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| - | June 25, 2009
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| - | WIKI CODING HERE
| + | * From the results, only one sequencing reaction was successful. The mutated site was successfully done. However, the forward and reverse reactions (both mutated and non-mutated) did not result in any sequencing. The lab is willing to repeat the sequencing reaction. |
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| + | Getting BBK LuxOD47E into the psB1AC3 Vector |
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| - | WIKI CODING HERE
| + | Objective: Now that I have LuxOD47E with BBK sites, I need to get it into the psB1AC3 vector, as it also has the biobrick sites in it. I did this through a restriction digest of the plasmid I isolated yesterday and the psB1AC3 vector using both EcoRI/PstI and XbaI/PstI for the insert as well as both EcorI/ PstI and XbaI/PstI for the vector. I left this to digest ovenright in the 37°C waterbath. |
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| | Today, I called up a couple of oil and gas companies to do a follow up on our sponsorship package. Most them rejected our sponsorship proposal. I will continue to contact more companies that me and Prima found. | | Today, I called up a couple of oil and gas companies to do a follow up on our sponsorship package. Most them rejected our sponsorship proposal. I will continue to contact more companies that me and Prima found. |
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| - | WIKI CODING HERE
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| + | Sequencing Results for luxOU-B0015 and B0015-R0040 |
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| - | WIKI CODING HERE
| + | Sequencing results came back for construction. Analysis included VecScreen and BLAST. Glycerol stocks were made (500uL overnight culture with 500uL of 50% glycerol). |
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| - | Purpose: to verify the presence of LuxPQ in a BBK vector. Colonies 8, 9, 10, and 11 of LuxPQ in psB1AK3 were screened by pTaq colony PCR with the following sets of forward and reverse primers: LuxPQ, BBK and BBK CP. The following conditions were used: 94ºC for 6 minutes; 36X (94ºC for 30 seconds; 52ºC for 45 seconds for LuxPQ-F/R primers (or 50ºC for 45 seconds for BBK F/R primers, or 52ºC for 45 seconds for BBK CP F/R primers); 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel, along with a restriction digest of colonies 1-7 with NotI (see below). | + | Purpose: to verify the presence of LuxPQ in a BBK vector. Colonies 8, 9, 10, and 11 of LuxPQ in psB1AK3 were screened by pTaq colony PCR with the following sets of forward and reverse primers: LuxPQ, BBK and BBK CP. The following conditions were used: 94ºC for 6 minutes; 36X (94ºC for 30 seconds; 52ºC for 45 seconds for LuxPQ-F/R primers (or 50ºC for 45 seconds for BBK F/R primers, or 52ºC for 45 seconds for BBK CP F/R primers); 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel, along with a restriction digest of colonies 1-7 with NotI (see below). Looking through all of the wells, it becomes evident that there are no bands at ~3.8kb (the size of LuxPQ), and as such, we must start back from linear LuxPQ with the BBk restriction sites and clone it back into a BBK vector. |
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| | [[Image:2009.06.24.LuxPQ_Col_PCR+BBK_Primer_Verif-1.png|700px]] | | [[Image:2009.06.24.LuxPQ_Col_PCR+BBK_Primer_Verif-1.png|700px]] |
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| - | KATIE
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| - | WIKI CODING HERE
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| - | KEVIN
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| + | Gel Electrophoresis in Second Life |
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| - | WIKI CODING HERE
| + | Today, I finished building necessary components and equipment for gel electrophoresis in Second Life. All that is left is the texture of the volt box and the pictures of the gels. I was also able to script the activity with Katie's PCR script as the basis of the inventory insert. |
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| + | The script has been tested and I troubleshot the GELBOX saying it had the gel inside when it didn't get, and got it to communicate with the computer in order for the computer to give the picture of the gel to the user. |
| - | MANDY
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| + | In addition, we learned how to check sequencing results for Lab in case we need to do this in the future. |
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| + | On the wiki, the sponsor page is fixed. |
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| - | WIKI CODING HERE
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| | I shadowed Jamie today. He showed me how to set up sequencing. HE was sending his B-R-LuxOU-B for colonies 1 and 7 to sequencing with BBK-CP-F primers. He pipetted a 10 microL of C1 and C7 in separate tubes and put the primers in a separate tube. We put primers in separately because if we had added it to the colonies, they wouldn't be able to take it out and redo it if something goes wrong. | | I shadowed Jamie today. He showed me how to set up sequencing. HE was sending his B-R-LuxOU-B for colonies 1 and 7 to sequencing with BBK-CP-F primers. He pipetted a 10 microL of C1 and C7 in separate tubes and put the primers in a separate tube. We put primers in separately because if we had added it to the colonies, they wouldn't be able to take it out and redo it if something goes wrong. |
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| - | STEFAN
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CAROL
Sequencing Results
- From the results, only one sequencing reaction was successful. The mutated site was successfully done. However, the forward and reverse reactions (both mutated and non-mutated) did not result in any sequencing. The lab is willing to repeat the sequencing reaction.
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EMILY
Getting BBK LuxOD47E into the psB1AC3 Vector
Objective: Now that I have LuxOD47E with BBK sites, I need to get it into the psB1AC3 vector, as it also has the biobrick sites in it. I did this through a restriction digest of the plasmid I isolated yesterday and the psB1AC3 vector using both EcoRI/PstI and XbaI/PstI for the insert as well as both EcorI/ PstI and XbaI/PstI for the vector. I left this to digest ovenright in the 37°C waterbath.
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FAHD
Marketing for June 25th 2009
Today, I called up a couple of oil and gas companies to do a follow up on our sponsorship package. Most them rejected our sponsorship proposal. I will continue to contact more companies that me and Prima found.
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JAMIE
Sequencing Results for luxOU-B0015 and B0015-R0040
Sequencing results came back for construction. Analysis included VecScreen and BLAST. Glycerol stocks were made (500uL overnight culture with 500uL of 50% glycerol).
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JEREMY
Screening more colonies of LuxPQ in psB1AK3
Purpose: to verify the presence of LuxPQ in a BBK vector. Colonies 8, 9, 10, and 11 of LuxPQ in psB1AK3 were screened by pTaq colony PCR with the following sets of forward and reverse primers: LuxPQ, BBK and BBK CP. The following conditions were used: 94ºC for 6 minutes; 36X (94ºC for 30 seconds; 52ºC for 45 seconds for LuxPQ-F/R primers (or 50ºC for 45 seconds for BBK F/R primers, or 52ºC for 45 seconds for BBK CP F/R primers); 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel, along with a restriction digest of colonies 1-7 with NotI (see below). Looking through all of the wells, it becomes evident that there are no bands at ~3.8kb (the size of LuxPQ), and as such, we must start back from linear LuxPQ with the BBk restriction sites and clone it back into a BBK vector.
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MANDY
Gel Electrophoresis in Second Life
Today, I finished building necessary components and equipment for gel electrophoresis in Second Life. All that is left is the texture of the volt box and the pictures of the gels. I was also able to script the activity with Katie's PCR script as the basis of the inventory insert.
The script has been tested and I troubleshot the GELBOX saying it had the gel inside when it didn't get, and got it to communicate with the computer in order for the computer to give the picture of the gel to the user.
In addition, we learned how to check sequencing results for Lab in case we need to do this in the future.
On the wiki, the sponsor page is fixed.
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PATRICK
Heads Up Display Screens
Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day.
Began planning out the navigation scheme for the HUD, what screens would be available for you to click through. Very quickly realized that displaying lots of text on a 15x8 character screen was a bad idea, long form communications would have to happen by giving the user notecards (text documents in second life).
Initially, I was sending the display messages to each of the 120 display tiles by name, using chat messages. This was extremely slow... and because each object only has a 100 message queue, and each object received every message whether it was the intended recipient or not, some tiles would have their message dropped! Naming all of the display tiles accurately also turned out to be obnoxious, as a handful of typos didn't become obvious until much later.
Also, updating the script contained in each display tile object for bugfixes or new functionality became a *major* hurdle. Either I would have to create a single new tile, copy it 119 times (easy, fast) and rename each tile with its coordinates (slow, painful), or I would have to go into each tile, delete the old script and replace it with the new one (slow, painful). This system would get rehauled for something a good deal better (though perhaps still not optimal).
The basic outlines of the HUD were established: a main menu, the unbind screen, the build screen a level selection screen, as well as 'count mode'. Count mode would be renamed interaction checker, the idea being that I could check whether the user had made an error in using the biobrick simulator by checking each object for interactions it could have with other objects. The idea is that if a reaction could happen, it should. If a gene is on but it has no gene product around, it should be expressed; or if a repressor is floating about with a compatible promoter still active, the promoter should be turned repressed. Or alternatively, if all copies of a gene are turned off, then none of their gene product should be present. This is another feature that has yet to make it into the simulator proper, but I've designed the sim's objects in a way that will make it easy to implement when I get the time.
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PRIMA
Setting up Sequencing
I shadowed Jamie today. He showed me how to set up sequencing. HE was sending his B-R-LuxOU-B for colonies 1 and 7 to sequencing with BBK-CP-F primers. He pipetted a 10 microL of C1 and C7 in separate tubes and put the primers in a separate tube. We put primers in separately because if we had added it to the colonies, they wouldn't be able to take it out and redo it if something goes wrong.
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VICKI
Construction of LuxOD47A BBk with either J13002 promoter or B0015 terminator sequences
Purpose: We have LuxOD47A BBk on psB1AC3. We are going to attempt to insert either the J13002 promoter in front of it, or the B0015 terminator behind it.
Protocol:
The construction technique (restriction digest, Antarctic phosphatase treatment and ligation) was conducted in accordance with the procedure outlined on the protocol page. 6 sets were prepared where J13002 was treated as the insert and LuxOD47A BBk was treated as the recipient. Another 6 sets were prepared where LuxOD47A BBk was treated as the insert and B0015 was treated as the recipient.
Transformation of constructed plasmids into competent TOP 10 cells
Purpose: to insert the constructed plasmids into TOP 10 cells so that we can acquire more copies of the construct in an environment designed for long-term genetic stability in the freezer
Protocol: The transformation was conducted in accordance with the protocol outlined on the protocol page. As we already established that the cells are competent, pBluescript was not used. Once the transformation took place, overnight plates were prepared, with the J13002 + LuxOD47A BBk on psB1AC3 cultured on C-laced plates and the LuxOD47A BBk + B0015 on psB1AK3 cultured on AK-laced plates.
Sequence reading from yesterday’s results
We submitted the LuxOD47A BBk samples for which there was a successful NotI restriction digest for sequencing. The results arrived today and are included below. They confirm that we do indeed have LuxOD47A in biobrick form.
The comparison results are right here:
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