1. Assignment 2
Write down your understanding on different types of RNA polymerase and compare their characteristics.
what will happen if you conduct a ligation on two DNA fragments, that one has Bgl II digested end and the other has BamH I digested end? After lygation, can Bgl II or BamH I chop the stitched DNA?

2. Isoschizomer （同裂酶）
Isoschizomers are pairs of REs specific recognize and cut to the same recognition sequence.
e.g. Sph I (CGTAC/G) and Bbu I (CGTAC/G)
The first enzyme to recognize and cut a given sequence is known as the prototype, all subsequent enzymes that recognize and cut that sequence are isoschizomers.
Isoschizomers are usually isolated from different strains of bacteria and therefore may require different reaction conditions.
An enzyme that recognizes the same sequence but cuts it differently is a neoschizomer. Neoschizomers are a specific type (subset) of Isoschizomers. For example, Sma I (CCC/GGG) and Xma I (C/CCGGG) are neoschizomers of each other.

3. Isocaudamer ( 同尾酶）
An enzyme that recognizes slightly different sequence, but produces the same ends is a isocaudomer.
Different optimum reaction conditioins and stabilities
e.g. isoschizomers MspI and HpaII
Recognition:
5'-CCGG-3'
MspI （＋）， HpaII（＋）
In some cases, only one out of a pair of isoschizomers can recognize both the methylated as well as unmethylated forms of restriction sites. In contrast, the other restriction enzyme can recognize only the unmethylated form of the restriction site. This property of some isoschizomers allows identification of methylation state of the restriction site while isolating it from a bacterial strain. For example, the restriction enzymes HpaII & MspI are isoschizomers, as they both recognize the sequence 5'-CCGG-3' when it is unmethylated. But when the second C of the sequence is methylated, only MspI can recognize both the forms while HpaII cannot.
5'-CCmGG-3'
MspI （＋）， HpaII（－）
This property can be used studying the DNA methylation status.

4. Chapter 3
vector 载体

5. In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell (host cell).
The four major types of vectors are
plasmids （质粒DNA） car
bacteriophages （噬菌体DNA） minivan
viruses, cosmids （病毒, 柯斯质粒） truck
artificial chromosomes （人工染色体） long vehicle
The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert DNA（gene） in the target cell.

6. Common to all engineered vectors are
（载体的共通性）
an origin of replication （复制起点）
a multicloning site （多克隆位点）
a selectable marker （筛选标记）
the copy numbers can be controlled （拷贝数可控）
can stably pass to the daughter cells （可稳定遗传）

7. 3.1 Plasmids and plasmid vectors
Plasmids are double-stranded generally circular and naked DNA sequences that are capable of automatically replicating in a host cell.
Can lead an independent existence in the bacterial cell.
Almost always carry one or more genes
These genes are responsible for an useful characteristic
displayed by the host bacterium.

8. e.g. Plasmid RP4
Plasmid RP4 carry genes for resistance to Ampicillin（氨苄）, Karamycin （卡那霉素Band Tetracyclne.
Only those E.coli cells that containing RP4 (or related plasmid) are able to survive in the medium that contains toxic amounts of one or all of above antibiotics.

9. 3.1.1 Properties of plasmid
Plasmids minimalistically consist of an origin of replication that allows for semi-independent replication of the plasmid in the host and also the transgene insert.
Size varies from 1 to over 1,000 kilobase pairs (kbp).
Generally, small plasmid make use of the host cell’s DNA replication enzymes to copy themselves.
Some of the larger plasmids carry genes that code for special enzymes for replication.

10. Non-integrative plasmid: Episomes::
A few types of plasmid are able to replicate by inserting themselves into the bacterial chromosome (episomes) .
Episomes may be stably maintained in this form through numerous cell divisions , but always are some stage exist as independent elements.
Non-integrative plasmid:
Episomes::
There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the left instance; whereas episomes, the right example, integrate into the host chromosome

11. The copy number refers to the number of plasmid molecules that normally found in a single bacteria. The number of identical plasmids within a single cell can range anywhere from one to even thousands under some circumstances. The factors that control copy number are not well understand.
Stringent: have a low copy number (for larger plasmid)
严密型
Relaxed: present in multiple copies for≧50
松弛型

12. Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.
Unlike viruses, plasmids do not encode genes necessary to encase (coat) the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer.
Plasmid host-to-host transfer requires direct, mechanical transfer by conjugation or changes in host gene expression allowing the intentional uptake of the genetic element by transformation

13. Plasmid transfer by conjugation
The donor and recipient cells attach to eachother by a pilus, a hollow appendage present on the surface of the donor cell. A copy of the plasmid is then passed to the recipient cell.

14. Conjugative plasmid: possess the ability to promote sexual conjugation
Non-conjugative plasmid: lacking of the set of transfer or tra genes that control conjugation and plasmid transfer.
A non-conjugative plasmid may, under some circumstances, be cotransferred alone with a conjugative plasmid when both are present in the same cell.

15. 3.1.2 Requirements of a vector
Only contain the necessary sequences of origin for maintenance in host cell ( to↑ loading capability)
Minimize the cleavage sites on its own sequence
Insert selectable markers (antibiotics, fluorescence proteins, etc) enable identification of cells that acquired
vector construct
Can replicate in limited host cells without recombination.
Some vectors contain inducible or tissue-specific promoters permitting controlled expression of introduced genes in transfected cells or transgenic animals.

16. One of the original plasmids used 4361 base pairs in length
3.1.3 Plasmid Vectors
pBR322
One of the original plasmids used
4361 base pairs in length
contains a replication region (source plasmid pMB1)
Two selectable markers (Amp and Tet resistance)
Several unique restriction sites scattered throughout plasmid (some lie within antibiotic resistance genes = means of screening for inserts)
Relaxed plasmid (Chloramphenicol 氯霉素 may induce up to 3000 copies per cell)
Created in 1977, it was named eponymously after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez

17. Map of plasmid pBR322
The plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie within the tetR gene. There are 2 sites for restriction enzymes HindIII and within the promoter of the tetR gene. There are 6 key restriction sites inside the ampR gene. The origin of replication or ori site in this plasmid is pMB1
The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the tet genes.

18. Insertional inactivation（插入失活）: Subcloning a DNA fragment into an active gene (usually a marker gene whose function can be easily detected) will disrupt the function of that gene. This can be detected by looking for colonies that no longer display that phenotype.
Can also been applied to select the cells host vector recombinant DNA:

19. pBR322 contains two antibiotic resistance genes, cloning an insert into one of these will disrupt that gene and inactivate the resistance to that antibiotic.
                                                                                                                                                    
For example, if DNA is cloned into the BamHI site in the Tetr gene, then transformed bacteria should be plate onto media containing the other antibiotic (ampicillin). Positive clones can then be selected by replica plating.
              

21. pUC18 Derivative of pBR322 Advantages over pBR322:
Smaller – so can accommodate larger DNA fragments during cloning (5-10kbp)
Higher copy # per cell (500 per cell = 5-10x more than pBR322)
Multiple cloning sites clustered in same location = “polylinker”

22. Interruptable gene encoding for enzyme beta galactosidase (lacZ)
Polylinker resides in the middle
Enzyme activity can be used as marker for gene insertion
Disrupted gene = nonfunctional
Intact gene = functional
Media containing XGAL used
(blue colonies = intact; white colonies = disrupted)
Amp resistance gene still present (= beta lactamase), Tet resistance gene omitted
XGAL: 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside

23. Map of PUC18/19 plasmids

24. -Galactosidase: enzyme involved in breakdown of lactose to glucose
and galactose.
encoding by lacZ that locates in the E. coli chromosome
Genetic engineered:
Usually, cloning vector posses a shortened derivative of lacZ (lacZ'), which encodes the N-terminal -peptide of  -galactosidase. A strain of E. coli is used that chromosome contain gene mutant encoding only the C-terminal portion of the enzyme,
The mutant E.coli can synthesize the complete enzyme only when they harbor the plasmid.
Foreign DNA insertion to the plasmid lacZ’ may lead -galactosidase function loss.

25. -galactosidase, which can also break down a derivative of lactose called X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, lactose analog). This produces a blue dye, which colours the colony blue.
IPTG (isopropyl-beta-D-thiogalactopyranoside) is a non-degradable lactose analogue which induce the lacZ gene expression. Transcription of lacZ can then occur and X-gal can be broken down.
If X-gal + IPTG + Amp adding in the culture medium agar, then the non-recombinant colonies will be colored blue, while the recombinant colonies with a disrupted lacZ’ gene and no -galactosidase inactivation will be white.

26. Cloning an insert into this site will disrupt the gene and recombinants will be unable to break down X-gal.
Colonies carrying plasmid with no insert will be coloured blue whereas colonies carrying recombinant plasmid will be white.
LBAagar plate:
X-gal + IPTG + Amp

27. most are phage promoters inserted plasmids
pUC derived plasmids
most are phage promoters inserted plasmids
provided the RNA polymerase recognize/binding sites
commonly used in RNA probe synthesis and protein expression
T7 promotere
Sp6 promotere
Amp promoter
pBR322 ori

28. 3.2 Cloning vector based on lambda bacteriophage
Lambda phage is a virus particle consisting of a head, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibers. The phage particle recognizes and binds to its host, E. coli, causing DNA in the head of the phage to be ejected through the tail into the cytoplasm of the bacterial cell.

29. definitions you should know:
Prophage(原噬菌体): a phage DNA integrated as a part of a bacterial chromosome.
lytic pathway（溶菌生长）: the type of bacteriophage infection involving phage multiplication (± 100) and release after lysis of the host cell..
lysogenic pathway（溶原生长）: the type of bacteriophage infection involving integration of phage DNA into a host chromosome.

30. icosahedron is a 20-face 3-D picture
icosahedron is a 20-face 3-D picture. The head of the lambda phage is an icosahedron.
Concatemer(多连联体) is a DNA sequence made of the succession of several genomes during phage replication. Each phage head receives one genome. Lambda phage concatemers are used as size markers in PFGE (Pulse Field Gel Electrophoresis)
multiplicity of infection (感染复数) is the ratio of phages to bacteria.
plaque is a zone of clearing on a lawn of bacteria caused by the lysis of the cells by infecting bacteriophages

31. Structure 3.2.1 Introduction to phage Composition Nucleic acid
Linear dsDNA
Protein
Protection
Infection
Head/Capsid
Contractile Sheath
Tail
Structure
Head or capsid
Icosahedral symmetry
Tail
Helical symmetry
Tail Fibers
Base Plate

32. Infection of Host Cells
Adsorption(吸附）
Irreversible attachment （可逆黏贴）
Sheath Contraction（尾鞘收缩）
Nucleic acid injection （核酸注入）

33. PHAGE LAMBDA - l - TEMPERATE LYTIC GROWTH OR LYSOGENY 48,502 bp
61 GENES locating
With functional
Clusters
Cos ends
COS
THE l CHROMOSOME
COS
COHESIVE SITE

34. Genome A to J early late Non-essential for lytic cycle
For foreign DNA substitution
structural protein encoding genes: from A to J, for head & tail
Recombination region: between J and N genes, for lysogen
Regulation region:
Cos site: 12nt sticky ends of 5’–GGGCGGCGACCT-3’
Repressor genes and early genes
λ-DNA Replication
Late genes

35. Linear Double Stranded
Lygase
Closed Circle
Cohesive Ends
Linear Double Stranded
Opened Circle
50: 50
excision
UV Induction
Two pathways of phage life cycle
Lysogeny
Lysis

36. Competition of lysis and lysogeny
Transcription of: Repressor gene and early genes
Translation produces: Repressor (CI wins)
Integrase
DNA Replicatioin (cro wins)
Repressor: Binds operators
Inhibits trasncription of genes in lytic growth
Stimulates own transcription
COMPETITION: REPRESSOR and LYTIC PROTEINS
Repressor wins: Shuts off lytic genes, continuous synthesis
Integration: Site specific recombination between phage
DNA and host DNA

37. l DNA IS NOW PROPHAGE; HOST IS NOW LYSOGEN
LYSOGEN, STABLE, LYSOGENY PASSIVELY REPLICATED
溶原生长
ATTACHMENT SITE
HOST CHROMOSOME
REPRESSOR
[REPRESSION]
GAL= GALACTOSE
BIO = BIOTIN OPERON
Immunity
INTEGRASE
PROPHAGE
l DNA IS NOW PROPHAGE; HOST IS NOW LYSOGEN
latent period

38. PROPHAGE INDUCTION 20 DNA DAMAGE REPRESSOR CLEAVAGE
LYTIC GENES NO LONGER INHIBITED
EXCISION, LYTIC GROWTH, PROGENY, LYSIS
~REPRESSOR FRAGMENT
EXCISIONASE
REPLICATION

39. Lytic Stages （溶菌生长） Synthesis of replication component Transcription
Translation
Energy
Precursors
Ribosomes
Assembly/ Maturation
Terminase cut concatemers
DNA packaged (size limitation)
Tails added
Inactive viruses produced
Lysis/Release
Lysozyme
peptidoglycan hydrolysis
viral mRNA
Viral DNA (RNA) chromsome
Viral structural proteins
Provided by host
ONE PHAGE-INFECTED CELL PRODUCES ~102 PROGENY IN ONE GROWTH CYCLE

40. package of virus particle :
Cos sites determine where specific cuts are made in the phage DNA sequence.
Packaging limit： the size of the DNA must fall between 78% and 105% of the wild-type phage DNA. No mater the DNA is original or manipulated
The distance between the cos sites can be lengthened by inserting a segment of non-phage passenger DNA.
If the distance between cos sites is too small or too large, the resultant phages will not be viable.

41. Lytic vs Lysogenic Cycle?
Role of CI repressor
Role of cro gene product
Role of proteases

42. 3.2.2 Construction of phage vector
Essential minimum phage DNA
- can maintain lytic cycle
- have less own cleavage sites (selection of natural
mutants, eg. EcoRI resistant mutant)
Central ~1/3 = “stuffer” fragment
can be substituted with any DNA fragment of similar size without affecting ability of lambda to package itself and infect E. coli
accommodates 5~25kbp of foreign DNA
(the package limitation)
foreign DNA either:
1) transfect into E. coli as naked DNA, or
2) package in vitro by combining with phage protein components (heads and tails) (more efficient, but labor intensive and expensive)

43. Screen for Mutant with No EcoRI sites

44. Insertion and replacement vectors（插入型和置换型载体）
An insertion vector: non-essential region deleted
two arms ligated with clone site.
The size of the foreign DNA fragment that an individual vector can carry depends on the extent to which the non-essential region has been deleted. Two popular insertion vectors are:
λg10: has a unique EcoRI site cI gene for a 8kb foreign DNA insertion
λZAPII: introduced a lacZ’ with a 6 RE sites. insertion DNA size up to 10 kb

46. Replacement vector :
two recognition sties for RE cloning
flanking a DNA segment (stuffer fragment)
could be replaced by the interest foreign DNA
containing additional RE sites for cut it into small pieces
Package size selection
λEMBL4: can carry up to 20 kb of inserted DNA. And the new DNA can be clioned using different RE sites.

47. The stuffer fragment get smaller, different sticky ends with unlikable re-lygation
The vector without new DNA insertion could not be packaged （too small）

48. Replacement of lambda DNA containing the red and gam genes in the lambda EMBL3 genomic DNA cloning vector with BamHI compatible DNA inserts of ~10-20 kb, permits lytic growth of recombinant phage in E. coli strains containing the P2 bacteriophage lysogen

49. Recombinant DNA package:
Cos sites determine where specific cuts are made in the phage DNA sequence, including the recombinant phage DNA.
The natural Packaging limits property allow the in vitro packaging be accomplished when DNA, appropriate enzymes, and cell extracts containing heads and tails proteins are combined. It takes advantage of viruses' natural ability to infect cells by creating infectious virus constructs capable of injecting DNA into a sensitive host cell.