
Background
Using synthetic biology methods, the Escherichia coli K-12 genome was reduced by making a series of planned, precise deletions. The multiple-deletion series (MDS™) strains (1), with genome reduction of up to 15%, were designed by identifying non-essential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving robust growth and protein production. Genome reduction also led to unanticipated beneficial properties, including high electroporation efficiency and accurate propagation of recombinant genes and plasmids that are unstable in other strains. Subsequent deletions and introduction of useful alleles produce strains suitable for many molecular biology applications. Recently, Scarab has built on the MDS™42 foundation strain, by creating the MDS™42 Meta ΔrecA strain. It improves the already high density fermentation of the MDS™42 strain. The MDS™42 Meta ΔrecA strain’s optimized metabolism permits fermentation ≥OD300 in minimal media in ~24 hrs without glucose spike or cell lysis resulting in >40 g/L of a test protein at the 10 liter scale. It produced ~700 mg/L of pGWIZ GFP test plasmid in minimal media at the 10 liter scale without a temperature shift.
Figures
Figure 1. High Plasmid Yield without a Temperature Shift using Minimal Media. The MDS™42 Meta ΔrecA strain produced ~700 mg/L of pGWIZ GFP test plasmid in 10 liter scale fed batch fermentation without a temperature shift using Scarab’s ultra minimal media.
Figure 2: Multiple Deletion Strains tolerate "deleterious” genes. A chimeric gene composed of VP60 of rabbit hemorrhagic disease virus fused to the B subunit of cholera toxin (CTX) was very unstable in E. coli. Individually, both genes were stable in E. coli HB101, C600 and DH10B, but pCTXVP60 carrying the fusion gene in the same hosts did not produce fusion protein and was recovered in low yields. All recovered plasmids contained mutations in the CTXVP60 open reading frame, virtually all resulting from IS insertions. In contrast, the recombinant plasmid was completely stable in MDS™; normal yields of plasmid DNA were obtained. Representative restriction patterns of pCTXVP60. (A) Plasmid DNA from MDS™42 was transformed and propagated in the indicated host, then digested with NcoI and EcoRI. A representative of each restriction pattern was purified and sequenced. M, molecular weight marker, 1 kbp ladder; 1, MDS™41, no insertion; 2, MDS™42, no insertion; 3, DH10B, IS10 insertion; 4, DH10B, IS10 insertion/deletion; 5, C600, IS5 insertion; 6, C600, IS1 insertion; 7, C600, IS1 insertion. (B) Relative position of the IS element insertion sites in the CTXVP60 reading frame determined for the five examples presented.
Figure 3: Plasmid stability in different host strains. Left: during four subcultures of pT-ITR, a plasmid with viral LTR segments; Lane 0, isolated plasmid DNA before subculture, lanes 1-4, successive subcultures. Plasmid DNA was digested with restriction enzymes and analyzed by agarose gel electrophoresis. KpnI cuts the plasmid at a single site, but in MG1655 two bands indicate a deletion in the plasmid. MscI cuts at two locations, but in MG1655 a third intermediate band confirms that the plasmid is deleted. Right: Stability of four variants of a Lentiviral expression plasmid in MDS™42 ΔrecA and Stbl3™ (Life Technologies), showing the proportion of transformants containing intact plasmids (Table 2 BioTechniques 43:466-470 (October 2007)(2).
Specifications
Kit Components MDS™42 Meta ΔrecA Electrocompetent Cell Kit pUC19 Control DNA (10 pg/µl) SOC Medium Genotypes MG1655 multiple-deletion strain (1) relA* Δrph ΔarpA ΔiclR ilvG+ ΔrecA(1819). Quality Control Transformation efficiency is tested using pUC19 Control DNA, in duplicate. Transformed cells are plated onto LB plates containing 50 µg/ml carbenicillin. Transformation efficiency is > 5 x 109 cfu/µg DNA. Storage Conditions Store components at –80°C. Do not store cells in liquid nitrogen.
Related Products
White Glove IS Detection Kit
Support
Product Manuals MDS™42 Meta ΔrecA Electrocompetent Cell Kit Papers
- Pósfai G, et al., (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044-6.
- Chacko S. Chakiath, CS & Esposito, D (2007): Improved recombinational stability of lentiviral expression vectors using reduced-genome Escherichia coli. BioTechniques 43:466-470.
Patents & Disclaimers
Products are sold for non-commercial use only, under Scarab Genomics limited use label license: Limited Label Use.Scarab is providing you with this Material subject to the non-transferable right to use the subject amount of the Material for your research at your academic institution. The Recipient agrees not to sell or otherwise transfer this Material, or anything derived or produced from the Material to a third party. NO RIGHTS ARE PROVIDED TO USE THE MATERIAL OR ANYTHING DERIVED OR PRODUCED FROM THE MATERIAL FOR COMMERCIAL PURPOSES. If the Recipient makes any changes to the chromosome of the Material that results in an invention in breach of this limited license, then Scarab will have a worldwide, exclusive, royalty-free license to such invention whether patentable or not. If the Recipient is not willing to accept the terms of this limited license, Scarab is willing to accept return of this product with a full refund, minus shipping and handling costs. For information on obtaining a license to this Material for purposes other than research, please contact Scarab’s Licensing Department. Scarab Genomics’ technology is covered by U.S. Pat. No. 6,989,265 and related foreign applications. Clean Genome® is a registered trademark of Scarab Genomics, LLC.
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支原体培养则是取样后在培养基上培养,看有多少支原体菌落会长出,是比较直观和可信的结果。
总体来讲,这两种检查手段可信度都较高,结合一起,不仅可以可靠的知道有无解脲支原体感染,还能知道感染是否严重。
RMgX + RX = R-R + MgX2.
这个反应需要的能量比生成格氏试剂的高,
因此降低反应温度是第一个选择。
其次, 增加镁得摩尔比, 让 RX与镁有更多机会反应, 而不是与RMgX。
第三, 降低RX的浓度, 即用更多的溶剂, 因为溶剂和格氏试剂有很显著的溶剂络合。
第四, 缓慢滴加RX., 即降低RX.在反应体系的浓度。
第五, 增加搅拌速率, 即, 让RX.与镁有更好的接触。
1. 姜黄素是一个典型的HAT抑制剂。
2. 针对P300: 在大约10年前,Cole和他的同事设计出了一种p300/CBP抑制剂,发表在nature杂志上。
希望能帮到你,望采纳!
Vanderbilt-Ingram癌症中心的主任HalMoses博士和他的实验室研究人员,于1985年辨识出TGF-b,这种物质具有生长刺激物和生长抑制的作用。从那段时间之后,TGF-b在直肠、乳房和其他癌症中所扮演的角色便获得研究人员的广泛研究。
如今Vanderbilt-Ingram癌症中心的一组研究人员,发现了TGF-b矛盾的生物学作用之线索。他们的研究结果将发表于2003年12月的NationalAcademyofScience网络版,网址为www.pnas.org,这篇研究将于2003年12月稍后发表于纸本中。
TGF-b通常会抑制细胞生长,但是,很多实质性肿瘤会过度表现TGF-b,而使细胞不完全被抑制,事实上,有时候它们因为TGF-b的讯息使肿瘤细胞的生长比正常细胞更快。
TGF-b使用多种标记途径将它的指令送达细胞核心,已知大约有至少四条途径以上。研究人员去除一种特殊蛋白质组成的TGF-b标记:Rho-ROCK。细胞便不再受到抑制,而再度开始生长。
[信息来源:中国科技信息网站]
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MaggotsFasterThanScalpelinWoundDebridement
December19,2011—Maggotdebridementtherapy(MDT)appearstobemoreeffectiveforwounddebridementcomparedwithconventionaltherapy,butonlyat1week;afterthattime,anothertypeofdressingshouldbeused,newresearchsuggests.
KristinaOpletalovà,MD,fromtheDepartmentofDermatology,UniversityofCaen,France,andcolleaguespublishedonlineDecember19intheArchivesofDermatology.
MedicalmaggotswereapprovedbytheUSFoodandDrugAdmiNISTrationasamedicaldeviceforwounddebridementin2004.Accordingtotheresearchers,useofmaggotsintreatingwoundsisassociatedwitheffectivewounddebridement,antibacterialeffects,andstimulationofwoundhealing.
However,theypointout,"[r]elativelyfewclinicalstudieshavebeenconductedandtheresultsarenotclear,partlyowingtomethodologicassessmentproblems."
InthecurrentProspective,randomizedcontrolled,phase3clinicaltrial,theresearcherssoughttodeterminetheefficacyofbaggedlarvaeonwounddebridementincomparisonwithconventionaltreatment.
TheprimaryobjectivewastocomparethemeanpercentageofsloughinwoundstreatedwithMDTwiththatofconventionaltreatmentatday15.Thestudyincluded119patientswithanonhealing,sloughywoundthatwas40cm2orsmallerandlessthan2cmdeep.Patientsalsohadananklebrachialindexof0.8orhigher.
Treatmentwasadministeredduringa2-weekhospitalstay.Conventionaltreatmentconsistedofsurgicaldebridement3timesaweekwithascalpel,withuseoftopicalanesthesia.TheMDTwasadministeredusinganencloseddressing(Vitapad,BioMondeLaboratories)containing80sterilemaggots.Atdischarge,aconventionaldressingwasapplied,andpatientswerefollowed-upatday30.
DebridementbyMDTwassignificantlyfasterthansurgicaldebridementduringthefirstweekoftreatment,reachingthesamelevelthecontrolgroupreachedatday15.NobenefitforMDTcomparedwithconventionaltreatmentinhealingrateswasobserved.Atday8,54.5%intheMDTgroupvs66.5%inthecontrolgroup(P=.04)hadevidenceofsloughandwoundhealing.However,byday15,themeanpercentageofsloughwas55.4%intheMDTgroupand53.8%inthecontrolgroup(P=.78).
"AthoughMDTshowsnosignificantbenefitatday15comparedwithconventionaltreatment,debridementbyMDTissignificantlyfasterandoccursduringthefirstweekoftreatment,"theresearchersconclude."Becausethereisnobenefitincontinuingthetreatmentafter1week,anothertypeofdressingshouldbeusedafter2or3applicationsofMDT."
Painscoresweresimilarandmildinbothgroups,althoughincontrasttoconventionaltreatment,MDTwasperformedwithouttopicalanesthesia.
Accordingtotheresearchers,noneofthepatientswerereticentaboutundergoingMDT."[A]crawlingsensationonthewoundwasrarelyandalmostequallynotedinbothgroups,revealingthatthesensationwassubjective,"Dr.Opletalovàandcolleaguespointout.
TwoquestionsregardingMDTremainunanswered,theauthorsnote."Candebridementbeimprovedusingmoremaggotsperdressing?Ifso,wouldthesedressingsbemorepainful?Furtherstudiesareneededtoanswerthesequestions."
ThestudywassupportedbygrantsfromtheClinicalResearchHospitalProgramandfromtheFrenchSocietyofDermatology.Theauthorshavedisclosednorelevantfinancialrelationships.
、
Cell-Based ELISA
的优点:
Cell-Based ELISA
(基于细胞的
ELISA
)是一种全新的
ELISA
技术,有两个最突出的优点:
1.1
不需要抽提蛋白、包被微孔板:细胞直接在微孔板里培养,待检测的时候,将细胞固定在微孔板上并
进行通透处理即可。这样就避免了抽提蛋白时,由于客观和主观上引起样品的损失而导致实验结果在一定
程度上偏离了实际情况。
同时不用包被微孔板,
简化了实验流程,
有助于提高效率。
科研人员在一个
96
孔
酶联板上,便能检测目标细胞蛋白经刺激或抑制作用后的表现。由于省去抽提蛋白和裂解细胞的步骤,样
本的损失也能降到最低,比起其他普通的
ELISA
测定方法,这项全新的
ELISA
技术能更快速、更方便
地一次检测大量的细胞内蛋白。
1.2
可同时检测两种不同蛋白:
封闭后加入两种抗不同蛋白且来源于不同宿主的一抗,
然后再加入不同的
二抗,加入两种荧光底物,检测两个波长。同时检测两种蛋白的好处是显而易见的,可以减少工作量,此
外还可以满足一些特殊的实验需要,例如,需要测定某个蛋白的磷酸化比例,就需要测定磷酸化蛋白的数
量和总蛋白的数量,这两个测定在同一次实验进行,有助于消除实验误差,得到更为精确的实验结果。
2
、
Cell-Based ELISA
的两种技术:
某些公司发展了双通道
Cell-Based ELISA
技术;
双通道与单通道
Cell-based ELISA
比较:
双通道
cell-based ELISA
,顾名思义,即一次可以同时检测两种目的蛋白,
R&D Systems
提供的
Cell-based ELISA
就是双通道
cell-based ELISA
,原理略:(需要荧光检测方式和相应仪器);
单通道
cell-based ELISA
,即一次只能检测一种目的蛋白,他和普通
ELISA
的主要区别在于样品处理过
程
3
、
cell-based ELISA
的应用:
该产品,最多发展起来的是用于检测磷酸化和非磷酸化蛋白的相对含量;
4
、有
cell-based ELISA
产品的公司:目前,多家
elisa
产品提供
商均
提供
cell
based
elisa
试剂盒,
如
Rnd
systems
,
raybiotech
,
ebioscience
,
millipore
等公司;
5
、如何自己进行
cell based elisa
实验?
事实上,根据单通道的
cell based elisa
原理,可以自己建立
cell based elisa
实验系统;
细胞加入培养板中;
加入刺激物或者抑制剂进行培养;
对细胞进行固定或者封闭;
加入第一抗体;
加入
HRP
结合的二抗(二抗的选择,同
western blot
);
加入底物进行显色;

