Lab Notebook - Tom

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B277 PCR of basic parts from construction file

6/23/09 /wiki/Construction_Files#PCR_Construction_of_.7B.3CespP.28beta.29.3E.7D

protocol used: Template:SBB-Protocols PCR2

all 2K55 cycling program, except B09ig328, run at 4K55

PCR Construction of {<espP(beta)>} PCR Construction of {<upaG_short>} PCR Construction of {<CPG_L2>} PCR Construction of {<CPG_L6>} PCR Construction of {<TshA>} PCR Construction of {<eCPX>} PCR Construction of {<ehaB>} Basic PCR construction of B09ig324 {<SOD>} Basic PCR construction of B09ig326 {<Tir-M>} Basic PCR construction of B09ig328 {<cel6A!}


PCR of basic parts from construction file (cont'd)

Cont'd PCR products (x10)from yesterday were zymocleaned, analyzed on gel, digested with Eco/Bam as per protocol, gel purified, Zymo column purif, and frozen. In addition, 3 ul of vector 9523-1439 obtained from V, was Eco/Bam digested for general use. Analytical gel showed all 10 PCR products in expected size range.


Elicia and Joey ligated the 10 digested,gp'd PCR products using 9523-1439 parent vector and transformed them into___ competent cells, plating on LB/AMP, needed LB/Spec, so they re-transformed 6/26.
Analytical gel.jpg
Analytical Gel

Streptavidin assay development


Transformation of DH10B with pBca9145-9494 for use as positive control in streptavidin assay= Done with protocol Lane modified Did both +/- plasmid. Used AMP plates from rm327, good XF. Using AMP plates from B144, lawns grew on both plates, suggesting no AMP on plates (sleeve of AMP plates tossed)


Picked colonies from pBca9145-9494 (putative +)plate into 3x 4ml cultures for SA assay Picked colonies from pBca9495-1393(putative -) plate into 3x 4ml cultures for SA assay


Ran above culture +/- arab using 35λ cells and 5λ SA at 40μg/mL using 384 well format (protocol: Results: high fluor in all wells, imaged upside down (USD) and right side up (RSU) with USD showing more plastic artifact brightness but also clearer pellet with spun cells. After transfer of sup to new wells(difficult) and adding PBS to cells, most fluor went with sups but still some in cells, tho inconsistent. This procedure does not seem accurate, nor feasible and a larger scale. Plan to try 96 conical , to allow packed pellet and more cells.


O/N DH10B cultures with pBca9145-9494 and pBca9495-1393 were diluted 1:100 (to max cell# at 5hrs induction) and 9494 cells only were induced with 100μg/mL arabinose for possible use this PM with new SA.


Tested ELESA protocol - Used ARA-induced (100 μg/ml for 5 hrs)pBca9145-9494 and uninduced pBca9495-1393 cells diluted 1:100 from O/N cultures. 96 well flat bottom plates used, one well each , no cell control. We may want to consider re-inducing culture at say, 3 hrs as cell # increases.


Re-innoc 9494 and 1363 1:1000 for ON.


SA assay using high (2mg/ml) and low (100ug/ml) arabinose induction. Used 25 and 100 ul of 9145-9494 and 9495-1363 in triplicate to test cell pellet size on fluor image. Plate imaged upside down so well A1 is top right of jpeg image.
Plate map File:063009SAassay.xls
Plate Image 063009 strep tag assay - cropped.jpg
Prelim. Results: It appears that a larger pellet reduces the fluorescence signal, as the 25ul cell wells look brighter than the 100ul, so more may not be better. Acidity of LB in high density cultures may inhibit binding of SA-PE (Terry).Low arab appears as good as high. OD600 show growth in high arabinose inhibited slightly compared to low and zero. AVE: -9494 HI 0.3265, LO 3809, CON 0.4276; -1363 HI 0.3072, LO 0.3580, CON 0.3452


SA imaging with varying cell number and LB vs PBS incubation with SA. 9494 and 1363 cells induced after 1:100 dilution of ON culture for 5 hrs with 100ug/ml arabinose. 100, 50, 25 or 0 ul cells transferred to v-bottom 96 well and q.s. to 100ul with PBS. Another aliquot of 1.5 mls of cells was spun 1 min. top speed ufuge, resus in 1.5 ml PBS and transferred the same way. SA added (5ul of 1:5 dil SA stock in PBS) and incub as usual, 37C, 30 min. Cells were spun (5000rpm, 5min), inverted with seal, and imaged. Plate Image 070209 SAassayTom edited.JPG
Results: All in triplicate, 9494 are top right and bottom left (LB samples on the left side of the plate are switched- 9494 on bottom instead of top as designed). LB does influence signal, perhaps due to SA binding inhibition under acidic conditions. Modifying protocol to add SA in PBS after spinning plate with 100ul sat'd cells/well.

Transformation of composite parts for testing. M10210 thru M10219, M10221, M10224, M10225 (13 total) plasmid obtained from Jenn. Transformed into TG1 competent cells as per protocol,<bk>


Picked colonies from 7/3 xf. LOTS/ plate. One /plate and re-picked 9494 and 1363 also. All on LB/AC except 9494 on amp only.


SA assay of composite parts as per protocol: Performed imaging and TECAN fluor at each wash step and normalized to OD.Media:070709 SA assay ave sd integ dens (ID) and ID over OD600-1.xls RESULTS: Positive staining cells were present in both assays for M10219 and M10224 as well as 9494 pos control. Nice to determine a cut-off for fluor/OD signal with more repeats. Tecan data bkgd looks good and data repeatable. Imaging protocol good for quick screen and confirmation studies.


SA assay on the following non-displayer parts (ON cultures obtained from Jenn) <CPG_L2!>, <upaG_short!>, <ehaB!>, <CPG_L6!>, <Ag43_short!>, <CPompX!>, <eCPX!>, <espP(Beta)!>, <TshA!><bk> Imaged and Tecaned at each wash step as per protocol:<bk> Data and normalized graphs show positive SA signals from <upaG_short!>, <ehaB!>, <CPompX!>, and <eCPX!>, with varied signal strength. Media:071009SA OD and Fl in LB edit.xls

Malaria (merozoite surface protein-1)

Felgner lab-Irvine: We'll ship PFI1475w-s2 plasmid on Monday (29 June) via FedEx. Also, PFI1475w-s2 is a 98 kDa protein and has a transmembrane domain at the C-terminal end DGIFCSSSNFLGISFLLILMLIL (length: 23).

Cellulase DNS Assay


Cellulase DNS assay with all cellulase part glycerol stocks. As per Patrick's note below and the paper

The protocol involves three steps:

1. Incubation of the cellulases with filter paper to allow for the release of glucose. -Add a uniformly sized piece of filter paper (about 7 mm in diameter) to each well of a 96 well PCR plate. I was planning on using a hole puncher to generate the filter paper circles but square pieces cut with scissors would work just as well. -Resuspend cells in the 96 well block using a vortexer and add 100 uL of liquid culture to the wells of the PCR plate. Make sure filter paper is submerged. -Cover, and incubate for 1 hour at 37 degrees in the thermocycler.

2. Reaction of the supernatant with the DNS reagent to allow for the development of color -Spin down PCR plate. -transfer 50ul of the supernatant to another 96 well PCR plate -Add 100 ul of DNS -React for 5 minutes at 95 degrees celcius in the thermocycler (check the program, but yesterday i edited to the program z on the black thermocycler behind the robot to have a 5 minute 95 degree heat followed by sixteen degrees forever). Be careful to allow it to cool down fully to sixteen degrees before opening as it is likely some of the wells will boil.

3. Transfer to another 96 well plate and measure absorbance at 540 nm. Media:081809 OD540 cellul assayDNS.xls


Made new cultures from glycerol stock plate and repeated above with following changes: Incubated in 96 well flat bottom with punch on bottom of well and 200 ul cells for 3 hours at 37C. Glucose control in trip at 5mM. Media:082009 OD540 cellul assayDNS 3hr inc.xls

RESULTS: Sheet 1 shows chart of OD, sheet 2 corresponding part name and well. Row C ODs seem to be consistently 5/100 of an OD below all the other rows. This could be a pipetting error or tecan error but it is likely not real. The yellow highlighed data do not contain cells and give a consistently slightly higher OD than wells with cells. With this three hour incubation, no wells gave OD above what is considered bkgd levels. 5mM glucose controls incubated in LB gave OD on average 1.26, Single enzymes may not be able to hydrolyze complex celluloses in paper significantly.