SBB09Ntbk-VaiUmesh: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
Line 25: | Line 25: | ||
==*'''[[User:Vaibhavi Umesh|Vaibhavi Umesh]] 4 February 2009 (EST)''':== | ==*'''[[User:Vaibhavi Umesh|Vaibhavi Umesh]] 4 February 2009 (EST)''':== | ||
<pre> | |||
- Everyone in class got Project Balls <br> | - Everyone in class got Project Balls <br> | ||
- Worked on Construction files for OmpX | - Worked on Construction files for OmpX | ||
</pre> | |||
=='''[[User:Vaibhavi Umesh|Vaibhavi Umesh]] 6 February 2009 (EST)''':== | |||
<pre> | |||
- Worked on designing oligos and the construction file for the project parts | |||
</pre> | |||
=='''[[User:Vaibhavi Umesh|Vaibhavi Umesh]] 9 February 2009 (EST)''':== | |||
<pre> | |||
- Worked on designing oligos and the construction file for the project parts | |||
- This is what I designed based on reading the paper Chris had suggested: | |||
<pre> | |||
1) Native N-terminus - OmpX {N.ompX!} | |||
PCR VU015F/VU016R on OmpX (529 bp, EcoRI/BamHI) | |||
Sub into pBca9145-Bca1144#5 (EcoRI/BamHI, 2057+910, L) | |||
Product is M10012 {N.ompX>} | |||
------------------------------------------- | |||
VU015F Construction of OmpX N term part | |||
ccaaaGAATTCatgAGATCTatgaaaaaaattgcatgtctttcagcactggccgc (40% GC content, length = 55 bp) | |||
VU016R Construction of OmpX N term part | |||
gcaaaGGATCCttaaccggcaatccaggtgcctacg (56% GC content, length = 36 bp) | |||
--------- | |||
2) Native C-terminus - OmpX { | |||
AGATCTgttggttaccgcttctaaGGATCC | |||
EIPCR VU017F /VU018R on pBca9145-Bca1144#5 (2090 bp, BglII) | |||
Product is M10013 { -------------------------------------------- | |||
VU017F EIPCR construction of {C.OmpX>} ccataAGATCTgttggttaccgcttctaaGGATCCtaaCTCGAGctgcag | |||
VU018R Reverse BglII oligo for His6 EIPCR CCAATAGATCTcatgaattccagaaatc | |||
------------- | |||
Assemble the complete part by SOEing | |||
Criteria for Linker between N and C terminus: | |||
From the paper, I was able to extract the following information: | |||
* The linker should be 6 peptides long | |||
* The first peptide should be Glycine | |||
* The third and sixth positions were restricted to R/K/S/H/Q/N | |||
* The substitution A165V resulted in improved display scaffolds | |||
* The substitution G166S resulted in improved display scaffolds | |||
* The display enhancing substitutions A165V and G166S are located immediately upstream of the native C-terminus of OmpX | |||
* The remaining positions (2 and 4) should be randomized | |||
keeping all this in mind I thought the following linker would be best suited: | |||
GSKNVS | |||
which has a nucleotide sequence of: GGTTCTAAAAATGTTTCT | |||
New N junction | |||
gttggttaccgcttc | |||
New C junction | |||
aaaaaaattgcatgtctttc | |||
Forward Oligo: | |||
Linker.C junction | |||
GGTTCTAAAAATGTTTCTaaaaaaattgcatgtctttc | |||
Reverse Oligo: | |||
N junction.Linker | |||
gttggttaccgcttcGGTTCTAAAAATGTTTCT | |||
Reverse Complement: | |||
AGAAACATTTTTAGAACCgaagcggtaaccaac | |||
PCR VU019F /VU016R on M10012 (527 bp, gp A) | |||
PCR VU017F/VU020R on M10013 (44 bp, gp B) | |||
PCR VU021F /VU016R on gp B + gp A (553 bp, EcoRI/BamHI) | |||
Sub into pBca9145-Bca1144#5 (EcoRI/BamHI, 2057+910, L) | |||
Product is M10014 { ------------------------- | |||
VU019F Forward SOEing oligo for cpOmpX | |||
GGTTCTAAAAATGTTTCTaaaaaaattgcatgtctttc | |||
VU020R Reverse SOEing oligo for cpOmpX | |||
AGAAACATTTTTAGAACCgaagcggtaaccaac | |||
VU021F Forward construction of C.OmpX | |||
ccataAGATCTgttggttaccgcttcGGTTCTAAAAATGTTTCT | |||
Note: VU021F had to be used instead of re-using VU017 because VU017 was used to construct {C.OmpX>} through EIPCR | |||
</pre> | |||
</pre> | |||
=='''[[User:Vaibhavi Umesh|Vaibhavi Umesh]] 10 February 2009 (EST)''':== | |||
<pre> | |||
- Project parts were due --> Contruction files | |||
- Oligos will be ordered this week | |||
- Chris said he would go through all the construction files and make corrections as necessary. | |||
</pre> | |||
=='''[[User:Vaibhavi Umesh|Vaibhavi Umesh]] 11 February 2009 (EST)''':== | |||
<pre> | |||
- Chris corrected the construction files: | |||
<pre> | |||
1) Native C-terminal portion of ompX {N.ompX} | |||
PCR Ovu015/Ovu016 on MG1655 gen. (322bp, EcoRI/BamHI) | |||
Sub into pBca9145-Bca1144#5 (EcoRI/BamHI, L) | |||
Product is pBca9145-M10012 {N.cpx} | |||
----------------------------------------------------------- | |||
Ovu015 Construction of OmpX N term part | |||
ctagaGAATTCatgAGATCTGGTCAGTCTggtgactacaacaaaaaccag | |||
Ovu016 Construction of OmpX N term part | |||
gacaaGGATCCgaagcggtaaccaacagaggcaatccaggtgcctac | |||
2) Native N-terminal portion of ompX {C.ompX} | |||
PCR Ovu017/Ovu018 on MG1655 gen. (196bp, EcoRI/BamHI) | |||
Sub into pBca9145-Bca1144#5 (EcoRI/BamHI, L) | |||
Product is pBca9145-M10013 {C.cpx} | |||
----------------------------------------------------------- | |||
Ovu017 Construction of OmpX C term part | |||
ctagaGAATTCatgAGATCTgcgacttctactgtaactgg | |||
Ovu018 Construction of OmpX C term part | |||
gacaaGGATCCttaagagcttgcagtacggcttttctcgg | |||
3) SOEing assembly of eCPX | |||
PCR Ovu015/Ovu019 on pBca9145-M10012 (334bp, EcoRI/BamHI = A) | |||
PCR Ovu020/Ovu018 on pBca9145-M10013 (194bp, EcoRI/BamHI = B) | |||
PCR Ovu015/Ovu018 on A+B (505bp, EcoRI/BamHI) | |||
Sub into pBca9145-Bca1144#5 (EcoRI/BamHI, L) | |||
Product is pBca9495KC-M10014 {<eCPX!} | |||
----------------------------------------------------------- | |||
Ovu019 SOEing of eCPX | |||
gtcgcTTTAGACTGTTTAGATCCgaagcggtaaccaacag | |||
Ovu020 SOEing of eCPX | |||
GGATCTAAACAGTCTAAAgcgacttctactgtaactgg | |||
</pre> | |||
</pre> |
Revision as of 12:18, 19 May 2009
~~!~~
Notes
to do
JCAnderson 13:51, 2 February 2009 (EST)
Today I learned how to change a wiki page and made my pages.
Vaibhavi Umesh 2 February 2009 (EST):
- Recieved project ball --> OmpX - Learned how to change a wiki page - Made/edited personal page Things to do - Make wiki notebook page - Make personal page - Read about circularly permuted plasmids - Read paper about OmpX - Begin working on construction file to design OmpX basic part
*Vaibhavi Umesh 4 February 2009 (EST):
- Everyone in class got Project Balls <br> - Worked on Construction files for OmpX
Vaibhavi Umesh 6 February 2009 (EST):
- Worked on designing oligos and the construction file for the project parts
Vaibhavi Umesh 9 February 2009 (EST):
- Worked on designing oligos and the construction file for the project parts - This is what I designed based on reading the paper Chris had suggested: <pre> 1) Native N-terminus - OmpX {N.ompX!} PCR VU015F/VU016R on OmpX (529 bp, EcoRI/BamHI) Sub into pBca9145-Bca1144#5 (EcoRI/BamHI, 2057+910, L) Product is M10012 {N.ompX>} ------------------------------------------- VU015F Construction of OmpX N term part ccaaaGAATTCatgAGATCTatgaaaaaaattgcatgtctttcagcactggccgc (40% GC content, length = 55 bp) VU016R Construction of OmpX N term part gcaaaGGATCCttaaccggcaatccaggtgcctacg (56% GC content, length = 36 bp) --------- 2) Native C-terminus - OmpX { AGATCTgttggttaccgcttctaaGGATCC EIPCR VU017F /VU018R on pBca9145-Bca1144#5 (2090 bp, BglII) Product is M10013 { -------------------------------------------- VU017F EIPCR construction of {C.OmpX>} ccataAGATCTgttggttaccgcttctaaGGATCCtaaCTCGAGctgcag VU018R Reverse BglII oligo for His6 EIPCR CCAATAGATCTcatgaattccagaaatc ------------- Assemble the complete part by SOEing Criteria for Linker between N and C terminus: From the paper, I was able to extract the following information: * The linker should be 6 peptides long * The first peptide should be Glycine * The third and sixth positions were restricted to R/K/S/H/Q/N * The substitution A165V resulted in improved display scaffolds * The substitution G166S resulted in improved display scaffolds * The display enhancing substitutions A165V and G166S are located immediately upstream of the native C-terminus of OmpX * The remaining positions (2 and 4) should be randomized keeping all this in mind I thought the following linker would be best suited: GSKNVS which has a nucleotide sequence of: GGTTCTAAAAATGTTTCT New N junction gttggttaccgcttc New C junction aaaaaaattgcatgtctttc Forward Oligo: Linker.C junction GGTTCTAAAAATGTTTCTaaaaaaattgcatgtctttc Reverse Oligo: N junction.Linker gttggttaccgcttcGGTTCTAAAAATGTTTCT Reverse Complement: AGAAACATTTTTAGAACCgaagcggtaaccaac PCR VU019F /VU016R on M10012 (527 bp, gp A) PCR VU017F/VU020R on M10013 (44 bp, gp B) PCR VU021F /VU016R on gp B + gp A (553 bp, EcoRI/BamHI) Sub into pBca9145-Bca1144#5 (EcoRI/BamHI, 2057+910, L) Product is M10014 { ------------------------- VU019F Forward SOEing oligo for cpOmpX GGTTCTAAAAATGTTTCTaaaaaaattgcatgtctttc VU020R Reverse SOEing oligo for cpOmpX AGAAACATTTTTAGAACCgaagcggtaaccaac VU021F Forward construction of C.OmpX ccataAGATCTgttggttaccgcttcGGTTCTAAAAATGTTTCT Note: VU021F had to be used instead of re-using VU017 because VU017 was used to construct {C.OmpX>} through EIPCR
Vaibhavi Umesh 10 February 2009 (EST):
- Project parts were due --> Contruction files - Oligos will be ordered this week - Chris said he would go through all the construction files and make corrections as necessary.
Vaibhavi Umesh 11 February 2009 (EST):
- Chris corrected the construction files: <pre> 1) Native C-terminal portion of ompX {N.ompX} PCR Ovu015/Ovu016 on MG1655 gen. (322bp, EcoRI/BamHI) Sub into pBca9145-Bca1144#5 (EcoRI/BamHI, L) Product is pBca9145-M10012 {N.cpx} ----------------------------------------------------------- Ovu015 Construction of OmpX N term part ctagaGAATTCatgAGATCTGGTCAGTCTggtgactacaacaaaaaccag Ovu016 Construction of OmpX N term part gacaaGGATCCgaagcggtaaccaacagaggcaatccaggtgcctac 2) Native N-terminal portion of ompX {C.ompX} PCR Ovu017/Ovu018 on MG1655 gen. (196bp, EcoRI/BamHI) Sub into pBca9145-Bca1144#5 (EcoRI/BamHI, L) Product is pBca9145-M10013 {C.cpx} ----------------------------------------------------------- Ovu017 Construction of OmpX C term part ctagaGAATTCatgAGATCTgcgacttctactgtaactgg Ovu018 Construction of OmpX C term part gacaaGGATCCttaagagcttgcagtacggcttttctcgg 3) SOEing assembly of eCPX PCR Ovu015/Ovu019 on pBca9145-M10012 (334bp, EcoRI/BamHI = A) PCR Ovu020/Ovu018 on pBca9145-M10013 (194bp, EcoRI/BamHI = B) PCR Ovu015/Ovu018 on A+B (505bp, EcoRI/BamHI) Sub into pBca9145-Bca1144#5 (EcoRI/BamHI, L) Product is pBca9495KC-M10014 {<eCPX!} ----------------------------------------------------------- Ovu019 SOEing of eCPX gtcgcTTTAGACTGTTTAGATCCgaagcggtaaccaacag Ovu020 SOEing of eCPX GGATCTAAACAGTCTAAAgcgacttctactgtaactgg