SBB09Ntbk-SusanChen

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Susan Chen 12:00, 06 May 2009 (PST)

Monday:

Grew more cells (in presence of arabinose)

Tuesday:

More incubations. TBS (4/24). fresh AgNO3. varied amount of cells and total volume of solution.

constructs 11-14 (1ml and 2ml) = 0.1mM AgNO3 in 200ul
constructs 15-17 and control (1ml) = 0.1mM AgNO3 in 1000ul
constructs 15-17 and control (2ml) = 1.0mM AgNO3 in 1000ul

incubate for 24 hours

Wednesday:

results:

Image:050609results table.jpg
note: the color that appeared were in solution, not in precipitate form.
Also, the lack of color from constructs 11-14 might be because of small volume since previous experiment had shown color and precipitate.



  • 2ml control, construct 15, construct 16, construct 17



control
construct15
construct16
construct17
no noticeable peaks or differences between the constructs. perhaps scattering signal is too high because of the presence of cells.



  • pelleted 2ml control, construct 15, construct 16, construct 17



control
construct15
construct16
construct17
noticeable increased absorbance around 440nm-500nm. To get rid of the scattering signal, cells were spun down, and this graph was generated using the respective supernatants.

Susan Chen 10:19, 30 April 2009 (PST)

Wednesday:

Results from incubation on Monday:
All of the tubes had some sort of reddish precipitate except the control. Construct 13 had the most precipitate and 16 had the least. 11 and 16 which did not have precipitate in the first experiment had precipitate.
tried to resuspend the reddish particles, but did not resuspend despite lots of vortexing and other attempts.
tried to - 1. add lysozyme 2. manually remove precipitate and put into water 3. lots of vortexing
Took absorbance measurements of control, 16 (lysozyme), 15 (in water), and 13 (vortexing). After normalizing the graph, the control had least absorbance, but there is no significant difference in signal in the 400-440nm range, so inconclusive results.

grew cells in 4ml LB and 4ul arabinose. used DH10B as control instead of 1363.


did not carry out this protocol

method to resuspend reddish particles (assumed to be silver nanoparticles): 
1. pellet cells and particles
2. pour out excess TBS and AgNO3
3. wash with water 2X and resuspend
4. react Ag (red particles) with nitric acid
5. react Ag ions with KBr. KBr(aq) + AgNO3(aq) → AgBr(s) + KNO3(aq)
6. soluble yellow product should form
7. measure absorbance of the yellow product
8. quantify the amount of silver (of the different constructs) formed from the intensity of the color.

Susan Chen 6:50, 28 April 2009 (PST)

Monday:

resuspended cells and incubated them in TBS and 0.1mM AgNO3 (new) at room temperature. put on rocker to agitate the solutions. (repeated experiment)

measured absorption of the various samples using the Tecan.
resuspended the pellets, but chunks of cells remained in solution. pipeted 100ul of each sample into column 9 of plate.
saved data as spectro ver1, ver2, etc
parameters: 250-800nm range. 10nm intervals. 96 well plate.

{graph of absorbance vs wavelength}

graph did not indicate differences between the control and samples with color. There could be too much scattering. could solubilize with base or proteinase to make solution clear. Alternatively, could get rid of cells and measure the total amount of silver bound to cells after washing out excess silver.


Susan Chen 9:46, 27 April 2009 (PST)

Friday:

washed (2X) the cells grown on Thursday in 200ul of TBS. Both the TBS and 1mM AgNO3 solutions were freshly made.
resuspended each of the pellets in 180ul of TBS and 20ul of AgNO3 (to make 0.1mM AgNO3)

Sunday:

checked the cells. noticed a noticeable color change (a reddish color) in the pellet for some of the tubes.

control 1363 - white
11 - white {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
12 - reddish* {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
13 - reddish {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
14 - reddish {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
15 - reddish {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}
16 - white {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
17 - reddish {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}

*deepest color change

grew more cells in the presence of arabinose (4ml LB, 4ul of 1000X arabinose, constructs 11-17 with control)

 
need to: 
1. repeat the experiment
2. find a way to quantify the color change - look in literature 
3. take picture of the solutions


Monday:
will do -
repeat the same experiment done on Friday
search literature for way to quantify results
carry out experiment in 96 well plate with more variations

So far:
1. when grown in presence of silver nitrate, no cell growth
2. when grown in the absence of arabinose, washed twice with TBS, and resuspended in 0.1mM AgNO3 and TBS, no color change in washed pellets
3. when grown in presence of arabinose, washed twice with TBS, and resuspended in 0.1mM AgNO3 and TBS, color change observed in washed pellets

Susan Chen 1:43, 23 April 2009 (PST)

Thursday:

The cells grown in AgNO3 on Tuesday seemed to have died. Perhaps the pH change killed the bacteria.

We washed the cells (we grew on Tues) with TBS, put them in 180ul of TBS and 20ul of 0.1mM AgNO3, and then put them on the shaker. no color change and not much apparent change.

We also grew the washed cells in 3ml LB and 0.1mM AgNO3
No cells seemed to have grown. silver is toxic to cells.

Chris put in formaldehyde to the cells. Cells should have turned black but didn't. not sure if enough silver nitrate was added.

Regrew cells in the presence of arabinose. Constructs 11-17 + control.
So hopefully, the cells will express the silver binding peptides and then on Friday, the TBS and the silver nitrate will be added.

Susan Chen 9:30, 22 April 2009 (PST)

Silver Binding Assay

Monday:

transformed constructs into bacteria
plated the cells
we will use constructs 11-17 and a pBca9145-Bca1363 control

Tuesday:

grew one colony from each sample (11-17) and control in 4ml LB (with CA)
grew one colony from each sample (11-17) and control in 4ml LB (with CA) and 0.1mM AgNO3

growing cells grid

Wednesday:

 make TBS, make AgNO3, measure OD 

cells of var. conc., arabinose, AgNO3 of var. conc., TBS

The cells grown in AgNO3 on Tuesday seemed to have died. Perhaps the pH change killed the bacteria.

We washed the cells (we grew on Tues) with TBS, put them in 180ul of TBS and 20ul of 0.1mM AgNO3, and then put them on the shaker. We also grew the washed cells in 3ml LB and 0.1mM AgNO3

Susan Chen 5:04, 21 March 2009 (PST)

more EhaB sequence analysis

  • There is a point mutation from T to C at base pair 848 from the model file. This is a silent mutation of the amino acid glycine. Therefore, the amino acid sequence should be the same as the original EhaB amino acids from 573-980.

EhaB aa573-980

reverse call file

Susan Chen 10:38, 09 March 2009 (PST)

more analysis of sequence

  • The point deletion in the call file is a misread. (see chromatogram)

The read is partial perfect. sbb024

  • note: in general, look at the width of the peak
  • the EcoRI restriction site in clone 1 is not from the template, but was probably a mutation from the pcr
to do: Seqresult follow up


Susan Chen 9:32, 08 March 2009 (PST)

analysis of clone 2 sequencing results from EhaB autotransporter

  • EcoRI and BglII restriction sites were present
  • the part sequence matched with the original exactly until 848
  • single nucleotide change from t to c at 848 (glycine). This is a silent mutation.
  • single nucleotide deletion at 874. This resulted in a frame shift.
  • chromatogram: good read from ~30 - 800 (not sure)

original EhaB autotransporter part

forward call file

Did it work? If everything beyond 874 is discarded, then the amino acid sequence matched exactly.

to do: sequence clone 2 from the other end

Susan Chen 12:00, 06 March 2009 (PST)

analysis of sequencing results from EhaB autotransporter part

The part will not work. sbb023

reasons:
1. An internal EcoRI restriction site was present (single nucleotide mutation from c to t at 38), either in the template or as a result of pcr, so the original part sequence was cut into two pieces (one piece from EcoRI to EcoRI and the other from EcoRI to BamHI). The DNA that was inserted into the vector plasmid was the longer EcoRI to BamHI piece, so the first ~35bps of the 5' end from the EhaB autotransporter was missing from the call file read.
2. There is a single nucleotide mutation from t to c at 844. The missense mutation changes the a.a. from Leu to Pro.
3. There is another single nucleotide change from a to g at 1198. The missense mutation changes the a.a. from Asp to Gly.

  • 50-700 usually good read

Original EhaB autotransporter Part Sequence

Call File forward

Call File reverse

to do: sequence clone 2 of EhaB autotransporter part to find out the source of the mutation
       rescue and make part again?


Susan Chen 6:29, 04 March 2009 (PST)

miniprep purification of plasmid DNA for M10052
Using the QIAGEN QIAPrep Spin Minipret kit

1. Pellet 1.5 mL saturated culture by spinning full speed, 30 seconds.
2. Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL)
3. Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
6. Spin in centrifuge at top speed for 5 minutes.
7. Label blue columns with an alcohol-resistant lab pen.
8. Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
10.Wash each column with 500 uL of PB buffer.
11.Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
12.Wash with 750uL of PE buffer (washes the salts off the resins).
13.Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
14.Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
15.Label new tubes and put columns in them.
16.Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
17.Spin in centrifuge at top speed for 30 seconds.
18.Take out columns and cap the tubes.

Mapping of M10052 with EcoRI/BamHI

  • pcr product is 1257bp
2ul plasmid DNA
6.5ul water
1ul Neb Buffer 2
.5ul EcoRI
.5ul XhoI
  • did this twice because had picked two clones
  • incubated at 37 degrees for 30 minutes
  • ran an analytical gel

Image:030409gelpic.jpg

  • the gel on the left

lane 0 = ladder
lane 3 = clone 1
lane 4 = clone 2

sequencing

  • 30ul of miniprepped plasmid DNA

clone 1 = plate 1 B2
clone 2 = plate 2 B2


sequencing results from M10053 part
The sequence was correct! sbb012

Original EILD Part Sequence

Call File

Susan Chen 9:44, 03 March 2009 (PST)

mapping of wobble product with EcoRI/XhoI

3ul DNA
5ul water
1ul Neb Buffer 2
.5ul EcoRI
.5ul XhoI
  • did this twice because had picked two clones
  • incubated at 37 degrees for 30 minutes
  • ran an analytical gel

analytical gel
lane 0 = ladder
lane 5 = clone 1
lane 6 = clone 2

The two clones had different size bands even though they are supposed to be the same. This means that one of them is wrong. It is most likely clone 2.

sequencing

pipetted 30ul of clone 1 and clone 2 into plate 1.
clone 1 = E1
clone 2 = F1


ligation of digested M10052 part

  6.5uL ddH2O
  1uL T4 DNA Ligase Buffer 
  1uL pBca9495KC-Bca1144#5 digest
  1uL M10052 digest
  0.5uL T4 DNA Ligase
  • Pound upside down on the bench to mix
  • Give it a quick spin to send it back to the bottom of the tube
  • Incubate on the benchtop for 30min
  • Put on ice and proceed to the transformation

transformation and plating of M10052 part

1. Thaw a 200 uL aliquot of cells on ice
2. Add 50 uL of water
3. Add 30 uL of KCM salts
4. Put ligation mixture on ice, let it cool a minute or two
5. Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
6. Let sit on ice for 10 min
7. Heat shock for 2 min at 42
8. Put back on ice for 1 min
9. Add 100uL of LB, let shake in the 37 degree incubator for 40 min
10.Plate 150ul of cells on KC antibiotics, let incubate overnight

to do:
miniprep M10052 (2x b/c two clones)
map digest
analytical gel
sequence


Susan Chen 11:16, 27 February 2009 (PST)

miniprep purification of DNA for wobble product
procedure for plasmid DNA purification using a QIAGEN QIAPrep Spin Miniprep kit

  • do this twice because picked two colonies

1. Pipet 2 mL of saturated culture in eppendorf. Pellet by spinning full speed, 30 seconds.
2. Dump supernatant, repeat to pellet another 2 mL
3. Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
6. Spin in centrifuge at top speed for 5 minutes.
7. Label blue columns with an alcohol-resistant lab pen.
8. Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
10.Wash each column with 500 uL of PB buffer.
11.Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
12.Wash with 750uL of PE buffer (washes the salts off the resins).
13.Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
14.Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
15.Label new tubes and put columns in them.
16.Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides) (pipet tip touched the resin)
17.Spin in centrifuge at top speed for 30 seconds.
18.Take out columns and cap the tubes.

to do: 
for the leucine EILD:
map with EcoRI/XhoI since part is 136bp (under 250bp)
    *if part is >250bp, then map with EcoRI/BamHI
run a gel 
sequence the part
for the EhaB:
ligate the digestion product
transform and plate

Susan Chen 10:53, 25 February 2009 (PST)

ligation of EcoRI/BamHI digestion product (wobble)

put in a 500ul eppendorf:
  6.5uL ddH2O
  1uL T4 DNA Ligase Buffer 
  1uL pBca9495AK vector digest
  1uL wobble product digest
  0.5uL T4 DNA Ligase
  • Pound upside down on the bench to mix
  • Give it a quick spin to send it back to the bottom of the tube
  • Incubate on the benchtop for 30min


transformation by heat shock and plating
1. Thaw a 200 uL aliquot of cells on ice
2. Add 50 uL of water
3. Add 30 uL of KCM salts
4. Put your ligation mixture on ice, let it cool a minute or two
5. Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
6. Let sit on ice for 10 min
7. Heat shock for 2 min at 42
8. Put back on ice for 1 min
10. Add 100uL of LB, let shake in the 37 degree incubator for 40 min
11. Plate on selective antibiotics, let incubate overnight

  • steps 1-3 were already done


digestion of PCR product with EcoRI/BamHI

  8uL of eluted PCR product
  1uL of NEB Buffer 2
  0.5uL EcoRI
  0.5uL BamHI
  • Incubate at 37 degrees on the thermocycler for 1hr

regular zymo cleanup remove the buffer and restriction enzymes from the restriction digest reaction.

1. Add 180 uL of Zymo ADB buffer 
2. Transfer into the Zymo column 
3. spin through, discard waste.
4. Add 200 uL of PE 
5. spin through, discard waste.
6. Add 200 uL of PE 
7. spin through, discard waste.
8. spin for 90 seconds, full speed to dry.
9. elute with water into a fresh Eppendorf tube

Susan Chen 3:20, 23 February 2009 (PST)

small-frag zymo clean up
Removed the polymerase, dNTPs, buffer, and most of the oligonucleotides from the wobble PCR reaction

1. Add 100 uL of Zymo ADB buffer to the 50ul reaction mixture.
2. Transfer into the Zymo column (put an eppendorf over it)
3. Add 500uL of Ethanol and pipette up and down to mix
4. spin through, discard waste.
5. Add 200 uL of PE 
6. spin through, discard waste.
7. Add 200 uL of PE 
8. spin through, discard waste.
9. spin for 90 seconds, full speed to dry.
10.elute with 50ul of water into a fresh Eppendorf tube
11.spin for 30 seconds and keep the eppendorf tube and label


EcoRI/BamHI digest of wobble product

In a PCR tube, put:
  50uL eluted DNA
  5.7uL NEB Buffer 2
  1uL EcoRI
  1uL BamHI

*Mix thoroughly by slamming the tube upside down on the table, 
and then a quick spin to move the liquid to the bottom of the tube.
*Incubate the reaction at 37 degrees on the thermocycler for about 40minutes


cleanup of digestion reaction
Using the small-frag zymo cleanup, remove the buffer and restriction enzymes from the digestion reaction

1. Add 100 uL of Zymo ADB buffer to the reaction.
2. Transfer into the Zymo column 
3. Add 500uL of Ethanol and pipette up and down to mix
4. spin through, discard waste.
5. Add 200 uL of PE 
6. spin through, discard waste.
7. Add 200 uL of PE 
8. spin through, discard waste.
9. spin for 90 seconds, full speed to dry.
10.elute with 50ul of water into a fresh Eppendorf tube
11.spin for 30 seconds. keep the eppendorf.
  • for this zymo cleanup. I had added PB buffer instead of PE buffer for both of the washes. I had also added

50ul of water for the elution, but did not elute yet. So steps 5 through 11 were repeated again. The DNA might
have washed out. But I kept the eppendorf after the washes with the correct buffer and elution.


analytical gel
shows the presence of product after the PCR cloning

  • added 5ul of product and 5ul of loading buffer/dye
  • pipet 5ul into well
  • 5ul of ladder (Fermentas Gene Ruler 1kb DNA Ladder Plus)
  • run at 100V for 20minutes


analytical gel
first lane = ladder
second lane = pcr product

  • The gel shows a green band slightly above the 1500bp mark (the bright band in the middle of the ladder).

The band should actually be below the bright band since the predicted PCR product is only 1257bp.


regular zymo clean up of PCR product
removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction

   1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 29ul reaction.
   2. Transfer into the Zymo column 
   3. spin through, discard waste.
   4. Add 200 uL of PE  
   5. spin through, discard waste.
   6. Add 200 uL of PE 
   7. spin through, discard waste.
   8. spin for 90 seconds, full speed to dry.
   9. elute (spin for 30 seconds) with 29ul of water into a fresh Eppendorf tube. keep eppendorf tube.


To Do: digest PCR product with EcoRI/BamHI 
       clean up the digestion
       ligate into vector


Susan Chen 9:06, 20 February 2009 (PST)

Wednesday was first day of lab. I got my oligos:

EhaB autotransporter
Osc0030F ggcctGAATTCattAGATCTggcttaatcgcaatgaactct
Osc0031R gtaccGGATCCttaccaggtatatttaacac

Leucine zipper EILD
Osc0028F TCCGGgaattcATGagatctGTTAAAGAACTGGAAGACAAAAACGAAGAACTGCTGTCTGAAATCTACCACCTGGACAACGAAG
Osc0029R CTGGTggatccGCAACCACCACGTTCACCAACCAGTTTTTTCAGACGAGCAACTTCGTTGTCCAGGTGGTAG

I prepared two sets of oligos for two PCR reactions, one wobble (leucine zipper EILD), one not (ehaB autotransporter):

procedures for wobble:
1. centrifuge the oligos down
2. add ddH2O to resuspend oligos and make 100uM stock. 531ul for Osc0028F (53.10nmol) and 599ul for Osc0029R (59.90nmol)
3. scrap tube with rack to vortex the oligos. also use vortexer.
4. centrifuge to spin down all liquids
5. take out a pcr tube, and label it
6. add 29ul H2O, 5ul expand buffer 2 (did I use a clean tip?), 5ul 10X dNTP, 5ul Osc0028F, 5ul Osc0029R, and .75ul 
expand polymerase 1
7. mix solution (with a pipet) and spin down
8. put in pcr machine (the gsis have already set the settings and will run it as well)

procedures for cloning by pcr:
1. centrifuge oligos down
2. add ddH2O to resuspend oligos and make 100uM stock. 269ul for Osc0030F (26.90nmol) and 329ul for Osc0031R (32.90nmol)
3. scrap tube with rack to vortex the oligos. also use vortexer.
4. centrifuge to spin down all liquids
5. dilute the 100uM stock to 10uM by adding 9ul H2O and 1ul 100uM oligo stock
6. take out pcr tube and label
7. to set up the reaction: add 24ul H2O, 3.3ul 10X expand buffer 2, 3.3ul dNTP, 1ul Osc0030F, 1ul Osc0031R, 
0.5ul expand polymerase I, 0.5ul DNA template (which was labeled EHEC...or something...also, I'm not sure if 
I added enough since the amount of template was really low)
8. mix solution and spin down
9. choose the 55 version of the reaction since the pcr product will be 1257bp long
10. put tube in pcr machine (again, the gsis will run it)

I stored the oligo stock in the green box afterwards.
I think the next thing to do is run a gel and clean up for the pcr reactions.

Susan Chen 12:52, 9 February 2009 (PST)

I worked on the construction files for the project, and put them on the lab wiki

to do: read the articles again
       check the construction files


Susan Chen 22:14, 4 February 2009 (PST)

I got my project ball today.

to do:
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