Plasmids involved in our transformations.

The following figure summarizes the construction of Car20ZT.2.  This plasmid provided the backbone for our first set of promoter constructs.  The selectable marker was rosy.  More recently (Spring 99), we've ended up subcloning many things into a white vector.  An example of a white vector derivative containing the wild-type hsp70 promoter is given here.  DNA strider files for some of plasmids have been posted here.

The following is a summary of the steps that went into making Car20ZT.2.

Below is a MacPlasmap rendition of Car20ZT.2.

Promoter constructs were made by inserting promoter fragments into the Sal1 site.  Generally, we subcloned XhoI/SalI fragments.  In retrospect, this was a difficult strategy because the XhoI and Sal1 ends could ligate together.  Nevertheless, we did manage to make several transgenic fly lines.

The major stumbling block was getting transformants.  The rosy line of flies was quite sickly and survival from the injection procedure was very poor.  Hongbing Tang decided to switch to the white system by subcloning the relevant part into pP[CaSpeR-4], which is shown below.
 
 

The subcloning is illustrated below:

An example of one of the resulting white constructs is provided below.  This construct contains the -194 version of the hsp70 promoter.  We've now got many versions of this including core promoter mutants and constructs with binding sites for Gal4 and the tet repressor located upstream from the TATA box.


-194//+84Z.T.W. (alias -194hsp70w):This plasmid was constructed in order to transfer our hsp70 promoter from a Car20/rosy based vector into into a CaSpeR/white vector.  In making the transfer, we kept a large portion of the rosy gene but deleted the promoter for rosy.  Our hope is that the large piece of rosy will help insulate the hsp70 promoter from extraneous influences by outside sequences.  It is our desire to have promoter activity be contingent on the sequences between -194 and +84.  The XhoI, NotI and EcoRI sites marked with double asterisks represent the junctions in the 3-way ligation used to construct this plasmid.


The following describes the strategy that has worked successfully to generate new promoter constructs in the white vector.  Many of our previous constructs were transferred from the rosy vector so they involved the approach described above which involved a 3 way ligation with a fragment from LacZT and pP[CaSpeR-4].  Fortunately, there is a unique XbaI site (position 8.92) immediately upstream from the hsp70 promoter in -194//+84Z.T.W (see above).  This XbaI site is also in most of our promoter mutations that reside in pUC13.  The promoter region is PCR amplified from the pUC13 vector using the forward sequencing primer and Cilli's NotI/SalI primer (slots 71 and 81 of Oligo box A) and then cut with Not1 and XbaI.  The fragment is gel purified and then subcloned into NotI/XbaI cut, gel purified -194//+84Z.T.W.  It is also possible to select a version of the white vector containing a promoter fragment that can be distinguished from the promoter fragment you are trying to substitute.  This allows one to screen for candidates that have the appropriate restriction site(s).