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What We've Accomplished

Primer Design

SO1415

Gene sequence: overhang of the actual gene

This sequence was found on http://www.ncbi.nlm.nih.gov/

Megablast was used to compare the designed primer against the s. oneidensis genome

The length, gc content, melt temp etc info was found on www.idtdna.com

The site used to find parameters of a well-designed primer: http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

SO_1415 gene sequence: 5’ GAATGAATAA ATGAAATGTCCTTCGGACTCCCTGTCCATTTTACGTTGTAATCAAATATTGGATGCGGCTGAAAAGCTCA TTGAGTCACAAGGTGTTGTATCTTTTAAGTTTTCTCAGCTTGCGCATGAGGTGGGATGCTCTACGGGTAC TTTATATAAATTTTTTGAACGTAAAGAAGATGTGTTGGTTTGTTTATTTTTAAGAAGCGCAACCTCAAAT CACTTACCGATATTTATCCATAAAAATCCAGAGTTAACTGCGCAAGAGAAGGTGCTGTTACCCATTTTAT TTACCTTTGAAACCATTAAGCGCAGTAGTAGCTTTTTTACGCTGCGTTCGGTGTCGGTCAATACCATGGT GTGGAAACTGGCCAGTGACGAAAAAGTGGAGCGGTTTAAAAAACGCATTAATGCTTTTTGGAGTTGGTTT ACAGACTCACTGCATTTAGCTGTTGAAAACGGCGAATTAGTGGCAACACCATTACAAATTAAAGAATTGG TCCAAGGGATAACGTTTTATTTAACAGGGTCTTTGACACAATTTGAAAGTCAATTGATTGCCCCAGAGTT TTTGTCTGATCGCCGTGAAACCTGTTATCGACATTTAGCAAACCTGATGGAGCGATACGAGTGGAAAAAG CCTTTAACTCTTGCGCTGTTTGATTCGTTAGAGGCGAGAACTATTAAGTTTTTTGACCAACATTATCGTG ACCATATGACCTGCGCGGCTTGTAGTGCGCTGTCAAATACCGACACTAAGACATCATCTCCCTGTACTCG TCAGTGTGGTTAG GGCGTCCTGC 3’

Primer 1:

TGA ATA AAT GAA ATG TCC TTC GGA CTC CCT G

LENGTH:31

GC CONTENT:41.9 %

MELT TEMP:59.9 ºC

MOLECULAR WEIGHT:9494.2 g/mole

EXTINCTION COEFFICIENT:298500 L/(mole·cm)

nmole/OD260:3.35

µg/OD260:31.81

Primer 2:

GAC GCC CTA ACC ACA CTG ACG

LENGTH:21

GC CONTENT:61.9 %

MELT TEMP:60.2 ºC

MOLECULAR WEIGHT:6345.2 g/mole

EXTINCTION COEFFICIENT:197000 L/(mole·cm)

nmole/OD260:5.08

µg/OD260:32.21

SO4157

5’ AAGGAAAACC ATGTCCACCATGCTGCCACTGTATTTAGTCGATGATGATGAAGCGATTCTCGACTCCTTAGGGTTTATGC TCAGGCAATTTGGTTACCAAGTACAAACCTTTAGCAGTGGACGGGATTTTTTAGCCCAATGTCCGTTAAC ACAGGCTGGCTGCGTGATTTTAGATAGCCGAATGCCGGAGATCACCGGCCAAGAAGTGCAGCAAAAACTA CTTGAAACCCAAAGCCCATTGGGAGTTATCTTTCTCACGGGGCACGGTGATTTGCCCATGGCATTAAGCG CCTTTCGTAAGGGTGCATGCGATTTTTTTCAAAAGCCGGTATCTGGCAAAGCCCTAGTACAAGCCATTAA AAAAGCGCATAAAGAAAGCCAAGCCAGCTTTGAGCAACAGAGTCTGCAGCATAAATTTGCCCAACTGACC GACCGTGAACAACAAGTGTTAGCCCATGTGGTTCAAGGTATGACCAACAAGCAGATCTCCGAGGCCATGT ATTTATCCTTAAGAACCATTGAAGTGCACCGCGCTAAGATCATGAAAAAGCTCGAAGTCAGTAATATGGC AGAATTAGTACAGCACTTAGCCCACCTAAATACACTCTTACCGGAGTAA TCCAATAAAC 3’

Primer 1:

AAA ACC ATG TCC ACC ATG CTG C

LENGTH:22

GC CONTENT:50.0 %

MELT TEMP:58.7 ºC

MOLECULAR WEIGHT:6648.4 g/mole

EXTINCTION COEFFICIENT:207700 L/(mole·cm)

nmole/OD260:4.81

µg/OD260:32.01

Primer 2: ATT GGA TTA CTC CGG TAA GAG TGT ATT TAG GT

LENGTH:32

GC CONTENT:37.5 %

MELT TEMP:58.6 ºC

MOLECULAR WEIGHT:9924.5 g/mole

EXTINCTION COEFFICIENT:320800 L/(mole·cm)

nmole/OD260:3.12

µg/OD260:30.94

hlyU

ATGAAAACCA TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG CCGATTGACTTAAGCCTAATTTTTTATTCATTTCGCCAACGCACATTTCTCCTTCATTCAATAAATAACA AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC ATCAATTCAATATTCAT TACGCACTTT

Primer 1: ATG AAA ACC ATT AAT GAC AAT AAA TAT TGT TCA ATA AAT GG

LENGTH:41

GC CONTENT:22.0 %

MELT TEMP:56.6 ºC

MOLECULAR WEIGHT:12655.3 g/mole

EXTINCTION COEFFICIENT:432600 L/(mole·cm)

nmole/OD260:2.31

µg/OD260:29.25

Primer 2: GTG CGT AAT GAA TAT TGA ATT GAT GCA GGA

LENGTH:30

GC CONTENT:36.7 %

MELT TEMP:57.8 ºC

MOLECULAR WEIGHT:9349.1 g/mole

EXTINCTION COEFFICIENT:309700 L/(mole·cm)

nmole/OD260:3.23

µg/OD260:30.19

Week's (Ambitious) Goals

Wednesday 5/30

Get all protocols
Identify materials/prepare order
Design Primers
Learn about budget/POs

Thursday 5/31

Do primer order
Start conjugation practice
Confirm restriction enzymes, ligases
Order confirmed/needed materials
Team Revew Meeting
Draft Thank-You Letters for our Sponsors

Friday 6/1

Evaluate/continue conjugation, practice electroporation for E. coli
Revise proposal to include possibility of screening with alginate beads and fluorocytometer
Meeting with Tim: Budgets/protocols, Pfizer/fundraising, iGEM registration, beads

Week of 6/4:

1. Evaluate the transformation that was done on Friday.

2. Confirm the correct plasmid (pJQ200)

3. Find appropriate restriction enzymes that cut by BLASTing all the plasmid's restriction enzyme sites onto the global transcription factors.

4. Re-design primers for the global transcription factors based on the restriction enzymes we have selected.

5. Order primers.

6. Practice regular (non-error prone) PCR with the primers to check that they work.

7. Incorporate the global transcription factors into the plasmid and transform this plasmid into the E.coli.

8. Conjugate this plasmid into Shewy.

Materials We Need

Primers: Need to Buy

Error-Prone PCR: Need to Buy

Plasmids: Need to Buy?

Restriction Enzymes: Need to Buy?

Ligases: Need to Buy?

Short-Term To-Do List

Lab Orientation: COMPLETED!

Lab Orientation Checklist

Lab Safety Training: COMPLETED!

Design of Primers: COMPLETED!

Ordering of Primers: COMPLETED!

Gathering of Protocols: Not Completed (Chris, please send me the protocols when they are gathered)

Ordering of Error-Prone PCR Materials: Not completed

Thank-You Letters sent to Pfizer: Not Completed

Thank-You Letters sent to BU ppl: Not completed

Protocols

"Calcium Chloride/Heat Shock Plasmid Transformations

Reagents to be Supplied by the User

LB plates with appropriate antibiotic concentration (for pET -25b(+), amipicillin at 50-100 microgram/mL) SOC media

1) Prepare one LB-Amp plate for each transformation, plus one plate for a negative (no plasmid) control. After storage at 2-8 degrees C, equilibrate to room temperature.

2) Centrifuge the tubes containing plasmid DNA to collect contents at the bottom of the tube. Add 1 microliter DNA to a sterile 1.5 mL tube on ice. Have one tube on ice with no DNA.

3) Add 50 microliters of competent cells (either freshly prepared, or frozen and thawed on ice). Avoid excessive pipetting, as cells are very fragile.

4) Gently flick the tubes to mix, and place on ice for 20 minutes.

5) Heat shock the cells for 45 seconds to 2 minutes in a water bath at exactly 42 degrees C. Do not shake.

6) Immediately return the tubes to ice for 2-10 minutes.

7) Add 950 microliters of room temperature SOC media to the tubes.

8) Incubate 1-1.5 hours at 37 degrees C with shaking (~150 rpm).

9) Plate 100 microliters of each transformation culture onto antibiotic plates.

10) Incubate the plates overnight (16-24 hours) at 37 degrees C.

See Making Heat Shock Competent Cells for more information. "

Negative Control: No plasmid

Trial 1:

We followed this protocol from step 3 in order to practice our transformation and conjugation techniques. The competent cells given to us by Joshua were E.coli sm10; the GFP plasmids (pMS291/lacI) were supplied by Ilaria. But we performed the protocol with four samples of plasmid DNA+E.coli. Each sample contained 1 microliter of 63.5 ng/microliter GFP plasmid DNA; 50 microliters of the competent E.coli cells; and 950 microliters of SOC media. 100 microliters of each sample tube were plated onto one kanamycin plate and one blank (non-antibiotic), control plate.

Trial 1 results:

A lawn of bacteria on each plate. No GFP fluorescence under UV illumination.

Trial 2:

Used E.coli sm10 cells, GFP plasmid pMS291 with lacZ promoter (stimulated by IPTG).

Sample 1 contained 50 microliter sm10 + 1 microliter mPS291.

Sample 2 contained 50 microliter sm10 + 1 microliter water.

Sample 3 contained 50 microliter E.coli Dh5-alpha + 1 microliter water.

Samples 2 and 3 served as negative controls (should show no colonies in the presence of kanamycin).

5 microliters of each of these samples were plated onto [LB]+[20microliters of 50X kanamycin]+[20microliters of 100micromolar IPTG].

50 microliters of each of these samples was also plated onto [LB]+[20microliters of 50X kanamycin]+[20microliters of 100micromolar IPTG].

Trial 2 results:

Colony growth on all plates. So the kanamycin is not killing any of the cells. Perhaps the kan plates were made incorrectly.

Trial 3:

Repeated Trial 2 but with double volume of kanamycin and double volume of IPTG (to ensure stimulation of lacZ promoter an thus GFP expression).

Trial 3 results:

5 microliters sm10 + water in IPTG and kan: lawn of colonies

50 microliters sm10 + water in IPTG and kan: less than 10 colonies

5 microliters sm10 + pMS291 in IPTG and kan: less than 10 colonies, fluorescent under UV illumination !

50 microliters sm10 + pMS291 in IPTG and kan: less than 10 colonies, no fluorescence

5 microliters Dh5-alpha + water in IPTG and kan: more than 100 colonies

5 microliters sm10 + water in kan: lawn of colonies

5 microliters sm10 + pMS291 in kan: lawn of colonies

5 microliters Dh5-alpha + water in kan: more than 100 isolated colonies

E.coli successfully transformed with pMS291 (showed fluorescence). These colonies will be grown out in SOC media for further examination. But kan still seems to be innefective.

[update] The succesfully transformed E.coli that fluoresced were grown out in SOC media and then centrifuged to obtain a pellet of cells. This pellet definitely fluoresced under UV confirming that the transformation was succesful.

Trial 4:

Two stocks were made: one with 20 microliters E.coli Dh5-alpha + pMS291 (lacZ), and the second with 20 microliters E.coli Dh5-alpha + water. Each of these two stocks were plated onto 4 different plates.

Plate 1 contained LB

Plate 2 contained LB + kanamycin

Plate 3 contained LB + kanamycin + IPTG

Plate 4 contained LB + kanamycin which was dripped onto the LB

Trial 4 Results:

20 microliters E.coli Dh5-alpha + pMS291 (lacZ)

Plate 1 containing LB: lawn of bacteria

Plate 2 containing LB + kanamycin: more than 100 colonies

Plate 3 containing LB + kanamycin + IPTG: more than 100 colonies and fluorescent!

Plate 4 containing LB + kanamycin which was dripped onto the LB: more than 100 colonies

20 microliters E.coli Dh5-alpha + water

Plate 1 contained LB: no colonies

Plate 2 contained LB + kanamycin: no colonies

Plate 3 contained LB + kanamycin + IPTG: no colonies

Plate 4 contained LB + kanamycin which was dripped onto the LB: no colonies

Therefore, the kanamycin worked correctly. E.coli without pMS291 (without kanamycin resistance) all died. Transformation was also successful

Question and Answer

Relevant Publications and Links

http://www.shewybase.bu.edu