IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/07/17: Difference between revisions
(fix raw html notebook nav) |
|||
| (8 intermediate revisions by 3 users not shown) | |||
| Line 1: | Line 1: | ||
{|{{table}} width="800" | {|{{table}} width="800" | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;"> Week Seven</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
==Lab== | ==Lab== | ||
Today's experiments were not totally relevant to the project. But because of our suspicions that our enzymes are not working properly, we decided to test them on J04450 and another plasmid called pSB, while waiting for the sequencing results to arrive. We | ===Floor Two=== | ||
Today's experiments were not totally relevant to the project. But because of our suspicions that our enzymes are not working properly, we decided to test them on J04450 and another plasmid called pSB, while waiting for the sequencing results to arrive. We tested Pst1, Nde1 and Xba1 enzymes. | |||
To continue protein expression half of the pellets (1a,1b,2a,2b) were resuspended (in 1X binding buffer), sonicated and centrifuged. The cells were then processed to give soluble and insoluble fractions of proteins. The samples were stored in the freezer. | |||
===Floor One=== | |||
Made up 600 ml of LB media (-NaCl). | |||
Performed transformations of <i>E. coli</i> cells using heat shock and electroporation. Two different vectors were used, pMS82 and pAU3-45 with different resistance genes and insertion sites. Each plasmid contains the biosensor (our reporter construct from Floor 2 which consists of the AntGP promoter + Neomycin gene for kanamycin resistance). After <i>E. coli</i> is transformed, the plasmids will be conjugated into <i>Streptomyces</i> sp. isolated from the soil samples. | |||
pMS82- Hygromycin (Hyg) resistance. | |||
pAU3-45 - Apramycin (Apr) resistance. | |||
Top10 cells were transformed via heat shock and ETpuz cells were electro-competent and transformed via electroporation. | |||
Plated on LB (-NaCl): | |||
+ Apr (pAU3-45) Top10 | |||
+ Apr + CM(Chloramphenicol) (pAU3-45) ETpuz | |||
+ Hyg (pMS82) Top10 | |||
+ Hyg + CM (pMS82) ETpuz | |||
<!-- ## Do not edit below this line unless you know what you are doing. ## --> | <!-- ## Do not edit below this line unless you know what you are doing. ## --> | ||
Latest revision as of 23:04, 26 September 2017
 Week Seven
|
 Main project page Previous entry Next entry
|
LabFloor TwoToday's experiments were not totally relevant to the project. But because of our suspicions that our enzymes are not working properly, we decided to test them on J04450 and another plasmid called pSB, while waiting for the sequencing results to arrive. We tested Pst1, Nde1 and Xba1 enzymes. To continue protein expression half of the pellets (1a,1b,2a,2b) were resuspended (in 1X binding buffer), sonicated and centrifuged. The cells were then processed to give soluble and insoluble fractions of proteins. The samples were stored in the freezer. Floor OneMade up 600 ml of LB media (-NaCl). Performed transformations of E. coli cells using heat shock and electroporation. Two different vectors were used, pMS82 and pAU3-45 with different resistance genes and insertion sites. Each plasmid contains the biosensor (our reporter construct from Floor 2 which consists of the AntGP promoter + Neomycin gene for kanamycin resistance). After E. coli is transformed, the plasmids will be conjugated into Streptomyces sp. isolated from the soil samples. pMS82- Hygromycin (Hyg) resistance. pAU3-45 - Apramycin (Apr) resistance. Top10 cells were transformed via heat shock and ETpuz cells were electro-competent and transformed via electroporation. Plated on LB (-NaCl): + Apr (pAU3-45) Top10 + Apr + CM(Chloramphenicol) (pAU3-45) ETpuz + Hyg (pMS82) Top10 + Hyg + CM (pMS82) ETpuz
| |
