Streak colony transforman and Cryo Preservation

Today, early in the morning I streak colony from desired-putative recombinant into another petri-dish, so in the evening they could be inoculated into 5 ml LB containing appropriate antibiotic for plasmid isolation tomorrow.

I got enough putative clone especially from PPICZalpha-AO6 and AO5, except for PPICZB-GFP EcoRi and PPICZalpha-AO1. I remember the latter of two, its vector came from different source. AO5 and AO6 I got by myself. however, we cannot sure until we digest to verify desired clones.

I usually make LA+kanamycin (50 ug/ml) for recombinant E.coli containing kanamycine resistance. for example we we want to use 300 ml of LA we must put 300 ul kanamycine from stock 50 mg/ml.

I also streak e.coli recombinant for cryopreservation I got from my previous experiment as follows:

1. pOKT-kan4R

2. pOKT-GFP+S (EcoRI)

3. pOKPldhSlpA-AO1

4. pOKPldhSlpA-A05

5. pOKPldhSlpA-A06

In the evening, it’s about 3.30 pm I prepare for inoculating putative recombinant that I have streak early this morning ( we select 6 of each petri dish representing all of clones) for plasmid isolation tomorrow.

some previous clones must be refresh today:

1. pGEMT ICPPFN

2. pAS900

3. pGEMTGFPUV+S1

4. pOKT-GFPUV+S1

5. pOKT-GFP+S (EcoRI)

Zeocin (25 ug/ml) in 5 ml LB, so we have to add 2.5 ul Zeocin from stock 100 mg/ml.

Choosing Competent Cell E.coli strain

Source: http://bitesizebio.com/2007/09/24/choosing-a-competent-ecoli-strain

Of all the of competent E. coli cell strains available, which one should you choose? The choice of strain to use in a given experiment is determined in large part by the nature of the experiment and the set of traits that best fit it. In this article I summarize some of the most important traits and their benefits in downstream applications.

Note that when writing out a strain’s genotype, one usually lists only the known mutations and everything else is assumed wild-type. The mutant alleles are not given a minus sign, e.g. endA describes the endA null phenotype. Deletions are indicated by a delta before the mutant allele.

This is the third of three articles on E. coli competent cells and transformation. Part 1, Part 2

Traits that maintain the integrity of the transformed plasmid
Genotype	Description	Benefit
endA	Knock-out mutation in non-specific endonuclease (Endonuclease I). Eliminates non-specific endonuclease activity.	Improved plasmid yield/quality
hsdR	Mutations in hsdR prevents restriction of unmethylated EcoKI sites	Efficient transformation of DNA generated from PCR reactions
dam/dcm	Mutations in dam/dam abolish adenine and cytosine methylation at specific recognition sequences.	Propagation of DNA for cleavage with methylation-sensitive restriction enzymes e.g. Ava II, Bcl I
mcrA, mcrBC,or mrr	Mutations in these genes prevents methylated DNA from other organisms from being recognized as foreign	Allows cloning of genomic DNA or methylated cDNA
recA	Mutation in recA reduces DNA recombination	Increased plasmid DNA stability
recBCD	recBCD encodes exonuclease V. Mutation in RecB or RecC reduces DNA recombination by a factor of 100.	Increased plasmid DNA stability
recJ/sbcC	Also involved in DNA recombination. Inactivation increases plasmid DNA stability	Increased plasmid DNA stability
uvcR/umuC	Involved in the UV and SOS DNA repair systems respectively. The inactivation of these genes increases the stability of plasmids carrying inverted repeats	Increased plasmid DNA stability

Traits for the identification of positive clones
Genotype	Description	Benefit
lacZ-delta-M15	Deletion of the N-terminal alpha-fragment from the LacZ gene, making ?-galactosidase function dependent on expresion for the lacZ-? fragment from another source (e.g. a plasmid)	Used for blue/white screening of recombinant plasmids carrying the lacZ-? fragment
lacI	The lac repressor. Inhibits expression from the Lac promoter in the absence of lactose/IPTG	A functional lacI is required for blue/white screening

Traits for improved transformation efficiency
Genotype	Description	Benefit
deoR	Deletion of a regulatory gene, allowing constitutive expression of deoxyribose synthesis gene	Increases the transformation efficiency for large plasmids
hee	Stands for “high electroporation efficiency”. Increases survival rate of cells during electroporation, leading to higher transformation efficiency.	High transformation efficiency
hte	Stands for “high transformation efficiency”.	High transformation efficiency.

Traits Related to Protein Expression
Genotype	Description	Benefit
lacIq	This mutated version of LacI gives high levels of lac repressor expression. This allows tighter regulation of gene expression from the lac promoter	Tightly regulated gene expression from lac promoter
DE3	Lysogen that encodes T7 RNA polymerase. Used to induce expression in T7-driven expression systems	Required for expression from the T7 promoter in E.coli
pLysS	pLysS is normally plasmid borne. It harbors T7 lysozyme, which destroys T7 polymerase produced from DE3. Used to reduce basal expression in T7-driven expression systems by inhibiting basal levels of T7 RNA polymerase	Tightly regulated gene expression from T7 promoter
lon	Defficiency in the Lon ATPase-dependent protease. Decreases the degradation of recombinant proteins; all B strains carry this mutation	Reduced degradation of recombinant proteins
ompT	Defficiency in an outer membrane protease.	Reduced degradation of recombinant proteins
araD/ara-14	Cannot metabolize arabinose	Enhances expression from araB promoter
dnaJ	dnaJ encoded chaparonin is inactivated	Improves folding of some heterologous proteins
gor	Mutation in glutathione reductase, which enhances disulphide bond formation	Improves folding of heterologous proteins requiring disulphide bonds

The table below has a few suggestions of competent cell strains to use for some applications. All of these strains are available from Invitrogen or Stratagene, although many other manufacturers make the same or equivalent strains:

Application	Strain
Routine Cloning/Sub-cloning, Blue/white screening	XLI-Blue, DH5-alpha, top10
Very high efficiency cloning e.g. for library construction	XL10-Gold, MegaX DH10b
Cloning of unmethylated DNA	XL1-Blue MR
Production of unmethylated DNA	JM110, INV110
Cloning of unstable plasmids	Sure, Stbl4
Expression from T7 promoter	BL21 (DE3)
Expression from T7 promoter, tight regulation	BL21 (DE3) pLysS
Expression from T7 promoter with codon bias correction	BL21 codon plus, Rosetta
Improved disulphide bond formation	Origami
Fast cloning (due to quick cell growth)	Mach1

Transformation PPICZalpha-PhyAO 1,5,6 , PhyAF, and PPICZB-GFP+S1 EcoRI into E. coli

Continuing previous experiment, today I’m going to transform gene encoding phytase from aspergillus oryzae, Aspergillus niger,  GFP+S1 (we mutated this gene through PCR and cloned into vector pGEMT easy, amazingly It glows up) into E.coli DH5alpha.

this morning we have subcultured E.coli 1% of total volume (1% of 25 ml=250uL) into fresh LB 25 ml, then incubated at 37 Celcius degree for about 3 hours. after 3 hour we prepared for making competent cells, heat shock, recovery cell, and plating onto Luria Agar containing antibiotic resistance.

if your protein is cytosolic and non-glycosilated, you may elect to express the protein intracellularly using one of the PPICZ vectors. PPICZ A,B,C (www.invitrogen.com) well-known are expression vector in yeast especially P. pastoris.

however if you wish your protein expressed outer cell membrane,glycosilated, or directed into an intracellular organelle, you may wish to try secreting your protein using on of the PPICZ alpha vector. invitrogene had recommended us to try both the native secretion signal and alpha-factor signal sequence in order to secrete our protein.

Finishing transformation is to plate e.coli recovered-cells onto appropriate media.

1. pOKBAD4-Sp/Sm  (LA+Km 50+Sp25)               6. PPICZB-GFP+S1 EcoRI (LA+Zc 25)

2. pOKBAD9-Sp/Sm  (LA+Km 50+Sp25)               7. PPICZ-alpha -AF new (LA+Zc 25)

3. pOKBAD13-Sp/Sm  (LA+Km 50+Sp25)             8. PPICZ-alpha -AO1 (LA+Zc 25)

4. pOK-E.coli hemB-Gm (LA+Km 50+Gm 10)       9. PPICZ-alpha -AO5 (LA+Zc 25)

5. pGLO-GFP+S1 EcoRI (LA+Ap100+Ara 0.2%)    10. PPICZ-alpha -AO6 (LA+Zc 25)

Maps of PPICZ can be viewed at www.genomex.com/vector_maps/ppicz_map.pdf

Transformation PPIZ A/B + GFP+S1 (EcoRI) and PPIZalpha+AO (Eco/XbaI)

Pagi hari udah bangun pagi-pagi “olah raga” nonton Piala Eropa di babak perempat final Spanyol vs Italia…eh udah ditungguin selama 2 jam, taunya cuma adu pinalti doang yang dimenagkan oleh Spanyol…bete banget..:(

pekerjaan di hari senin ini adalah geneclean DNA fragment hasil digest dengan beberapa enzyme, saya akan melakukan kloning gen GFP dan Phytase dari Aspergillus oryzae ke dalam yeast (pichia pastoris).

vektornya tidak lupa di tambahkan CIP 0.5 uL lalu diinkubasi lagi selama kurang lebih 2 jam, lalu di elektrophoresis dech….100v 40 menit tut..tut (waktu elektroforesis tergantung panjang nya gel) jangan sampe kita lengah,, takut ngga taunya lolos deh DNA yang kita elektrophoresis…jadi sia2 bukan pekerjaan kita jadinya kalo begitu??

udah dapet digeneclean kita check hasilnya untuk memastikan bener2 fragmentnya 1 pita dan ketebalan pita mempengaruhi berapa banyak kita akan meligasi..

setelah oke kita lakukan ligasi vektor dengan insertnya dengan enzyme T4 ligase (promega) dilanjutkan dengan pembuatan media untuk transformasi dan juga inokulasi e.coli DH5a juga sudah ke media LB.

Display of alpha amylase on the surface of lactobacillus casey cells by use of the PgsA Anchor protein, and Production of lactic acid from starch