IGEM:Harvard/2007/Laboratory Notebooks/Quorum Sensing/Week 5

Colony PCR of R-P constructs
Stephanie Colony PCR'ed the following:
 * R0052<P0140 colony #1 (worked)
 * R0052<P0140 colony #2
 * R0051<P0140 colony #1 (worked)
 * R0051<P0140 colony #2 (worked)
 * R0051<P0340 colony #1 (faint)
 * R0051<P0340 colony #2
 * R0011<P0340 colony #1
 * R0011<P0340 colony #2 (worked)
 * R0052<P0340 colony #1 (worked)
 * R0052<P0340 colony #2
 * R0011<P0140 colony #1

Stephanie's notebook entry

Plate Reader (Light Scattering)
Stephanie set up another plate reader for fluorescence. One of the things she is testing is choosing ridiculous wavelengths to see if we can get light scattering data that correlates to cell growth.

Stephanie's notebook entry Results posted by Stephanie

Miniprep of the J04450 and JT constructs
In preparation for building the constitutive RFP + JT construct, George miniprepped the following parts which were grown overnight:
 * J04450
 * J23039>T9002

George's notebook entry

Digesting the J04450 and J-T
George digested the J04450 with SpeI and PstI; he also digested the J23039>T9002 with XbaI and PstI.

George's notebook entry

Sequencing the Promoters: R0011, R0051, R0052
George and Stephanie sent the R0011, R0051, and the R0052 for sequencing.

Dephosphorylating the J04450 and J-T
George dephosphorylated the J04450 and the J-T with Antarctic Phosphotase and then heat inactivated the AP. Silly George, AP is for vectors not inserts. Redo it tomorrow.

George's notebook entry

Liquid Cultures of R-P constructs and J-T
George grew liquid cultures of the following:
 * R0011 <P0340 colony #2
 * R0051 <P0140 colony #1
 * R0051 <P0340 colony #1
 * R0052 <P0140 colony #2 Lower Right
 * R0052 <P0340 colony #1 Upper Right
 * R0052 <P0340 colony #1 Lower Right
 * J23039 <T9002

The Upper/Lower Right is due to the fact that the colony PCR plate had duplicates of those colonies and it was unclear which colony was correct (at least, the PCR and gel was correct). So, George took both samples and labeled one as coming from the upper right of the plate (when viewed with the handwriting going the correct way) versus lower right.

George's notebook entry

Miniprepping the R-P and JT
George miniprepped the R-P and J23039 <T9002 constructs that were grown overnight in preparation for building the RFP+JT and tetR+JT constructs.

George's notebook entry

Digesting the R-P and JT
George digested the R-P constructs with SpeI and PstI; he also digested the J23039 <T9002 with XbaI and PstI in preparation for building the RFP+JT and tetR+JT constructs.

George's notebook entry

Sequencing the R-P constructs
George sent off sequences for the R-P constructs

George's notebook entry

Dephosphorylating the R-P constructs
George dephosphorylated the R-P constructs with Antarctic Phosphotase and then he heat inactivated the AP.

George's notebook entry

Clonewell and Vacufuge of R-P constructs and JT
George clonewelled and vacufuged the R-P constructs. Unfortunately, he forgot to include JT and had to re-use the same gel.

George's notebook entry

Ligation and Transformation tetR-JT and RFP-JT constructs
George Roche ligated the following:
 * (R0051 <P0140) <(J23039 <T9002)
 * J04450 <(J23039 <T9002)

and then transformed them into BL21.

User:GeorgeXu

Colony PCR of tetR-JT and RFP-JT
George colony PCR'ed the tetR-JT and RFP-JT constructs.

Liquid Cultures of R-P and JT parts
George grew liquid cultures of all the working R-P parts (according to the colony PCR). He discovered that the R52's were incorrect (sequences + wrong selection plate). The parts he grew up were:
 * R0051 <P0140 colony #1 in Top10
 * R0051 <P0340 colony #1 in Top10
 * R0011 <P0340 colony #2 in Top10
 * J23039 <T9002 in BL21

Miniprep of R-P and JT
Stephanie miniprepped the parts that were grown in liquid culture.

Stephanie's notebook entry

Digestion of R-P and JT
Stephanie digested the parts that were miniprepped.

Stephanie's notebook entry

Dephosphorylation of R-P and JT
George dephosphorylated the JT with Antarctic Phosphotase.

George's notebook entry

Clonewell and Vacufuge of R-P and JT
George clonewelled and vacufuged the R-P and JT

George's notebook entry

Colony PCR of RP-JT parts
George colony PCR'ed the RP-JT parts for the third time. Unfortunately, the results were horrible.

|George's notebook entry