Exam 181122 Flashcards
3.2 (4p) What expression system (E. coli, BEVS or HEK cells) would you recommend (AND WHY) for the production of:
a.) a human secreted protein that requires autocatalytic removal of a pro-domain for function?
I would choose the HEK cell line, as the post-translational modifications required for human proteins are likely expressed or at the least, present in the HEK genome. My second choice would be the BEVS expression system, as it’s cheaper and hosts quite a bit of post-translational modifications.
3.2 (4p) What expression system (E. coli, BEVS or HEK cells) would you recommend (AND WHY) for the production of:
b) a human cytosolic protein with no posttranslational modifications?
E.coli or BEVS. Since there are no post-translational modifications, a human cell line isn’t necessary. The issue at hand is that e.coli is not generally regarded as safe (gras) due to the preence of LPS. BEVS have previously not been GRAS either, however, the tide is turning. If the protein is supposed to be used pharmaceutically, I would consider the HEK cell line.
Explain flashBac
Flashbac is based on homologous recombination.
1. You transform your bacteria with a flashbac plasmid. The plasmid contains a chi-sequence (which helps it evade recBCD degradation) as well as the BAC gene, which makes it use the bacterial replication system to stay stable. Importantly, the plasmid also contains a lef2 gene and ORF1629 (dysfunctional). The plasmid also hosts the entire viral genome.
2. You transform a donor plasmid to the e.coli. The donor plasmid contains your GOI, lef2 and functional ORF1629.
3. Wait for homologous recombination between the lef2, GOI / BAC, functional/dysfunctional ORF1629.
4. Extract DNA, transfect insect cells.
5. Only the plasmids which have been homologously recombined will be able to propagate due to ORF1629 being dysfunctional in the non-homologously recombined plasmids.
Explain flashBac
Flashbac is based on homologous recombination.
1. You transform your bacteria with a flashbac plasmid. The plasmid contains a chi-sequence (which helps it evade recBCD degradation) as well as the BAC gene, which makes it use the bacterial replication system to stay stable. Importantly, the plasmid also contains a lef2 gene and ORF1629 (dysfunctional). The plasmid also hosts the entire viral genome.
2. You transform a donor plasmid to the e.coli. The donor plasmid contains your GOI, lef2 and functional ORF1629.
3. Wait for homologous recombination between the lef2, GOI / BAC, functional/dysfunctional ORF1629.
4. Extract DNA, transfect insect cells.
5. Only the plasmids which have been homologously recombined will be able to propagate due to ORF1629 being dysfunctional in the non-homologously recombined plasmids.
Explain BacToBac.
Bac to bac is a transposition-based system.
1. You have bacmid competent e.coli. This means that there’s a stable bacmid inside of the e.coli which contains the entire BEVS viral genome as well as lacI. Within LacI, you’ll find two transposition sites.
2. You generate a donor plasmid which contains the gene of interest flanked by two transposition sites.
3. You transform your competent e.coli with your donor plasmid.
4. You incubate the culture, wait for transposition to happen. If transposition happens, it will disrupt the lacI gene, which in turns makes the cells unable to metabolise x.gal, and when blue/white screening them, you’ll see that some colonies are white.
5. The white colonies are selected, grown further, then the bacmid is extracted.
6. The bacmid is transfected into insect cells, in which, it will create viruses.
(4p WK) BEVS: This text is copied from a recent publication about Baculovirus-driven protein expression in insect:
“Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™)”
Discuss or describe at 2 different aspects the two different system with pro and cons, comparing them to each other.
BacToBac pros
1. Robust selection method with blue/white screening.
2. Cheap
BacToBac cons
1. Time consuming.
FlashBac pros
1. Quicker.
2. Intrinsic selection method.
FlashBac cons
1. Expensive. You have to buy the donor plasmid (it’s a complicated plasmid compared to bac-to-bac’s plasmid).
What’s a nemogram?
RCF, radius, RPM graph.
Know two, get the third for free!
What’s the difference between northern- and southern blotting?
Northern blot: RNA analysis.
Southern blot: DNA analysis.
Both of the methods includes:
1. Running agarose gel.
2. Transferring contents to membrane.
3. Immobilization nucleic acids by heat / x-ray.
4. Fluorescence / x-ray probing.
Why does positive ions contribute to DNA stability?
Positive ions neutralize the negative phosphate groups in DNA’s backbone, this makes the DNA strands less repellent of one another.
- (3p, CvW) Proteins are often analyzed with UV/vis spectroscopy. Which are the three types of chromophores that are relevant for UV/vis spectroscopy of a protein sample?
There are four amino acids with aromatic rings.
Tyrosine
Tryptophan
Histidine
Phenylalanine
Out of these four, tyr, trp, his absorb light at 280nm.
(6 p, KF). Imagine that you want to use CRISPR/Cas9 to introduce mutations in the coding region of a gene in a mammalian cell line. In a typical scenario, CRISPR/Cas9 is first used to introduce a double-strand break at the specific site in the genome. (6 p)
b) b) Assume that you are happy to get any kind of mutation in your gene of interest, for example a small deletion that gives a frame-shift mutation. Explain which mechanism in the cell could give rise to such mutation after the dsDNA-break is introduced by CRISPR/Cas9!
In order to get a mutation of any kind, you can create a gRNA that causes a dsDNA break repaired by NHEJ. You would then screen the cells for frameshift mutations.
(CJS/9p) Describe what happens in nature (not in the lab) when Agrobacterium infects a plant. Describe the steps and interactions between Agrobacterium and the plant on a cellular and molecular level.
- agrobacteria sense phenolic substances that are secreted by wounded plant tissue by vir-genes.
- Vir-proteins and ss-T-DNA are produed from the T-DNA plasmid.
- vir-proteins form a complex with the T-DNA which translocates into the plant cell’s nucleus.
- the T-DNA is transcribed in the presence of several vir-proteins.
Interestingly, T-DNA can be transformed into almost any kind of eukaryote.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866890/
(CJS/9p) Describe what happens in nature (not in the lab) when Agrobacterium infects a plant. Describe the steps and interactions between Agrobacterium and the plant on a cellular and molecular level.
- agrobacteria sense phenolic substances that are secreted by wounded plant tissue by vir-genes.
- Vir-proteins and ss-T-DNA are produed from the T-DNA plasmid.
- vir-proteins form a complex with the T-DNA which translocates into the plant cell’s nucleus.
- the T-DNA is transcribed in the presence of several vir-proteins.
Interestingly, T-DNA can be transformed into almost any kind of eukaryote.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866890/
What does the term spallation mean?
Spallation is when fragments of material (spall) is expunged from an atom when it’s exposed to stress. Proton bombardment or hard collisions can generate neutrons.
What does the term perdeuteration mean?
Perdeuteration is when hydrogen is exchanged for deuterium. This is beneficial in x-ray crystal chromatography.
