Team:Caltech/Notebook/Degradation

Degradation Notebook

June 22, 2012
Created a spreadsheet detailing various possible bacterial strains and plasmids to be used for complex polymer degradation
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June 25 - 29, 2012
Sent out requests for bacterial strains and plasmids based on spreadsheet
Emailed Dr. Jared Leadbetter asking if he has strains + protocol for lignocellulose plates
Made carb. R plates
Made minimal media agar plates, bottom layer of overlay plates
Met with Professor Leadbetter to discuss difficulties with creating enrichment plates and finding bacterial samples
Will be following up on some of his suggestions

Notes from Meeting with Professor Jared Leadbetter from June 28th

Overlay Plate Options
- Top layer with minimal media, although either minimal media or just water + agarose should work
- Embed bacteria into top layer mix
- Use filter paper made of chosen carbon sources

Bacterial Strains
- Ponds around campus
- Beach
- Teredinibacter turnerae gen: helps degrade wood in shipworms (http://ijs.sgmjournals.org/content/52/6/2261.abstract)

May also choose a different host organism for transformation
- Instead of using E. coli can try using yeast (simplifies problem to only of degradation, and not also ethanol synthesis)
- Can also try bacteria found by Aztecs to make alcohol (Zymomonas mobilis)

Emailed for samples of nitrocellulose, latex, and teflon membranes.
Researched Z. mobilis.
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July 2-6, 2012
Researched zymomonas mobilis, narrowed down focus to strain ZM4
Gathered more information on protocols specific to that strain.

Looked up additional information about potential vectors to use to transform z. mobilis
Requested p42-0119 plasmid for expression z. mobilis from the Oak Ridge National Laboratory

Made 20% glucose solution stock and RM plates for Z. mobilis
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July 9-13, 2012
Ordered strains from the ATCC.
Discussed Biolog plates idea

Research z mobilis degradation 5 and 6 carbon sugars

Researched for genetic sequences for sugar degradation enzymes.

Sent in request for oligos sequences for alpha-gluctosidase gene (can degrade maltose)
Made tetracycline plates
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July 16-20, 2012
The pMQ30 is a deletion plasmid. We are using this plasmid to knock out expression of NADH dehydrogenase. According to the paper ''Respiratory chain analysis of high ethanol producing Zymomonas mobilis mutants,'' a mutant strain of Z. mobilis lacking NADH dehydrogenase had increased ethanol production. However, pMQ30 has gentamicin resistance, and Zymomonas is already resistant to gentamicin, so we are replacing it with tetracycline resistance from vector backbone pSB1T3.

The pLAFR5 plasmid is being used for conjugation from E. coli into Z. mobilis.
DKN79 (DH5alpha + pLAFR5) has been plated to grow more copies of pLAFR5.
DKN1005 (BW20427 [WM3064] + pLAFR5) has been plated to be used for testing conjugation into Z. mobilis ZM4.
The pMQ95 and pMQ97 plasmids are to be expressed in Zymomonas; however they contain antibiotic resistance genes that Z. mobilis is already naturally resistant to. So, we will need to switch out the resistances on the plasmids to tetracycline.

Electroporated pSB1T3 into competent E coli cells, incubated in LB for an hour, and streaked transformants onto tetra plates
Streaked DH5alpha + pMQ30 onto a gentamicin 20 ug/mL plate
Streaked Z. mobilis ZM4 from ATCC onto a RM plate and liquid culture.
Streaked out DKN79 (DH5alpha + pLAFR5) onto a tetracycline plate
Streaked out DKN1005 (BW20427 [WM3064] + pLAFR5) onto a tetracycline plate
Made LB media

No growth with pSB1T3 transformants, so retransformed and plated E coli + pSB1T3
Kept a liquid culture of E coli + pSB1T3
Ordered primers as part of switching out the pSB1T3 plasmid's resistance to tetracycline
Z. mobilis plate has no growth
Sent DNAWorks requests for als genes (alsBACE)
Re-streaked out DH5alpha + pMQ30 because first plate's growth was poor
Streaked out pMQ95 and pMQ97

There was no growth on the pSB1T3 plates. I kept a liquid culture of the electroporated cells that were left over after plating the on the tetra plates, and the tube looked very cloudy. The liquid culture did not have any antibiotics, but the plates had tetracycline. So it is possible that the gene is broken or its the wrong part or something. We decided to get the tetra resistance gene from a different source on the registry plates. So, I transformed BBa_K274002 and BBa_K274003 into E. coli from 2012 Kit Plate 3 12B, 2012 Kit Plate 4 20D, and 2012 Kit Plate 3 22D. I was originally trying to use BBa_J04450 from Plate 1, well 7A.
I was able to liquid cultures of pMQ30, pMQ95, pMQ97, DKN79 (DH5alpha + pLAFR5), and DKN1005 (BW20427 [WM3064] + pLAFR5). There has been no growth on the Z. mobilis plates yet. There seems to be growth in the Z. mobilis liquid culture, but not enough to make a glycerol stock. I designed primers for switching out resistance on pMQ95 or pMQ97 and for getting the tet resistance gene from the plated electroporated cells.

Growth has appeared on the Z. mobilis plates!!
Cleaned out the lab space
Made glycerol stock of Z. mobilis (one without gentamicin, one with gent)
Made liquid cultures of DKN1005 (BW20427 [WM3064] + pLAFR5), part from 2012 registry plate 1 7A, and 2012 registry part from plate 4 20A
Made glycerol stocks from liquid cultures of DKN1005 (BW20427 [WM3064] + pLAFR5) and part from plate 1 7A
Made more LB + tetracycline plates

Z. mobilis liquid cultures currently growing:
1. original (only Rich Media)
2. RM + amp (taken from original)
3. RM + amp (taken from original)
4. RM + gent (taken from plate)

Ordered primers for amplifying out the desired products from the plasmids (tetracycline gene from pSB1T3 and everything but the antibiotics gene from pMQ30, pMQ95, and pMQ97)
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July 23-27, 2012
Made liquid cultures of strains carrying pMQ30, pMQ95, pMQ97, and pSB1T3

Made Rich Media + tetracycline (25ug/mL) plates
Mini prep'ed the plasmids from the liquid cultures
Ran PCR on the mini preps

Ran a gel of the PCR products -> indicated that the plasmid backbones did not PCR correctly
Had another PCR run -> gel indicated that the correct plasmid backbones were produced

'''Constructing plasmids to be expressed in Z. mobilis ZM4'''
''There are three plasmids that we worked on synthesizing this week. We would like to use pMQ30, pMQ97, and pMQ95 in Z. mobilis. These plasmids carry gene resistances that Z. mobilis is already resistant. Therefore, we need to switch out the resistances to tetracycline. For the first two plasmids, we used Gibson assembly. Plasmid pMQ95 was altered by using yeast to assemble the fragments together. We could not use Gibson assembly for pMQ95 because the primers used for PCR amplification of the various DNA fragments would interfere with the promoter region for the new gene for tetracycline resistance.''
Purified the PCR products with QIAquick PCR purification kit of the tet gene DNA insert from pSB1T3 into the plasmid pMQ30 (i30), the tet gene DNA insert from pSB1T3 into the plasmid pMQ95 (i95), the backbone of pMQ30 (bb30), the backbone of pMQ97 (bb97), and the backbone of pMQ95 (bb95)
Digested i30 and bb95 with restriction enzyme DpnI and purified these products again
Ran gibson to build two plasmids: one made of bb30 and i30, and the other made of bb97 and i30
These plasmids were electroporated into E. coli and plated on tetracycline plates
Synthesized a plasmid made from bb95 and i95 in yeast
Made a liquid culture of DKN1005, which is the donor conjugation strain carrying pLAFR5

Tested conjugation of pLAFR5 into Z. mobilis ZM4 from DKN1005
Electroporated E. coli cells with pMQ30 and pMQ97 did not grow -> Plated a new set of electroporated cells with pMQ30 and pMQ97
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