User:Mradha
Module 3: Yeast
SAGA subunits, S. cerevisiae
| Ada subunits | size,chromosome,null p-type | notes |
|---|---|---|
| Ada1 (aka HFI1, SUP110, SRM12, GAN1) | 1.467 kb/489 aa, Chr. XVI, viable |
|
| Ada2 (aka SWI8) | 1.305 kb/434aa, Chr. IV, viable |
|
| Ada3(aka NGG1, SWI7) | 2.109 kb/702aa, Chr. IV, viable |
|
| Gcn5 (aka ADA4, SWI9) | 1.32 kb/439aa, Chr. VII, viable |
|
| Ada5 (aka SPT20) | 1.815 kb/604aa, Chr. XV, viable |
| Spt subunits | size, chromosome, null p-type | notes |
|---|---|---|
| Spt3 | 1.014 kb/337aa, Chr. IV, viable |
|
| Spt7(aka GIT2) | 3.999 kb/1332aa, Chr. II, viable |
|
| Spt8 | 1.809 kb/602aa, Chr. XII, viable |
|
| Spt20 (aka Ada5) | 1.815 kb/604aa, Chr. XV, viable |
| TAF subunits | size, chromosome, null p-type | notes |
|---|---|---|
| TAF5 (aka TAF90) | 2.397 kb/798aa, Chr. II, inviable | |
| TAF6 (aka TAF60) | 1.551 kb/516aa, Chr. VII, inviable | |
| TAF9 (aka TAF17) | 0.474 kb/157aa, Chr. XIII, inviable | |
| TAF10 (aka TAF23, TAF25) | 0.621 kb/206aa, Chr. IV, inviable | |
| TAF12(aka TAF61, TAF68) | 1.620 kb/539aa, Chr. IV, inviable |
| Tra1 subunit | size, chromosome, null p-type | notes |
|---|---|---|
| Tra1 | 11.235 kb/3744aa, Chr. VIII, inviable |
| other subunits | size, chromosome, null p-type | notes |
|---|---|---|
| Sgf73 | 1.974 kb/657aa, Chr. VII , viable |
|
| Sgf29 | 0.779 kb/259aa, Chr. III, viable |
|
| Sgf11 | 0.3 kb/99aa, Chr.XVI, viable |
|
| Ubp8 | 1.416 kb/471aa, Chr. XIII, viable |
|
| Sus1 | gene with intron, Chr. II, viable |
Module 1
Protein Modifications
| protein | function |
|---|---|
| I | Modify protein so that similar proteins, other than pVIII can bind to the surface. |
| II | Keep track of the number of proteins that it nicks. |
| III | Make the proteins bigger so that they can bind to objects easier and so that they can also bind to bigger objects. |
| IV | Modify protein so that similar proteins, other than pVIII can bind to the surface. |
| V | Put tag on the protein so that you can track the bacteria stages. |
| VI | Make it bind more tightly to p3, so that p3 can be modified easily and so that p3 is much more flexible. |
| VII | Make is bind more tightly to p9 to allow for more flexibility. |
| VIII | Make fewer copies so that the proteins can be more flexible. |
| IX | Change the function so that it now lyse the bacteria. |
| X | Increase the number of proteins so that the phage can produce more double strands. |
| XI | Modify protein so that similar proteins, other than pVIII can bind to the surface. |
Results of Ligation Reactions
| protein | function |
|---|---|
| Digest 1: Candidate 1 | One band visible. |
| Digest 1: Candidate 2 | One band visible. |
| Digest 1: Candidate 3 | Two visible bands. |
| Digest 1: Candidate 4 | Two visible bands. |
| Digest 2: Candidate 1 | Four visible bands. |
| Digest 2: Candidate 2 | Three visible bands. |
| Digest 2: Candidate 3 | Three vibible bands. |
| Digest 2: Candidate 4 | Two bands visible but one might just be noise. |
Refactoring M13
Alterations Made
| Changes Made | Method/Reason |
|---|---|
| Changed the Hpal to BamH1 | aspartic acid and a tyrosine to a cystine. |
| Gene VII | I deleted this gene and left the RBS for gene 9 and the promoter for gene 8 intact. |
| Gene X | I separated gene X from gene II and placed its promoter and RBS in front of it and created a new part. This new sequence contains gene X's promoter, RBS and then the gene itself. |
| Gene II | I disrupted the gene X promoter and RBS located within this gene to prevent homologous recombination. I used the degeneracies of the existing amino acids to create new codons without altering the sequence of the protein. |
I created three new parts on the Registry of Standard Biological Parts BBa_M31000, M31001, and M31002. M31000 contains the entire DNA sequence from the Hpal site to the BamH1 site. I removed the Hpal site and changed it into another BamH1 site. This allows for this portion of the plasmid to cut up and removed from the rest of the genome. It will also have blunt ends but the ends will anneal to the each other. I removed gene 7 and left gene 8's promoter and gene 9's RBS. In M31001 I added gene X's promoter and RBS to the front of gene X and made it a compact and independent gene. Then in M31002 I changed gene 2 so that gene 10's promoter and RBS were disrupted and unfunctional. This made it so that gene 2 can be manipulated individually compared to gene 10. A few of the problems I ran into were that it was extremely hard to annotate each of the genes without changing the numerical sequence of the genes following it. The parts registry did not make the necessary changes which resulted in me making multiple individual gene parts.
Module 4
