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Contents

Start-up Genome Engineering

Protein Function Re-engineering Ideas
I & XI assembly The genes can be modified to be sensitive to the size and content of the material being secreted, in order to increase the quality and ensure viability of each product being excreted. This can be done by allowing communication (through chemical signaling, for example) to call for proteins necessary to make a finished product. For example, p3's may be missing and the product would get "stuck". Also, improving communication with p4 to resize the channel when necessary would quicken the export rate.
II replication of DNA + strand Test if increasing the attraction of host enzymes is beneficial to accelerate production of more double stranded phage DNA.
III phage tail protein (5 copies) This protein can be modified to change shape by sensing the membrane composition (chemical/structural) of the host to prepare itself for efficient infection.
IV assembly Allow communication with p1 and p11 to be more sensitive to size and content of material being exported.
V binds ssDNA One can test if allowing/increasing p5-p10 interaction optimizes the production of new phages.
VI phage tail protein (5 copies) Add the ability to extend or retract p3 by allowing communication (with p3) during infection.
VII & IX phage head protein (5 copies) Switch the order of p7 and p9 in its genome to learn about their differences in function in M13.
VIII phage coat protein (2700 copies) Mark its interaction with p5 to see how ssDNA is handed off before excretion.
X DNA replication As a regulatory protein, it can optimize how many double stranded DNA should be made by allocating how much DNA should be spent on making proteins for production or for the final product to carry. One can test if increasing p10's interaction with p5 optimizes the process.

Understanding of Biology of M13 - Question 2

  • Would you expect the phage to tolerate p8 modifications that
    • make the protein neutral rather than charged at the C-terminus?

No, because charge at the ends of proteins can be essential to its interaction with other proteins.

    • encode all Leucines with the CTA codon instead of the CTG codon?

I think it would depend on the context of the codons. If one is statistically more predominant than the other, then it may be an indication that a particular gene may have a preference. And so this would require an overall inspection of all the Leucine codons in the gene.

    • double the size of the protein?

This may limit the ability of the finish product to exit through the channels (p4, p1, p11).

  • Would you expect the phage to tolerate these same modifications to p3?

The answers are essentially the same, except it is more detrimental for p3's structure to be modified either in size or at the C-terminus.

  • Would you expect the phage to tolerate transcriptional terminators that are
    • 2X stronger - Yes. The materials and means for production may be limited, but still tolerable.
    • 100X stronger - No. If any one of the components are missing, production would shut down.
    • 2X weaker - Yes. A more disorderly working environment would still be tolerable.
    • 100X weaker - No, because disorder can cause a traffic jam or crash in the system.

M13 Relatives - Question 3

Nature often preserves functionally critical genomic elements, and evolutionary cousins can help us identify which genetic elements are disposable, which are interchangeable, and which are essential. Who are M13's closest evolutionary relatives and how do they differ from the phage you're working with?

  • Two of M13's closest evolutionary relatives are Lambda phages and T4. They differ in their life cycles, target infection area of E.Coli, and specific means of replicating DNA. The Lambda phage goes through both lysogenic and lytic lifecycles where as T4 is lytic, and M13 is lysogenic. They differ in what structure of E.Coli they infect. Lambda phages bind to a receptor protein, whereas the M13 infects the f-pilus of E.Coli. Also, Lambda phages integrate its DNA into the host genome where as T4 or M13's do not. Although there are differences, these stem from common goals. There are at least three (common) goals that seem essential: infection/entrance, synthesis, and reproduction. And so these point to genomic elements that are essential, while varieties of methods that achieve these three goals are interchangeable, and I would expect disposable genetic elements to be eliminated to make the genome as concise as possible.

Module 1: Day 4 For Next Time

M13 Refactoring

A major part of refactoring this section of the M13K07 genome (HpaI site - BamHI site) was decoupling overlapping genes and inserting handles in between them to make insertions and deletions easier. I made a part for each gene, where a part usually consisted of restriction site A, the promoter (if there is one), the RBS, the coding region, then restriction site A again. Each part had different restriction sites that were added both in the beginning and the end. The reason for having two of the same restriction sites within a part is so that the gene can easily be deleted and the backbone ligated. The reason for having different restriction sites for different parts was to allow insertions in between parts of interest. So the general layout of this section was (E1=restriction site 1): ---HpaI-g2\\E1 g10 E1//\\E2 g5 E2//\\E3 g7 E3//\\E4 g9 E4//\\E5 g8 E5//g3-BamHI--- For each gene, I identified the RBS and/or promoter region and included it in the part. For genes that had RBS and/or promoter sequences of another gene, I changed the wobble positions of every codon to prevent homolous recombination between the repeated sequence, as well as to individualize a function of a part to a single gene. In addition, I removed (via silent mutation) start codons of an overlapping gene. Also, I deleted DNA sequences in between the RBS and promoter region to make each part concise.


The refactored genome is Part:BBa_M30333 on the Registry of Standard Biological Parts


Part Elements Modifications (for most: new restriction sites)
BBa_M31112 HpaI (---AAC), rest of gene2 The promoter and RBS regions for gene 10 were modified via silent mutations.
BBa_M31010 SphI (GCATGC), g10-promoter, g10-RBS, g10 coding region, SphI The DNA sequence between the promoter and RBS was deleted.
BBa_M31005 XbaI (TCTAGA), g5-promoter, g5-RBS, g5 coding region, XbaI RBS of gene 7 still needs to be modified.
BBa_M31007 SpeI (ACTAGT), g7-RBS, g7-coding region, SpeI ATG site for gene 9 was changed to gTa (gene 7 still encodes same amino acids). RBS of gene 9 still needs to be modified.
BBa_M31979 NcoI (CCATGG), g9-RBS, g9-coding region, NcoI ATG site for gene 8 was changed to gTa (gene 9 still encodes same amino acids). Promoter and RBS of gene 8 still needs to be modified.
BBa_M31998 SalI (GTCGAC), g8-promoter, g8-RBS, g8-coding region, SalI The region between the promoter and RBS was deleted.
BBa_M31999 g3-promoter, g3-RBS, incomplete coding region of g3, BamHI (GGATCC) The region between the promoer and RBS remain .


Module 4 Research Proposal

Topic

Regulation of gene expression by assembly/disassembly of TFIID/TFTC/STAGA complexes through SMAT activation/deactivation

Background

RNA Pol II needs general transcription factors such as TFIID, which includes TATA-binding proteins and TBP-associated factors (TAFs). TFIID allows the assembly of site specific preinitiation complex. TFIID have TAFs in common with complexes such as SAGA, SPT3-TAF9-GCN5 containing complex (STAGA), TBP-free TAF-containing complex (TFTC), and p300/CBP associated factor (PCAF) complex. Both TFIID and SAGA have subunit interactions which are mediated by histone fold (HF) motifs. Both complexes are also similar in alignment in their spatial distribution of TAF5, TAF6, and TAF10. There has been a lot of focus on the composition of these complexes, however, we want to address how these complexes are assembled and dissembled. Specifically, we want to see how TAF interactions regulate TFIID, TFTC, and STAGA formation. Researchers found a small TAF complex (SMAT), which is composed of TAF8, TAF10, and SPT7L, which may be an important regulator of composition of TFIID and TFTC-type complexes. TAF8 is required for TAF10 to become part of a higher order TAF, but TAF8 itself was not found in TFTC/STAGA complexes, which suggest regulatory function related to this small complex. There are many hypotheses about the exact role of SMAT and more research needs to be done in order to confirm them. One hypothesis is that cellular signals may induce a cascade of events that would modify SMAT, perhaps phosphorylate it. The phosphorylated SMAT may modify and release one or more factors and become incorporated into a large TAF-containing complex, such as TFIID, STAGA, or TFTC, which then begins transcription. Essentially, transcription may depend on the activation of SMAT. Thus, it is to our interest to explore what causes the activation of SMAT.

Idea

First, we need to verify that the phosphorylated components of TAF-containing complexes are released factors of SMAT. We will need to find a way to track subunits of SMAT. We can also experiment with different amounts of activated SMAT and the corresponding gene expression or the amount of TAF-containing complexes containing SMAT subunits. Then, we can explore how SMAT becomes activated, such as which enzymes are involved and what chemicals or intra and extra-cellular conditions activate the process.

Source

Demény, M., Soutoglou, E., Nagy, Z. et al. Identification of a Small TAF Complex and Its Role in the Assembly of TAF-Containing Complexes (2007) http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&rendertype=abstract&artid=1820849

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