Team:Chalmers-Gothenburg/Attachment
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Yeast to cancer cell attachment
For this part of the labwork, three different plasmids were assembled. The first plasmid presented here was the “final product” for the cancer cell attachment, a plasmid containing the yeast anchoring protein Aga2 as well as the colorectal cancer cell surface attaching protein HlpA. To control the function of this fusion protein, yeast to cancer cell binding was tested in vitro.
The second plasmid presented here was assembled to confirm that the final product was expressed correctly, and in the desired location. To confirm this, GFP was fused to the assembled Aga2-HlpA protein and expressed in yeast. The GFP expression and location of the expressed protein was then examined under microscope.
The third plasmid presented was assembled to confirm the function of the cell surface display system, namely the function of Aga2. This was done through fusing only GFP to Aga2, after which the GFP expression and location was examined under a microscope.
General workflow
An overview of the general workflow, from amplification of genes to cloning of plasmids into yeast, is presented in Labwork-Protocols .
The only plasmid backbone used for expression of proteins in this part of the lab-work was p416TEF, digested with XbaI and XhoI. The plasmids consecutive promoter, TEF1, as well as terminator, CYC1, were both kept and used for the expression of our protein. The plasmid also contains the bacterial ampR gene, as well as a the yeast URA3 marker gene. The yeast strain used was PK113-11C, which carries the auxotrophic his3 and ura3 markers. Since only the plasmid p416TEF was used, all minimal media used had to be supplemented with Histidine.
In the following sections, the construction of the parts mentioned above, the methods used for checking the protein function as well as the results will be presented. Note that the exact mechanisms of function for the proteins will not be presented here, please go to Project Overview-Project-Anchoring for detailed description. The week by week lab journal is attached in the end of this chapter.
p416TEF-Kozak-Aga2-Linker-HlpA
Below the gBlock and primer design is described, as well as the methods used to testing the protein function. The assembly itself is not desctibed here, since the description can be found in the Protocols section.
gBlock construction
Due to that Aga2 attaches to the yeast cell with the N-terminal of the protein, HlpA was fused to the C-terminal of Aga2. In terms of gBlock construction, this means that HlpA was fused to the 5’-end of Aga2. The stop codon of Aga2 was removed as well as the start codon of HlpA. To avoid sterical hindrance of the function of both proteins, a flexible linker was placed in between the genes. To get a better protein expression a Kozak sequence was added before Aga2. Non native yeast sequences were optimized for S. cerevisiae. The constructed gBlock was synthesized by IDT. The full names of the proteins as well as the source of the gene sequence are listed in the table below.
| Protein | Full name | Native organism | Sequence source |
|---|---|---|---|
| Aga2 | Alpha-Agglutin 2 | Saccharomyces cerevisiae | Uniprot |
| HlpA | Histone like protein A | Streptococcus gallolyticus | Homology BLAST in genome of S. gallolyticus, with HlpA gene from Streptococcus pyrogenes |
| (GGGS)x4 | Flexible linker | Synthetic sequence | SysBio lab Chalmers |
| Kozak sequence | Kozak sequence | Synthetic sequence | SysBio lab Chalmers |
Primer design
Primers were designed with about 20 bp overlap to gBlock, and an about 30 bp long tail that overlaps with the ends of the XhoI/XbaI linearized p416TEF backbone. This allows for efficient Gibson assembly. Primers are listed in file "HlpAPrimerList" below.
Methods for testing protein function
For testing the colon cancer cells binding with S.cerevisiae engineered with Aga2-HlpA the following method was followed: RKO cells were grown in Eagle's Minimum Essential Medium (EMEM) + FBS 10%, by one of our supervisors in the university's cancer cell lab. Cells were grown both in a 42 wells plate and subsequently, after trypsin treatment, were also placed in falcon tubes in PBS buffer solution. S.cerevisiae was grown in Delft media + His overnight both at 30°C and 22°C. A negative control was also made with S.cerevisiae containing only the empty p416Tef plasmid, the negative control was grown in the same conditions. Yeast was centrifuged, then the media was removed and the cells were resuspended in PBS buffer. Resuspended cancer cells and cells in the wells plate attached to the surface were incubated with yeast (both negative control and test) in a 30°C room. After 30 minutes, the wells plate was washed with PBS couple of times, than the surface was scratched with a pipette tip and PBS was added, half the amount was checked under the microscope and the other half was plated in a YPD plate. The liquid culture was also checked under microscope. For detailed method please check the Lab Notebook.
Results of cancer cell attachment
The result from plating was not as expected, in fact, in both negative control and test, yeast cells grew. This can be because the PBS washing was not effective or because the yeast did not attached.
Figure n°x: Negative control: 11C S.cerevisiae + p416tef plasmid after cancer cells binding The results from the microscope are showed in the figures below, we saw that cancer cells were dying and we could not conclude that the yeast was considerably binding to the cancer cells. However, the only binding we could see was to cells that were still alive but still the quantity was so small that it is not considered interesting. FIGURESSSSSSSSSImportant for lab journal
In lab journal the Aga2-Linker-HlpA gBlock is often referred to as “HlpA” only.
p416TEF-Kozak-Aga2-Linker-HlpA-Linker-GFP
Instead of synthesizing a new gBlock with the new fusion protein, the previously synthesized Kozak-Aga2-HlpA was used as a template. GFP was amplified from the plasmid p413TEF-GFP available in our lab. Below the primer design and origin of new parts are described, as well as the methods used to test the protein function. Please see previous chapter for origin of previously mentioned genes.
Origin of new genes
The new parts in this plasmid are the second linker and the GFP. The full names of the proteins as well as the source of the gene sequence are listed in the table below.
| Protein | Full name | Native organism | Sequence source |
|---|---|---|---|
| (GGGS)x2 | Flexible Linker | Synthetic sequence | Article (Chen et al., 2013) |
| GFP | Green Fluorescent Protein | Optimized for Saccharomyces cerevisiae | Plasmid in SysBio lab at Chalmers |
Primer design
Primers were designed for Gibson assembly of Kozak-Aga2-HlpA and GFP into the XhoI/XbaI linearized p416TEF. In addition to this, the primers were designed to add a linker in between the HlpA and GFP to allow for better protein function, and to remove the stop codon of HlpA as well as start codon of GFP. Since The GFP was amplified from a plasmid, the plasmid template had to be digested with DnpI before purification of PCR-product. Primers are listed in file "HlpAPrimerList" below.
Methods for testing protein function
The purpose of GFP-tagging Kozak-Aga2-HlpA was to see that the protein was expressed properly, and at the correct place. In order to check this, 3 colonies of 11C yeast containing p416TEF-Kozak-Aga2-HlpA-Linker-GFP were inockuIated in Delft+His media and were grown in a 30 degree C overnight. A negative control of 11C yeast was grown in Delft+His+Ura media in the same way. The the results of the protein expression were then checked under microscope. The same experiment was also repeated in room temperature to help with protein folding and repeated several times.
Results recombinant protein expression
Even with several replicates of the experiment, and with yeast grown in room temperature, no GFP showed under the microscope. There are three possible explanations for this, since the protein sequences are correct based on the sequencing. The protein could be expressed, but misfolded. In this case HlpA is the most likely the cause of the misfolding, since it has never been expressed in yeast before while the other proteins have. It is also possible that the error occurred on an transcriptional level, however we did not have time or resources to look further into this. The third possibility is that the protein is secreted, but that Aga2 failed to adhere to Aga1 on the yeast surface. This is also something that we lacked the resources to confirm. Moving on with what we had, checking the function of our anchor was the next course of action.
p416TEF-Kozak-Aga2-Linker-GFP
Primer design
Primers were designed for Gibson assembly of Kozak-Aga2 and GFP into the XhoI/XbaI linearized p416TEF. Just like in the previous case, the primers were designed to add a linker in between the Aga2 and GFP to allow for better protein function.the Aga2 sequence already lacked a stop codon, but the start codon of the GFP still had to be removed. Since the GFP was amplified from a plasmid, the plasmid template had to be digested with DnpI before purification of PCR-product. Primers are listed in file "HlpAPrimerList" below.
Methods for testing cell surface binding
In order to see if Aga2 and GFP were properly expressed and functioning, the cell surface binding was once again tested under microscope. The plasmid p416TEF-Kozak-Aga2-Linker-GFP was transformed into yeast 11C, after which 3 colonies were picked and inoculated in Delft-His minimal media. As a negative control a 11C colony without the plasmid was inoculated overnight in Delft-His-Ura minimal media, and as a positive control 11C containing p413TEF-GFP was inoculated overnight in Delft-Ura minimal media. All inoculated cultures were grown overnight in 30 degrees celsius, after which the GFP expression and location was checked under microscope. This experiment was also repeated with incubation in room temperature.
Results recombinant protein expression
The results were not what we expected. Since the anchoring sequence has been shown to work with GFP before, and since the GFP expression foom the GFP template plasmid works well by itself the results should have been GFP expressed at the surface at the cell. However, in our cells expressing Aga2-GFP the fluorescence was weak compared to when the GFP alone was expressed. It also seemed like the Aga2 did not attach to the surface of the cell, but was located close to the nucleus of the cell. One theory is that the Aga2-GFP is expressed and secreted, but does not attach to the cell surface. If time had allowed a follow-up experiment would have been to overexpress Aga1, the yeast cell-wall protein that Aga2 attaches to, to see if this would make a difference.