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pGEM

-Qiagen DNeasy Kit was used to extract genomic DNA from E.Coli pellets.

PCR

-PCR is performed with DinB forward (Tm: 60C) and DinB reverse (Tm: 62C) primers.

-PCR

-PCR product -

PCR product successful

Ligation and Tansformation (1)

-Transformation was unsuccessful

-No colonies grew on plate with what should have had DinB insert.

-Only blue colonies grew on control plate.

-Because the control came from the kit, white colonies should have grown.

-It was undetermined why there was no growth and why the control was inaccurate

-Unsuccessful control results means PCR sample needed to be re-ligated and re-transformed.

-DinB Inserted Plate: 2 white, 5 blue

-Control Plate: 4 white, 3 blue

-The next steps are to determine whether or not the white colonies that grew are our inserted f13s

Further Testing

-Resuspended a white colony from pGEM + DinB plate in order to

run a PCR. Running this PCR would tell if colonies were of desired

product (pGEM with the DinB insert)

-PCR is performed with SP6 (Tm: 51.5C) and T7 (Tm: 56.3C) primers.

-PCR

Primers SP6 and T7 are used because they will cleave the insert out of the vector

20 cycles:

-Denaturation: 95C for 5:30 min; 30 sec for subsequent

-Annealing: 53C for 1 min

-Extension: 72C for 1 min

-Final Extension: 72C for 10 min

-DinB insertion was successful

-Cell cultures made from two white colonies

-Plasmid DNA purified with Qiagen DNeasy Kit

-EcoRI and BamHI used

-Digestion Failed; no enzyme cut -

Gel Electrophoresis

-Digestion Failed; concluded that purified DNA was not

completely clean, under which conditions the enzymes would

not activate -

Gel Electrophoresis

-Mini-dialysis to clean DNA

-Re-digestion (again with EcoRI and BamHI)

Gel Electrophoresis

**Noticed: Cells in culture were not healthy. Something about the growth environment was toxic and making calls sick.

Inoculation

-PCR shows successful insertion so there should should be digestion

-To investigate why the cells were unhealthy, each colony was inoculated with and without ampicillin; expected to see normal growth on plates without ampicillin and no growth on plates with amp because vectors with insert were being selected against.

-All plates grew as lawns and no individual colonies were distinguishable.

-Re-inoculated; expected to see normal growth on plates without ampicillin and no growth on plates with amp because vectors with insert were being selected against.

-Results as expected. Healthy and unheathy colonies seen on all plates.

-A healthy and an unhealthy colony were picked to be re-inoculated, respectively; expecting to see some healthy and some unhealthy colonies on the same plate. Healthy (white round colonies) are mutated to survive the toxicity of the environment. Unhealthy colonies are transparent and flat

-Results were as expected; most colonies were unhealthy

-More cells grew on plates without ampicillin than on plates with

Conclusions

-f13s inserted

-Healthy cells: mutated cells

-Unhealthy cells

Could be any combination of vector/insert of just vector.

-Whatever caused the cells to become sick remains undetermined. We suspect that the inserted f13s mutation was causing the colonies to be sick.

Next Steps

-Test both healthy and unhealthy colonies to see if they have insert. Make culture of inserted and not inserted. Observe culture to see if the cells with f13s insert get sick and the cells without it remain healthy. This will prove that the f13s is the cause of toxicity.

pBAD33

- Obtained low-copy plasmid DNA pBAD33 from E.Coli cells with QIAprep Miniprep Kit

Double Digest of pBAD33 with EcoRI and BamHI

- PCR was performed with our own designed forward primer and reverse primers

* Designed for EcoRI and BamHI digest

- MJ forward primer- Tm: 72.9C

5’-GGACGCGAGTACCGCTATCATGAAT

TCATGCGTAAAATGATTCATGTGGA-3’

-MJ reverse primer- Tm: 74.7C

5’-GGATCCTAATCCCAGCACCAGTTGG

TGGTGATGGTGATGATGTCA-3’

-DNA resource: F13S TLS deficient cell suspension

20 cycles:

- Denaturation: 95C for 5 min; 30 sec for subsequent

- Annealing: 55C for first three cycles (specific Tm)

67C for the rest cycles (Tm)

- Extension: 72C for 1 min

72C for 10 min for the last cycle

* Gel picture was lost, but we did get bands for PCR products

* It was also confirmed on NanoDrop; the NanoDrop gave us the PCR products had DNA concentration at ~28ng/uL

-used QIAquick PCR purification kit to purify PCR product

-F13S PCR product was ligased into pBAD33 vector which has been cut by EcoRI and BamHI

-Transferred only ligation-1 and ligation-2 tubes

-Transformed cells were plated on Chloramphenicol plates for selecting successful gene insertion.

Further tests

-8 colonies out of 157 white colonies from the plate were chosen and made suspension for each colony

- Added phenol chloroform (break cells, destroy proteins, expose all DNA and RNA)

-Extracted DNA phase and ran gel electrophoresis

8 selected colonies were re-inoculated for future use

gel from dirty prep colonies

1~8 & uncut pBAD33

-ran PCR with DinB forward and DinB reverse primers

-gel eletrophoresis

3) Extract plasmid and double digest with EcoRI and BamHI

(colony 2 and colony 6 were used for inoculation)

Conclusion

- Dirty prep didn't show obvious evidence about insertion

- PCR with DinBF and DinBR showed a band, but this band didn't show correct size (1kb)

- Double digest didn't cut the plasmid into right sizes; rather, it showed "gross re-arrangement"

Concluded that cells from colony 1 there was no successful insertion.

Results from dirty prep were not very reliable and obvious. We may want to inoculate more colony suspension for PCR and double digest test. We may see some colonies have successful insertion, while some don't (or may be gross-arranged).

Protein Overexpression

DinBC66A-Chis

-5 tubes prpared with LB/Ampicillin solution

-Tube 1 is introduced with 100µL of IPTG

solution is diluted down each tube by 100µL

IPTG:

Isopropyl -D-1-thiogalactopyranoside

-Samples are prepared and for sodium dodecyl sulfate polyacrylamide gel electrophoresis.

-Gel Results

When LB/DinBC66A-CHis cell culture was at

OD 0.711, ITPG was added at 1.25mmol

-Cells were harvested and proteins extracted according to the Novagen Bugbuster Protein Extraction Reagent Kit

-SDS determines the size of the proteins

-Protein should be seen in column 3 and 6

- We expect our protein size to be ~30-35 kDa

-Wash 1 should show a lot of debris, but no protein in colums 4 and 6

-Last wash sould show nothing in columns 5 and 8

-The load should show everything including protein column 9

-Gel Results

-The IPTG was successful in eliminating the lac repression and it successfully expressed the proteins.

Biochemistry Methods Laboratory

-DNA recombination technology for

Protein Overexpression

Plasmid vector extraction

- Ran extracted pBAD 33 on gel electrophoresis

* Lost the gel picture

Cells are resuspended and a culture

is grown

pGEM PCR Product

Gel run on 2.17 with

2.10 PCR

Induces lac operon expression in E. coli by binding to the lacI

repressor, preventing repression of lacZ.

PCR

Product

20 cycles:

-Denaturation: 95C for 1 min; 30 sec for subsequent

-Annealing: 55C for 1 min

-Extension: 72C for 1 min

-Final extension: 72C for 10 min

Painful memory of numerous unsuccessful digests --Yeah~! Finally!

Serial Dilution

Gel Electrophoresis

BamHI

BamHI & EcoRI double digest

uncut

BamHI

EcoRI

EcoRI & BamHI

double digest

EcoRI

2.10

Gel for EcoRI and BamHI

double digest (1)

Gel for EcoRI and BamHI

double digest (2)

2.24

3.7

Gel for EcoRI and BamHI

double digest

-Promega ligation protocol followed

-f13s PCR product from 2/17 ligated into the pGEM and the control from kit is used

Results

SDS Page

PCR

Lane 1 & 8: Ladder

Lane 2-7: Highest to lowest IPTG concentration

Lane 4 (second dilution) used as concentraion for culture

Conclusion

Culture

Protein Extraction and Purification

Ligation and Transformation (2)

-Magnetic agarose beads are used to bind the proteins while all cellular debris and dna is washed away (4 washes)

Results

SDS Page

PCR

Well layout

1) BIO-RAD precision plus protein unstained standard--Ladder

2) Skip (ladder spilt out)

3) Protein elution 1

4) First wash of Protein elution 1

5) Last wash of Protein elution 1

6) Protein elution 2

7) First wash of Protein elution 2

8) Last wash of Protein elution 2

9) Load

-PCR setting:

Gel run on 4.4

Dyed and imaged on 4.5

-PCR product

-Gel eletrophoresis

pGEM PCR Product

Gel run on 3.10

Conclusion

DinB insert

-PCR Product-

Gel Electrophoresis

Culture

PCR Production Modification

Double Digestion

Gel run on 3.14

Double Digest

-Re-digested (again with EcoRI and BamHI)

Double Digestion

Gel run on 3.17

Ligation

-Digestion Failed -

Double Digestion

2036 bp

1636 bp

Gel run on 3.21 with

3.17 digest

150 ng : 50 ng

50 ng : 16 ng

ligation-1

ligation-2

ligation-3

1:3 (pBAD33:PCR product)

3:1 (pBAD33:PCR product)

no PCR product--control

* pBAD33: PCR product is the ratio in nano-gram

Transformation

Plating

Colonies plated on 3.21

Growth observed on 3.24

Imaged on 4.7

Healthy

Unhealthy

1) Dirty prep (trash prep)

Ampicillin No Ampicillin

what we expect from

dirty-prep gel

-Different types of cells

As seen in colony growth

heavy dye

gel from dirty prep colonies

1~8 & uncut pBAD33

***dye washed***

2) PCR (colony 1 cell suspension)

PCR of ligated pBAD33

"Gross Re-arrangement"

Gel of double

digested pBAD33

4/4/11

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