Aim Three
Using the methods described in Hmelo et al. (2015), we would take the E. coli developed from Aims #1 and #2 and engineer them so they would encode for the Bacillus subtilis sacB gene. By coding the sacB gene, as well as the modifications developed and described under Aims #1 and #2, the E. coli bacteria would be able to develop a growth-no growth screening system when grown on a 5% sucrose agar plate which can determine whether or not the patient’s blood sample contains cancerous c-Myc mRNA (E. coli grows) or does not contain cancerous c-Myc mRNA (E. coli does not grow).
This is because the sacB gene will code for an enzyme called levansucrase, which will be lethal to E. coli in the presence of sucrose, but with our additions from Aims #1 and #2, levansucrase will be made conditionally lethal and will only be expressed in the presence of c-Myc mRNA from a patient’s blood sample. We have yet to complete this aim due to the time constraints related to our project, but we have concrete steps toward it as shown below.
Linear Map for SacB
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Plasmid Map
Download PDFReference the Experiments Page to view a complete list of the protocols used for each aim.
At the current stage of our research, we have successfully been able to ligate the new plasmid using a Gibson assembly protocol. This new plasmid includes an insert from a sacB gene (ordered from IDT as gBlock) and the backbone which includes a Kanamycin resistant gene (mini prepped from pCB705). We were able to confirm that the ligation accurately cut at the correct sites (NheI and XhoI) by performing a gel electrophoresis. After cut sites were confirmed, a gel extraction was performed to isolate the plasmid which subsequently was then used for a gibson assembly. This new plasmid was then transformed into a LacIq e. Coli cells (stored at -80 degrees Celsius) and incubated overnight. Once we confirmed the cell was transformed into the cell, we then attempted to transform the 872 and 12XX series plasmids into the same cell. This is where we have left off and have continued to research. There was difficulty to co-transform using an electroporation into a cell that has already been heat shocked.
[Figure 3a] Aim #3 includes the cloning of a plasmid and using negative selection to "sense" for the target gene. The plasmid pCB705 (from Collins et al.) contains a plasma that codes for kanamycin resistance in its backbone and was used as the vector. The insert was a gBlock (IDT) containing the sacB gene and the LAC operator.
LacI inhibits LacO, which then inhibits sacB from killing the cell. When a toxin is added, this inhibits LacI, causing sacB to be produced. Cas12a will only cut the plasmid in the presence of the correct trigger RNA, resulting in a growth of colonies
Negative selection of the sacB² enzyme will allow for the detection of trigger RNA based on the growth on a 5% Sucrose plate.
[Figure 3b] There are cuts at NheI and XhoI at the appropriate base part pairings. This gel image confirms that there are the correct cuts based on the size of the bands seen in this picture. There are four wells, which all were loaded with 24uL of the sample (contains the plasmid with sacB and the 705 backbone). In this case, all wells resulted in a positive result.