided into three sections. On the upper left site, DH5α was plated as the negative control, on the upper right site the colonies containing both plasmids and on the lower site a colony containing pSB1C3 (link) gene. The maximal chloramphenicol concentration on which DH5α grows is 0.1 μg/mL. The section for the split-marker was additionally divided into two segments, for the two different split point of chloramphenicol. On the left part the CmA version and on the right part the CmB version was plated. Until a concentration of 1 µg/mL and in other two repeats till a concentration of 5 µg/mL the two split system versions were providing the cell resistance against chloramphenicol. Furthermore, it can be observed from the figure that the CmB version works better than the other one. This was to assume, because of the extein sequence flanking the split Npu DnaE intein.
For the hygromycin B resistance the concentrations that were prepared are from the upper left to the lower right site 30, 40, 60 80, 100, 200, 500 and 1000 μg/mL. Each plate was divided into three sections. On the upper left site, DH5α was plated as the negative control, on the upper right site the colonies containing both plasmids and on the lower site a colony containing plasmid with a hygromycin resistance gene. For DH5α the used concentrations were too high to grow. The positive control did not have any problems growing on hygromycin B. The cells with the split antibiotic resistance plasmids easily grew on the plates till a concentration of 500 μg/mL and with some difficulties on 1000 μg/mL.

Regarding the ampicillin resistance the concentrations that were prepared are from the upper left to the lower right site 5, 30, 60, 80, 90, 100, 150, 200 and 300 μg/mL (Fig.16). The plate was divided into three sections. On the upper left site, DH5α was plated as the negative control, on the upper right site the colonies containing both plasmids and on the lower site a colony containing pTXB1. DH5α could not grow on any of these concentrations. The positive control was also able to grow on any ampicillin concentration. The cells with the split antibiotic resistance plasmids easily grew on the plates till a concentration of 200 μg/mL.

For the kanamycin resistance the concentrations that were prepared are from the upper left to the lower right site 35, 40, 45, 50, 60, 70, 80 and 90μg/mL (Fig.17). As before too, the plate was divided into three sections. On the upper left site, DH5α was plated as the negative control, on the upper right site the colonies containing both plasmids and on the lower site a colony containing pSB1K3. For DH5α the used concentrations were too high to grow, except of the 40µg/ml plate, where two colonies can be seen. The positive control was also able to grow on any ampicillin concentration. The cells with the split antibiotic resistance plasmids easily grew on the plates till a concentration of 90 μg/mL.

In order to test the functionality of our split resistance genes, we conducted growth experiments with DH5α containing the two-plasmid system. The Escherichia colis (E. colis) were cultivated in liquid LB media and all tested plasmids were transformed in DH5α. For every growth curve there were five samples. For the split kanamycin curve the first sample was DH5α cultivated in LB-media without antibiotic, the second DH5α in LB-kanamycin, the third DH5α + pSB1K3 in LB-kanamycin, the fourth DH5α + pSB1C3 + pSB3T5 in LB-kanamycin-tetracyclin, the fifth DH5α + pSB1Ka3 + pSB3Kb5 in LB-kanamycin.
DH5α with antibiotic did not grow in comparison to DH5α without antibiotic that showed the expected curve. The cells containing pSB1K3 grew the best and the split antibiotic system followed . It could also be observed, that the cells containing two plasmids with each one antibiotic resistance did grow less efficient than the split kanamycin cells.

The same procedure was conducted to test the functionality of the split ampicillin system. The Escherichia colis (E. colis) were also cultivated in liquid LB media and all tested plasmids were transformed in DH5α. The first sample was DH5α cultivated in LB-media without antibiotic, the second DH5α in LB-kanamycin, the third DH5α + pTXB1 in LB-ampicillin, the fourth DH5α + pSB1C3 + pSB3T5 in LB-chloramphenicol-tetracyclin, the fifth DH5α + pSB1Aa3 + pSB3Ab5 also in LB-ampicillin.
DH5α with antibiotic did not grow in comparison to DH5α without antibiotic, as also seen before. The cells containing pSB1K3 grew as good as the cells with the split antibiotic system. DH5α with two antibiotic resistance genes did grow better than DH5α with antibiotic, but worser than the others.

We aimed to split the protein conveying resistance to chloramphenicol, chloramphenicol acetyltransferase (BBa_J3105). The resistance protein is commonly used within iGEM, thus making a split Chloramphenicol acetyltransferasa a vluable addition to the iGEM partsreg. The protein sequence of this part can be found here. According to our modeling, the best split point to use when introducing Npu DnaE was VAQ-CTY in the positions 28-33, the B(_VAQCTY) is 56.95.
Even though this split point is located at the second amino acid of a beta-sheet, we decided to test whether splitting at this position could lead to Chloramphenicol acetyltransferase fragments that could be reconstituted by intein-mediated protein splicing.
Similar to our modeling of the split kanamycin resistance, we used the homology modeling software MODELLER (Sali & Webb, 1989) to visualize each part of the chloramphenicol acetyltransferase fused to the Npu DnaE intein.

The amino acid sequences given to the software were:

Part 1:
MEKKITGYTTVDISQWHRKEHFEAFQSVAQCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNNLEGHHHHHH

Part 2:
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCCTYNQTVQLDITAFLKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA

The templates used for homology modeling were: