Month: October 2019

Microbe Machine

Molecular cloning with Gibson Assembly

Molecular cloning is a technique established in the early 1970s that made introducing specific genes into a host possible [1]. This significantly advanced research because it allowed us to isolate and study individual genes. One method cloning has made possible is protein purification. By introducing a gene to a bacterial vector, the encoding protein is able to be overexpressed and then subjected to biochemical analyses such as crystallization or enzymatic assays to elucidate the function of the protein [2].

I am using this exact technique in my senior thesis at Reed College to study CG11811, a protein novelly indicated as a non-muscle myosin II (NMII) regulator. NMII is an actin binding protein that when activated, constricts actin filaments to change cell shape [3]. Our previous studies found that when CG11811 was depleted using RNAi, there were decreased contractility events. However, we do not know how exactly CG11811 regulates NMII. Therefore, I am cloning CG11811 into a strain of E. coli designed to mass produce proteins in order to purify it and run enzymatic assays to reveal some of its biochemical properties. The first step of this cloning process is creating the recombinant DNA plasmid to transform into the bacteria.

A very efficient technique for this purpose is Gibson Assembly, designed by Dr. Daniel Gibson in 2009 [4]. With Gibson cloning, any two sequences (the gene of interest and the vector) can be joined together without the need for restriction enzyme sites (Figure 1A). The basis of this technique is to design two sets of primers that overlap the gene and the vector (Figure 1B). When these sets of primers are used in PCR, the vector has a section at the end that is complementary to the insert and the insert has a section at the end that is complementary to the vector (Figure 1B). These two PCR products are then combined in the Gibson reaction with an exonuclease that will chew back the overlaps to create “sticky” ends that will anneal to each other, polymerase to fill in any gaps in base pairs in the overlaps, and ligase to join the fragments together (Figure 2). The resulting product is a contiguous plasmid that can then be transformed into the protein expression E. coli to get mass quantities of CG11811.

Figure 1. Primers with overlaps sections produces a PCR product with ends that can anneal the insert and vector together. By designing primers with added end portions of the insert or vector is supposed to join, “sticky” ends are created that can be annealed in a Gibson Assembly reaction.

Figure 2. Gibson Assembly creates complementary ends that anneal to each other to create a contiguous plasmid. In the reaction, an exonuclease chews up the ends that then anneal to the complementary sequences of the other fragment ends. A polymerase then fills in any missing bases and then the ligase completes the plasmid.








Citations

1. Jackson, D. A., Symons, R. H., & Berg, P. (1972). Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 69(10), 2904–2909. doi:10.1073/pnas.69.10.2904
2. Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in microbiology, 5, 172. doi:10.3389/fmicb.2014.00172
3. Martin, A. C., Kaschube, M., & Wieschaus, E. F. (2009). Pulsed contractions of an actin–myosin network drive apical constriction. Nature, 457(7228), 495–499. https://doi.org/10.1038/nature07522
4. Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345. https://doi.org/10.1038/nmeth.1318

Scratch-wound Assays

The scratch-wound assay is a common method for assessing protein distribution and directionality in collectively migrating cells (Cory, 2011). This method is advantageous because it does not require chemical attractants in order to induce migration. Instead, the scratch-wound assay procedure typically involves culturing a sheet of cells on top of extracellular matrix. The matrix operates like tracks that allow the cells to migrate. A needle with a fine filamentous tip is then used to etch a wound onto the sheet of tissue. The wound is then left to heal via cellular proliferation and migration, and the direction of migration can be inferred as into the wound.

Figure 1. Shot depleted RasV12;WTSRNAi epithelial sheet with scratched wound at 0 min. Wound is outlined in yellow. Image was taken with Phase-contrast microscopy at Western blot analysis of acetylated and alpha tubulin Cell lysates of 40x.

For my thesis, I will be using scratch-wound assays in order to study orientation of microtubule growth in migrating Drosophila cells. I am currently culturing sheets of sheets of RasV12;WTSRNAi cells. Some of the cultures have depleted levels of either the protein Short-stop (Shot) or Pickled eggs (Pigs), both of which crosslink actin and microtubules. Into all of these cultures, I have transfected in a DNA construct that expresses a protein called Eb1, which marks the growing ends of microtubule filaments, that is tagged with a red fluorescent molecule. After performing a scratch-wound assay, I can take three minute timelapses of the migrating cells using Total Internal Reflection Fluorescence (TIRF) microscopy. TIRF microscopy essentially uses a laser to excite the red fluorescent molecule attached to the Eb1 proteins on the growing ends of microtubules. The results are short movies in which we can see red comets marking microtubule growth. One of the goals of my thesis is to calculate the angles at which the microtubules are growing relative to the line of the wound and see if there is a difference in orientation of growth between conditions in which proteins have been depleted in cells.

References:

Cory, Giles. “Scratch-wound assay.” Cell Migration. Humana Press, 2011. 25-30.