7 - Cloning Genes for Synthetic Biology

Question 1

Molecular cloning

Answer 1

isolation of individual genes or other segments of DNA and moving them into an extrachromosomal DNA from another organism

2 stages: isolation, then insertion

Question 2

chimera

Answer 2

Hybrid molecule of DNA that has DNA from more than one source or organism

Question 3

cloning vector

Answer 3

Any molecule of DNA that can replicate itself inside a cell and is used for carrying cloned genes or segments of DNA. Usually a small multicopy plasmid or a modified virus

Question 4

vector

Answer 4

in molecular biololy: a molecyle of DNA that can replicate and is used to carry cloned genes or DNA fragments

in biology in general: an organosm that carries and distributes a disease-causing microorganism

Question 5

synthetic biology

Answer 5

branch of biology that genetically engineers microorganisms to create a new function.

Question 6

properties of cloning vectors

Answer 6

usually bacterial plasmids

example: ColE1 - basis of many vectors used in molbio. Modified - do not release toxins, introduce resistance to antibiotics
1) should be a reasonably small and manageable DNA molecule
2) moving the vector from one organism to another should be relatively easy
3) generating and purifying large amounts of vector DNA should be straightforward

For convenience:
1) a mechanism to select host cells containing the vector

2) ability to insert genes into the vector
3) detect the prescence of an inserted gene into the vector

Question 7

Key features for cloning vectors

Answer 7

Antibiotic reststance gene

selectable marker

promoter region

origin of replication (ORI) = ensure that it is replicated and inherited to daughter cells

multiple cloning site (MCS) or polylinker = series of RE sites that are used to connect the DNA fragment or GOI to plasmid

insert = the GOI that has been joined into the plasmid

primer binding site = complementary to PCR primer

terminators = ensures that only the GOI is transcribed

Question 8

origin of replication

Answer 8

contains the seq that are essential for the bac to rep the plasmid

controls how many copies are present, and when it is replicated

stringent plasmids = replicate during cell division

relaxed plasmids = replicate whenever

also determines whether two or more plasmids can be maintained in the same bacterial cell. Plasmids with the same type of origin are incompatible, and usually one is rejected.

Question 9

promoters

Answer 9

includes Rpol binding site + binding sites for reg proteins

must be compatible with host.

can be designed so the gene is only expressed under certain conditions, or expressed constitituvely

inducible promoter = a promoter that only turns on a gene under certain conditions

repressible promoters = promoter that turns off the gene expression under certain conditions

tissue-specific promoters = turns on genes specific to the type of tissue in which the cell funcitons.

Question 10

terminator/polyA seq

Answer 10

polyA tail = series of A found on the 3’ end of mRNA, stabilizes the mRNA in the cell

read-through = when Rpol does not stop trc at the terminator and continues to trc the DNA

the polyA tail is very important in euks as it stabilizes the RNA and makes sure it is not degraded

Question 11

Adding inserts to a vector (210 ish)

Answer 11

Key: overhang!!!!

5 methods:

1) RE CLONING
combines insert with cloning vector using REs to make compatible ends. If blunt ends are crates, T4 ligase is used to ligase, otherwise DNA ligase. most vectors have MCS/polylinkers (=contains cut sites for 7 or 8 commonly used REs)

2) TA CLONING OF PCR PRODUCTS
TA cloning uses Taq polymerase to generate single A overhangs, cloned with vector with mathcing T overhangs.
terminal transferase = enzyme that rec 3’ end, adds 1 nt. Taq pol has this ability (adds A to 3’ of dsDNA)
TA cloning vector = vector with single T overhang, used in TA cloning

can make a TA cloning vector by cutting vector with blund end, mix with terminal transferase + ddTTP

TA coning begins with PCR. Then mix with vector + DNA ligase

TOPO-TA removes ligation step. PCR first. Overhang of vector created with topoisomerase I. Stays on DNA until a complementary segment attaches, and ligates them

3) RECOMBINEERING INCREASES THE SPEED
homology = 2 or more pieces of DNA with similar seq at same position

recombineering = uses enzymes for homologous recombination from lambda in otder to combine an insert + vector.

lysogeny = state in which a virus replicates its genome in step with the host cell without making virus particles or destroying the host cell.
Advantages:
- Red (rebombination system) proteins only need a small region of homology (45bp) to integrate DNA into vector
- quick and easy procedure
- the system rec short ss oligio-nt and longer DNA fragments to initiate a recomb event. can therefore be used to create small deletions, insertions, or even single nt changes in any gene, even in the host genome.

Red system as three proteins (Gam, Beta, Exo) and is from lambda

The insert must have at least 50bp on each side homologous to the site on vector (can be created with tailed PCR primers).

step 1: get the empty vector + linear insert into the host bacteria’s cytoplam.

2: shift host bacteria to higher growth
3: lambda recombination enzyme genes are trc and trn into proteins, which rec the linear DNA fragment ends and integrate them into the vector.
4: bacteria shifted back to lower temp to stop Red enzyme production
5: recombineered vectors are removed and propagated in an E. coli strain without Red enzyme genes (preventing accidental recombination events)

4) ISOTHERMAL/GIBSON DNA ASSEMBLY
generates compementary ss overhangs between insert an dvectors, but uses a 5’ exonuclease to create them. The three enzymes needed are DNA exonuclease, DNA polymerase, DNA ligase. the exonuclease removes nts from 5’ end, leaving a ss 3’ overhang. the complementary sequences anneal (with ss gaps DNA polymerase fills in), and ligase ligates.

can be used on large inserts. Can be used to combine multiple fragments into one vector.

5) GATEWAY CLONING
relies on lambda, as the vectors use integration and exsition sites from the lambda genome for adding an insert.

a seq (attP) from the lambda phage DNA rec and combines with attB in bacterial genome. integrase from the phage directs the recomb event, and results in the attP being split to attL and attR on either side of the insertion. integration is reversible.

two steps:

1) BP reaction - creates an entry clone containing the DNA insert flanked by two attL sites.
2) LR reaction - creates an expression clone containing the DNA insert flanked by two attB sites.

BP reaction:
includes the int protein. Used to excise a DNA fragment flanked by attB and insert the DNA fragment into a vector with attP sequence.

Question 12

Adding vectors to host organisms with transformation

Answer 12

construct = noun used for the final assembled insert containing the GOI with the vector.

the construct is inserted into bacterial cytoplasm using transformation.

transformation = the procedure that uses calcium ions and temperature changes to get a plasmid DNA into the cytoplasm without killing the bacterium.

1: a tube of bacterial cells in solution containing calcium chloride is kept on ice. the ions cause bacteria to swell, making them competent for uptake of the plasmid.
2: add construct to bacteria solution
3: tube transferred to warm bath, allowing the constructs to enter bacterial cell through small pores that develop with temp change.
4: brief recovery in normal temp
5: cells plated onto nutrient agar to grow overnight. must contain the right antibiotic to select the bacteria with plasmids.

Question 13

Detecting inserts in vectors

Answer 13

have to know if the plasmid took up the desired insert.

one method is inserting resitance against two antibiotics, one for plasmid uptake-screening, one for insertion screening. The RE site must be in the second antibiotic restistance gene, causing disruption in the gene if the insert is inserted (= insertional inactivation). The bacteria with “empty” plasmids will be resistant to both antibiotics, the other only for one of them.

Reporter genes = genes used in the genetic analysis because its product is convenient to assay or easy to detect.

LacZ gene encodes beta-galactosidase. Two possible ways to monitor beta-galactosidase in bacterial colonies is bu allowing acces to X-gal and ONPG (turns blue and yellow, respectively, if beta-galactosidase is active.

alpha complementation = assembly of functional beta-agalactosidase from N-terminal alpha fragment plus rest of protein

alpha fragment = N-terminal fragment of Beta-galactosidase.

X-gal -> D-galactose and -bromo-4-chloro-3-indoxyl (unstable, reacts with oxygen in air)-> indigo type dye

Another way is to use ccdB from E.coli, encodes a toxin that blocks DNA gyrase (crucial for DNA replication during growth), leading to death in those bacteria with ccdB. ccdA encodes an antitoxin, inactivating the toxin.

GalK encodes calactose kinase, essential enzyme for growth on galactose, and which converts 2-DOG into a toxin that kills the bacteria. Typically used in the recombineering system (found on the vector before the insertion).

Vector with no insert = growth on galactose nutrient media, not 2-DOG.

Vector with insert = growth on 2-DOG, not galactose

Question 14

Types of cloning vectors

Answer 14

Shuttle vectors

bacteriophage lambda vectors

cosmid vectors

yeast artificual chromosomes

bacterial and P1 artificial chromosomes

expression vectors

mammalian expression vectors

Question 15

shuttle vectors

Answer 15

moving genes among organisms

can survive in and moved between more than one type of host cell

earliest were designed to shuttle between bacteria and yeast

the vector needs 3 things:

1) an origin of replication that works in yeast. Prok replication origins fo not work in euks (and vice versa), but between euks they are somewhat similar, and the ones for yeat may work in higher organisms
2) a centromere (Cen) seq to allow correct partition of the plasmid in yeast. Must be segregated correctly in cell division. the Cen seq will be rec by the microtubules that drag the chromosomes apart.
3) a gene to select for the plasmid in yeast. auxotrophic (=organism that harbours a defective essential gene and therefore has an additional nutritional requirement not founf in the other organisms of that species) yeast strains are often used. The corresponding biosynthetic gene is present in the vector. in the absense of the plasmid, the yeast will die due to the lack of essential organic compound

Question 16

bacteriophage lambda vectors

Answer 16

easy to propagate

cos seq = labmda cohesive ends = complementary 12 bp long overhang found at each end of the linear form of the lambda genome. after the infection of a bacterial cell, the ends ligate to form a circulat dna.

foreign DNA is inserted into the middle of lambda. inserted into E. coli by in vitro packaging (= procedure in which virus proteins are mixed with DNA to assemble infectious virus particles).

infecting two different E. coli cultures with two diff defect lambda mutants generates the necessary lambda proteins. each of the two mutants lacks head protein and cannot form particles containing its own DNA. A mix of the two lysates = full set of lambda proteins, if mixed with lamda DNA can generate infectious phage particles.

Question 17

Cosmid vectors

Answer 17

cosmid = small multicopy plasmid that carries lambda cos sites and can carry around 45 kbp of cloned DNA

fill the lambda head with cloned DNA.

The cosmid is first linearized so each end has cos. the cosmid and GOI are cut with RE to give identical sticky ends. The target DNA is often only partially digested. This allows large segments of the genome to be isolated, and if a cut site lies within the GOI, some fragments will still carry the intact gene.

The constructed cosmid can be packaged into lambda particles and used to infect E. coli

Question 18

Yeast artificial chromosomes

Answer 18

most euk genes are too long for bacterial plasmid vectors.

Huge segments of DNA (up to 2000 kbp) can be carried on YACs.

must have yeast specific origin of replication and a Cen seq. Telomere seq are also present on both ends (needed for euk).

Question 19

Bacterial and P1 artificial chromosomes

Answer 19

multicopy vectors give higher yield than single copy vectors. however, there are disadvantages, including unstable inserts (esp if long and contains repeated seq).

cloning of large segments for bacteria are done in BACs (=bacterial artificial chromosomes, single copy vector).

Another cloning vector for larger euk DNA seg is the PAC (P1 artificial chromosome, also single copy).

Question 20

expression vectors

Answer 20

vectors specifically designed to place a cloned gene under control of a plasmid-borne promoter

lamda left promoter (P_L) = one of the promoters repressed by binding of the lambda repressor or cI protein

lambda repressor (cI protein) = repressor protein responsible for maintaining the bacteriophage lambda in the lysogenic state

tet operon = bac genes that produce proteins that confer resistance to the antibiotix tetracycline

bacteriophage T7 = bacteriophage that infects E. coli, whise promoters are only rec by its own RNA polymerase

In practice, the GOI is normally first cloned by a general cloning vector and then transferred to an expression vector.

two basic alternatives for promoters: strong, tigthly regulated. Strong = if a lot of product is desired. Tightly regulated = in physiological experiments where the effects of gene expression are to be tested under varying conditions.

Question 21

mammalian expression vectors

Answer 21

study GOI in cultured mammalian cells.

mammalian cell lines = mammalian cells that are grown in a dish with nutrient media and housed in a special incubator to hold the cells at a specific temp. The air in the incubator also contains CO2 and O2 to imitate the natural environment of the cell. can be derived from any tissue.

introducing a vector into a host mammalian cell line is called transfection (not transformation)

transient transfection = process of transfecting a plasmid into a mammalian cell line without selecting for cells that have integraed the plasmid into the mammalian genome

stable transfection = process of transfecting a mammalian cell by selecting the cells that have integrated the plasmid into their genome.

Question 22

synthetic biology standardizes vector contruction

Answer 22

biological parts = term coined by synthetic biologists to describe DNA segments that encode the various regions of a vector, including promoter, terminator, coding seq, etc.

Question 23

DNA library

Answer 23

aka gene library

collection of cloned segments of DNA that is big enough to contain at least one copy of every gene from a particular organism

metagenomic library = collection of cloned segments of DNA that has genes from multiple organisms found in a particular enviroment.

Making:
using 4 base specific RE to cut the genomic DNA. Cuts about every 256 bases on average. this is shorter than an average gene, but the DNA is only partially digested (done by only allowing a short amount of time for the RE to cut DNA). This generates a mixture of fragments with various lengths, many of which will still have restriction sites for the enzyme. The hope is that an intect copy of every gene will be present on at least some of the fragments.

Question 24

screening DNA libraries

Answer 24

library screening is less relevant now that many species have had their genome sequenced.

whole exome sequencing = the process of sequencing only the exons from a genome. The process requires separation of exon sequences from the remaining genomic sequences.

1) clone all possible genes from an organism into a library
2) identidy GOI. Cloned DNA from a related organism is often used to screen a library. could also synthetize an artificial probe.

Target DNA is denatured to be single stranded. bacterial colonies containing the target DNA are first attached to a nylon membrane, and lysed open to the DNA adheres to said membrane. the probe is also denatured to be single stranded. When two ssDNA are mixed, the probe can anneal to its complemenrary seq in taget DNA. detection can be done in many ways.

Instead of looking for DNA/DNA hybrids, immunological screening can be used to identify GOI.

immunological screening = screening procedure that relies on Ab binding to protein.

The bacteria expressing the library inserts are grown on master plates, and samples of each bacterial colony are transferred to a suitable membrane. The cells are lysed and the released proteins are attached to the cel membrane, which is then treated with an Ab solution. After excess primary Ab is washed away, a second Ab specific for the first Ab is added. This will bind any primary Av it encounters. This secondary Ab carries the detection system, such as an alkaline phosphatase, which converts a colorless substrase to a colored product.

Question 25

cloning complementary DNA avoids introns

Answer 25

one problem when cloning euk genes: introns. Makes the gene longer (unnecessary), and the bacteria cannot process the RNA in the right way.

cDNA (complementary DNA) solves this problem. Made from mRNA + reverse transcriptase.